Evidence Report/Technology Assessment 

Number 150 

Hereditary Nonpolyposis Colorectal Cancer: 

Diagnostic Strategies and Their Implications 

Prepared for: 

Agency for Healthcare Research and Quality 

U.S. Department of Health and Human Services 

540 Gaither Road 

Rockville, MD 20850 

www.ahrq.gov 

Contract No. 290-02-0022 

Prepared by: 

Tufts-New England Medical Center Evidence Based Practice Center, Boston, Massachusetts

Investigators 

Peter A. Bonis, M.D., Principal Investigator 

Thomas A. Trikalinos, M.D., Investigator 

Mei Chung, M.P.H., Investigator 

Priscilla Chew, M.P.H., Investigator 

Stanley Ip, M.D., Investigator 

Deirdre A. DeVine, M.Litt., Project Manager 

Joseph Lau, M.D., Project Director 

AHRQ Publication No. 07-E008 

May 2007
This report is based on research conducted by the Tufts-New England Medical Center Evidencebased Practice Center (EPC) under contract to the Agency for Healthcare Research and Quality 

(AHRQ), Rockville, MD (Contract No. 290-02-0022). The findings and conclusions in this 

document are those of the author(s), who are responsible for its contents, and do not necessarily 

represent the views of AHRQ. No statement in this report should be construed as an official 

position of AHRQ or of the U.S. Department of Health and Human Services. 

The information in this report is intended to help clinicians, employers, policymakers, and others 

make informed decisions about the provision of health care services. This report is intended as a 

reference and not as a substitute for clinical judgment. 

This report may be used, in whole or in part, as the basis for development of clinical practice 

guidelines and other quality enhancement tools, or as a basis for reimbursement and coverage 

policies. AHRQ or U.S. Department of Health and Human Services endorsement of such 

derivative products may not be stated or implied. 
ii 

This document is in the public domain and may be used and reprinted without permission except 

those copyrighted materials noted for which further reproduction is prohibited without the 

specific permission of copyright holders. 

Suggested Citation: 

Bonis PA, Trikalinos TA, Chung M, Chew P, Ip S, DeVine D, Lau J. Hereditary Nonpolyposis 

Colorectal Cancer: Diagnostic Strategies and Their Implications. Evidence Report/Technology 

Assessment No. 150 (Prepared by Tufts-New England Medical Center Evidence-based Practice 

Center under Contract No. 290-02-0022). AHRQ Publication No. 07-E008. Rockville, MD: 

Agency for Healthcare Research and Quality. May 2007. 

None of the investigators has any affiliations or financial involvement that conflicts 

with the material presented in this report. 
iii

Preface 

 The Agency for Healthcare Research and Quality (AHRQ), through its Evidence-Based 

Practice Centers (EPCs), sponsors the development of evidence reports and technology 

assessments to assist public- and private-sector organizations in their efforts to improve the 

quality of health care in the United States. The Centers for Disease Control and Prevention 

(CDC) requested and provided funding for this report. The reports and assessments provide 

organizations with comprehensive, science-based information on common, costly medical 

conditions and new health care technologies. The EPCs systematically review the relevant 

scientific literature on topics assigned to them by AHRQ and conduct additional analyses when 

appropriate prior to developing their reports and assessments. 

 To bring the broadest range of experts into the development of evidence reports and health 

technology assessments, AHRQ encourages the EPCs to form partnerships and enter into 

collaborations with other medical and research organizations. The EPCs work with these partner 

organizations to ensure that the evidence reports and technology assessments they produce will 

become building blocks for health care quality improvement projects throughout the Nation. The 

reports undergo peer review prior to their release. 

 AHRQ expects that the EPC evidence reports and technology assessments will inform 

individual health plans, providers, and purchasers as well as the health care system as a whole by 

providing important information to help improve health care quality. 

 We welcome comments on this evidence report. They may be sent by mail to the Task Order 

Officer named below at: Agency for Healthcare Research and Quality, 540 Gaither Road, 

Rockville, MD 20850, or by e-mail to epc@ahrq.gov. 

Carolyn M. Clancy, M.D. 

Director 

Agency for Healthcare Research and Quality 

Jean Slutsky, P.A., M.S.P.H. 

Director, Center for Outcomes and Evidence 

Agency for Healthcare Research and Quality 

Julie Louise Gerberding, M.D., M.P.H. 

Director 

Centers for Disease Control and Prevention 

Beth A. Collins-Sharp, Ph.D., R.N. 

Director, EPC Program 

Agency for Healthcare Research and Quality 

 Gurvaneet Randhawa, M.D., M.P.H. 

EPC Program Task Order Officer 

Agency for Healthcare Research and Quality 
iv

Acknowledgments 

We would like to acknowledge the Centers for Disease Control and Prevention for its funding of 

the evidence review project. We also acknowledge with appreciation the following members of 

the Technical Expert Panel for their advice and consultation to the Evidence-based Practice 

Center during preparation of this report: 

Linda Bradley, Ph.D. 

Evaluation of Genomic Applications 

 in Practice and Prevention (EGAPP) 

Office of Genomics and Disease Prevention 

Centers for Disease Control and Prevention 

Atlanta, Georgia 

Robert H. Fletcher, M.D., M.Sc. 

University of North Carolina 

Chapel Hill, North Carolina 

James Haddow, M.D. 

Women & Infants Hospital 

Providence, Rhode Island 

Celia Kaye, M.D., Ph.D. 

University of Colorado School of Medicine 

Denver, Colorado 

National Newborn Screening and Genetics 

 Resource Center 

University of Texas Health Science Center 

San Antonio, Texas 

Carolyn Sue Richards, Ph.D., F.A.C.M.G. 

Molecular Diagnostic Center 

Oregon Health & Science University 

Portland, Oregon 
v

Structured Abstract 

Objectives: Hereditary Nonpolyposis Colorectal Cancer (HNPCC) has been defined clinically 

and genetically. The disorder has traditionally been recognized in kindreds with a clustering of 

related cancers in association with mutations in DNA mismatch repair genes. HNPCC is 

associated with a substantially increased risk for several forms of malignancy but particularly 

colorectal and endometrial cancer. 

 There were three main objectives of this report: (1) to assess the sensitivity, specificity, and 

reliability of laboratory and genetic tests commonly used in evaluating patients for HNPCC 

(analytic validity); (2) to summarize the accuracy of commonly used clinical and laboratory 

characteristics for predicting the presence of HNPCC in patients with colorectal cancer (clinical 

validity) and use these estimates to describe the efficiency of various strategies for identifying 

patients with a mismatch repair mutation; (3) to describe the benefits and harms related to 

screening and testing patients with colorectal cancer and their family members for HNPCC. 

Data Sources: Published literature identified through an electronic search (through April 2006), 

review of relevant bibliographies, and suggestions from technical experts. 

Review Methods: We evaluated studies critically and summarized the data qualitatively or by 

meta-analysis when studies used similar methodology and endpoints. We used decision trees to 

describe the efficiency of various strategies for identifying patients with HNPCC from a 

hypothetical population of patients with colorectal cancer. 

Results: We included a total of 104 studies of which 40 addressed issues related to clinical 

validity, 3 to analytic validity, and 61 to benefits and harms. 

 We identified only three studies on analytic validity and thus there exists a major gap in the 

published literature with regard to the accuracy and reliability of specific tests used in the 

evaluation of HNPCC. 

 Among unselected patients with colorectal cancer who fulfilled the Amsterdam I criteria, 44% 

(95% CI: 35, 52%) carried pathogenic mismatch repair mutations (mainly in the MLH1 and 

MSH2 genes). The proportion was somewhat higher (51% [95% CI: 35, 66%]) among studies 

that performed sequencing on all available samples. The prevalence of MMR mutation carriers 

may be higher when genetic testing includes evaluation for large genomic 

deletions/rearrangements and when testing is also performed on MSH6 and PMS2. 

Approximately 71% (95% CI 63, 78%) of colorectal cancers from patients who fulfilled the 

Amsterdam I criteria demonstrated microsatellite instability while 40% (95% CI: 28, 53%) 

demonstrated loss of protein expression by immunohistochemistry. 

 Of nine clinical strategies considered for detecting the presence of mismatch repair mutations 

in patients with colorectal cancer, the combination of three clinical predictors (age less than 50 

years old at diagnosis; or a history of colorectal or endometrial cancer in a first degree family 

member; or the presence of multiple, synchronous or metachronous colorectal or endometrial 

cancers in the proband) combined with either immunohistochemistry (IHC) or MSI testing of 

tumor tissue identified a similar number of patients with mismatch repair mutations as other 

more complex strategies. 
vi

 There was little published information regarding potential harms associated with screening 

individuals with HNPCC-related cancers using clinical criteria (e.g., the Amsterdam criteria), 

MSI or IHC testing. Limited data suggested that testing probands for MMR mutations was not 

associated with severe psychological impact following formal counseling. Pre-test genetic 

counseling had good efficacy in improving knowledge about HNPCC and resulted in a high 

likelihood of proceeding with genetic testing, satisfaction in the decision to undergo genetic 

testing, and decreasing depression and distress levels among family members of HNPCC 

probands with cancer and among asymptomatic individuals from HNPCC families. 

 Identification of HNPCC mutations was associated with an increase in the likelihood that 

family members of probands with CRC would undergo cancer-screening procedures. HNPCC 

family members who underwent cancer-screening procedures had a lower risk of developing 

HNPCC-related cancers and lower mortality rates than those who did not take actions. However, 

all of the relevant studies suggesting these benefits had important limitations. Survival was 

increased among asymptomatic HNPCC family members who received colonoscopy screening, 

regardless of their mutation status. There was limited direct evidence related to harms of the 

cancer-screening procedures in family members of probands with HNPCC. However, 

complication rates associated with these procedures in other settings are probably similar. 

Conclusions: This report characterizes the accuracy of clinical and laboratory predictors of 

MMR mutations that can be used to identify patients with an increased risk of having MMR 

mutations. However, the sensitivity, specificity, and reliability of the tests used to evaluate 

individuals for suspected HNPCC is not known confidently. Data regarding the net benefits and 

harms associated with predictive genetic testing in patients with HNPCC-related cancers and 

their families members is incomplete but suggest that such testing improves compliance with 

screening procedures. At-risk family members who undergo screening colonoscopy have a 

reduced risk of developing HNPCC-related cancers and lower mortality. However, all studies 

supporting these benefits had important limitations. 
vii

Contents 

Executive Summary.........................................................................................................................1 

Evidence Report...........................................................................................................................15

Chapter 1. Introduction .................................................................................................................17 

 Background..............................................................................................................................17 

 Who Should be Screened? .......................................................................................................18 

 How Should HNPCC be Defined?...........................................................................................19 

 Identifying HNPCC in Patients With Colorectal Cancer.........................................................21 

 Clinical Criteria That Suggest the Diagnosis.....................................................................21 

Testing All Cancers Regardless of the Family History .....................................................23 

Combinations of Family History and Laboratory Testing.................................................23 

 Analytic Validity......................................................................................................................24 

 Benefits and Harms of Screening, Testing, and Subsequent Management Strategies.............25 

Chapter 2. Methods.......................................................................................................................27 

 Key Questions Addressed in This Report................................................................................27 

 Literature Search Strategy........................................................................................................30 

 Inclusion/Exclusion Criteria and Data Extraction ...................................................................30 

 Studies Related to Analytic Validity..................................................................................30 

 Studies Related to Clinical Validity...................................................................................31 

 Studies Related to Benefits and Harms..............................................................................33 

 Evaluation of Study Quality.....................................................................................................33 

 Data Synthesis, Analysis and Reporting..................................................................................39 

 Questions Related to Analytic Validity .............................................................................39 

 Questions Related to Clinical Validity ..............................................................................39 

 Questions Related to Benefits and Harms .........................................................................42 

 Evidence and Summary Tables..........................................................................................43 

Chapter 3. Results .........................................................................................................................45 

 Overview..................................................................................................................................45 

 Analytic Validity......................................................................................................................46 

Key Question 4: What is Known About the Analytic (Sensitivity, Specificity, 

Reproducibility, Reliability) and Clinical Validity of Tests That Identify 

HNPCC Mutations? .....................................................................................................46 

Definition ...........................................................................................................................46 

Laboratory Tests Used in HNPCC.....................................................................................46 

Limitations of a Literature-Based Approach to Analytic Validity ....................................51 

 Clinical Validity.......................................................................................................................59 

 Key Question 2 and Pertinent Subquestions......................................................................59 

Key question 2a: Assuming a Clinical Definition of the Lynch Syndrome, What 

Proportion of Patients has a Mismatch Repair Mutation? ...........................................59 
viii

Key Question 2b: Assuming a Clinical Definition of the Lynch Syndrome, What 

Proportion of Patients has MSI? ..................................................................................67 

Key Question 2c: Assuming a Clinical Definition of the Lynch Syndrome, What 

Proportion of Patients has Abnormal Protein Expression 

by Immunohistochemistry?..........................................................................................70 

Key Question 2: How Accurate Are Various Predictors Assuming a Genetic 

Definition of the Lynch Syndrome? ............................................................................72 

Overview of Clinical Predictors Among Unselected Patients With CRC .........................75 

Amsterdam I Criteria .........................................................................................................76

Amsterdam II Criteria........................................................................................................80

Modified Amsterdam Criteria............................................................................................83 

Bethesda Guidelines...........................................................................................................84 

Revised Bethesda Guidelines.............................................................................................85 

Young Age of Onset ..........................................................................................................85 

Familial Cancer History.....................................................................................................85 

Multiple CRC or Endometrial Cancer in the Same Patient ...............................................87 

Familial History of Cancer, Age of Onset Less Than 50 Years, or Multiple 

Cancers in the Same Proband ............................................................................................87 

MSI ....................................................................................................................................87 

IHC.....................................................................................................................................91 

Expected Outcomes With Different Testing Strategies...........................................................93 

 Outline of the Problem.......................................................................................................93 

 Brief Description of Strategies and Values Used in the Decision Tree.............................94 

 Results With the Different Strategies ..............................................................................102 

Benefits and Harms................................................................................................................105 

Key Question 1: Does Risk Assessment and HNPCC Mutation Testing in Patients 

With Newly Diagnosed CRC Lead to Improved Outcomes for the Patient or 

Family Members, or is it Useful in Medical, Personal, or Public Health 

Decision Making? (Overarching Question)...............................................................105 

Key Question 3: What Are the Harms Associated With Screening High-Risk 

Individuals for HNPCC?............................................................................................106 

Key Question 5: What Are the Harms Associated With Screening for 

High-Risk Individuals? ..............................................................................................106 

Key Question 6a: What Are the Management Options for CRC Patients Who Are 

HNPCC Positive? b: Does the Identification of HNPCC Mutations Lead 

to Improved Patient Outcomes in Terms of Early Detection, Mortality/ 

Morbidity, or Management Decisions (e.g., Counseling, Surveillance, 

Treatment, Other Decision Making) by Patients and Providers? ..............................110 

Key Question 7: What Are the Harms Associated With Subsequent Management 

Options After Identification of HNPCC Mutations in CRC Patients? ......................114 

Key Question 8b: What is the Accuracy of HNPCC Testing in Family Members 

in Predicting the Risk of CRC?..................................................................................117 

Key Question 8c: Do Other Factors, Such as Race/Ethnicity, Age, Gender, 

or Co-Morbidities Affect the Accuracy of Testing?..................................................117 

Key Question 9: What Are the Harms Associated With Informing/Counseling 

Family Members or With Subsequent Testing for HNPCC Mutations? ...................126 
ix

Key Question 8a: What is the Efficacy of Pre-Test Genetic Counseling for 

Informing Family Members of Potential Risks and Benefits of Testing? .................126 

Key Question 10a: What Are the Management Options for Family Members 

of CRC Patients Who Have a Positive HNPCC Test? b1: Does the 

Identification of HNPCC Mutations Lead to Improved Outcomes 

in Terms of Decision Making by Patients, Family Members and 

Providers, or Public Health Policy? b2: Does the Identification 

of HNPCC Mutations Lead to Improved Outcomes in Terms of 

Early Detection and Mortality/Morbidity of Patients, and 

Family Members? ......................................................................................................137 

Key Question 11: What Are the Harms Associated With Subsequent Actions 

or Interventions for Family Members? ......................................................................144 

Chapter 4. Discussion .................................................................................................................151 

 Analytic Validity....................................................................................................................151 

 Clinical Validity.....................................................................................................................151 

 Benefits and Harms................................................................................................................153 

 Summary................................................................................................................................153 

 Implications for Future Research...........................................................................................156

References and Included Studies .................................................................................................159 

List of Acronyms/Abbreviations..................................................................................................179 

Figures 

Figure 1. Analytic framework that served as the basis of the key questions proposed 

by the CDC Office of Genomics and Disease Prevention 2005 ..................................28 

Figure 2. Literature search results ...............................................................................................45 

Figure 3. Prevalence of mismatch repair gene mutations among colorectal cancer 

patients fulfilling the Amsterdam I criteria..................................................................61 

Figure 4. Prevalence of mismatch repair gene mutations among colorectal cancer 

patients fulfilling the Amsterdam II criteria ................................................................63 

Figure 5. Prevalence of MSI in colorectal cancer tumors from patients fulfilling the 

Amsterdam I criteria ....................................................................................................68 

Figure 6. Prevalence of MSI-H in colorectal cancer tumors from patients fulfilling the 

Amsterdam II criteria...................................................................................................70 

Figure 7. Prevalence of suggestive IHC in colorectal cancer tumors from patients 

fulfilling the Amsterdam I criteria ...............................................................................71 

Figure 8. Relationship between the CRC populations defined by different 

clinical predictors.........................................................................................................72 

Figure 9. Sensitivity and specificity of clinical criteria for detecting mismatch repair 

mutations among unselected patients with CRC .........................................................76 

Figure 10. Sensitivity and specificity of the Amsterdam I criteria for detecting 

mismatch repair mutations in patients with CRC ........................................................77 
x

Figure 11. Sensitivity and specificity of the Amsterdam I criteria for detecting mismatch 

repair mutations: study subgroups according to various factors.................................78 

Figure 12. Sensitivity and specificity of the Amsterdam I criteria for detecting 

mismatch repair gene mutations among populations with an 

increasing likelihood of having HNPCC .....................................................................79 

Figure 13. Sensitivity and specificity of the Amsterdam II criteria for detecting 

mismatch repair gene mutations ..................................................................................81 

Figure 14. Sensitivity and specificity of the Amsterdam II criteria for detecting mismatch 

repair mutations: study subgroups according to various factors.................................81 

Figure 15. Sensitivity and specificity of the Amsterdam II criteria for detecting mismatch 

repair mutations among patients selected after MSI or IHC testing............................82 

Figure 16. Sensitivity and specificity of the Amsterdam II criteria for detecting mismatch 

repair mutations among populations with an increasing likelihood for HNPCC ........83 

Figure 17. Sensitivity and specificity of the Bethesda guidelines for detecting 

mismatch repair gene mutations: all available studies................................................84 

Figure 18. Sensitivity and specificity of familial cancer history for detecting mismatch 

repair gene mutations: all available studies ................................................................86 

Figure 19. Sensitivity and specificity of MSI-H to identify mismatch repair 

gene mutations .............................................................................................................88 

Figure 20. Sensitivity and specificity of MSI-H to identify mismatch repair gene 

mutations based upon study characteristic...................................................................90 

Figure 21. Summary ROC curve for the diagnostic ability of MSI-H and MSI-L 

to identify mismatch repair gene mutations.................................................................90 

Figure 22. Sensitivity and specificity of IHC to identify mismatch repair gene 

mutations based on study characteristics .....................................................................92 

Figure 23. Summary ROC curve for the diagnostic ability of IHC to identify 

mismatch repair gene mutations ..................................................................................93 

Figure 24. Decision tree model used to calculate the impact of different testing strategies .........96 

Tables 

Table 1. Lifetime cancer risk in HNPCC...................................................................................17 

Table 2. 2x2 table for test characteristics when considering the presence of a mismatch 

repair mutation as the reference standard ....................................................................20 

Table 3. 2x2 table for test characteristics when considering Amsterdam criteria I as the 

reference standard ........................................................................................................21 

Table 4. Original and revised Amsterdam criteria.....................................................................22 

Table 5. Original and revised Bethesda guidelines....................................................................22 

Table 6. Summary of key questions...........................................................................................29 

Table 7. Interpretation of overall quality grading of individual studies ....................................34 

Table 8. Quality criteria used to characterize studies of clinical validity..................................35 

Table 9. Quality criteria used to characterize studies of analytic validity .................................36 

Table 10. Quality criteria for studies on benefits and harms .......................................................37 

Table 11. Genetic testing strategies used by studies included in quantitative analyses...............47 

Table 12. North American laboratories offering clinical testing for Hereditary 

Nonpolyposis Colon Cancer (Lynch Syndrome).........................................................54 
xi

Table 13. Summary estimates of the prevalence of MMR mutations among CRC 

fulfilling Amsterdam I criteria.....................................................................................62 

Table 14. Summary estimates of the prevalence of MMR mutations among CRC 

fulfilling Amsterdam II criteria....................................................................................64 

Table 15. Summary estimates of the prevalence of MSI-H in colorectal cancer tumors 

from patients fulfilling Amsterdam I criteria...............................................................69 

Table 16. Overview of available evidence on the sensitivity and specificity of various 

predictors for MMR .....................................................................................................73 

Table 17. Sensitivity and specificity of various predictors for detecting MMR 

gene mutations .............................................................................................................74 

Table 18. Probabilities and parameters used in the decision trees for different strategies 

to detect MMR mutations ..........................................................................................100 

Table 19. Expected number of MMR, MSI or IHC tests and expected MMR testing 

results with the nine strategies, assuming a population of 100,000 

incident cases of CRC................................................................................................104 

Table 20. Overall sensitivity and specificity for each of the nine strategies .............................104 

Table 21. Key Question 5. What are the harms associated with genetic testing 

for HNPCC mutations?..............................................................................................108 

Table 22. Key Question 6b. Does the identification of HNPCC mutations lead to improved 

patient outcomes in terms of early detection, mortality/morbidity or management 

decisions (e.g., counseling, surveillance, treatment, other decision making) by 

patients and providers? ..............................................................................................112 

Table 23. Key Question 7. What are the harms associated with subsequent management 

options after identification of HNPCC mutations in CRC patients? .........................116 

Table 24. Key Question 8b. What is the accuracy of HNPCC testing in family 

members in predicting the risk of CRC? Key Question 8c. Do other 

factors, such as race/ethnicity, age, gender, or co-morbidities affect 

the accuracy of the testing?........................................................................................121 

Table 25. Key Questions 8b and 8c in kindreds with HNPCC based on clinical 

and/or genetic criteria ................................................................................................123 

Table 26. Key Question 8a. What is the efficacy of pre-test genetic counseling for 

informing family members of potential risks and benefits of testing? ......................129 

Table 27. Key Question 9. What are the harms associated with informing/counseling 

family members or with subsequent testing for HNPCC mutations?........................130 

Table 28. Key Question 10b1. Does the identification of HNPCC mutations lead 

to improved outcomes in terms of decision making by patients, family 

members and providers, or public health policy? ......................................................141 

Table 29. Key Question 10b2. Does the identification of HNPCC mutations lead 

to improved outcomes in terms of early detection and mortality/ 

morbidity, of patients, family members? ...................................................................143 

Table 30. Key Question 11. What are the harms associated with subsequent actions or 

interventions for family members? ............................................................................146 
xii

Overview Tables 

Overview Table 1: Key Question 6b ..........................................................................................111

Overview Table 2. Key Questions 8b and 8c .............................................................................118 

Overview Table 3. Key Questions 8a and 9 ...............................................................................127 

Overview Table 4. Key Question 10...........................................................................................139 

Overview Table 5. Key Question 11...........................................................................................145 

Extra Tables 

Extra Table 1. Studies examined the factors that might affect the acceptance 

of genetic testing....................................................................................................................135 

Extra Table 2. Surveys of genetic centers of insurance providers..............................................137 

Extra Table 3. Factors that might affect the compliance with CRC screening...........................144 

Appendixes 

Appendix A: Search Strategy 

Appendix B: Sample Data Abstraction Forms 

Appendix C: Evidence Tables 

Appendix D: List of Excluded Studies 

Appendix E: Peer Reviewers 

Appendix F: Summary Tables for Clinical Validity 

Appendix G: One-Way Sensitivity Analyses for the Decision Trees 

Appendixes and Evidence Tables for this report are provided electronically at 

http://www.ahrq.gov/downloads/pub/evidence/pdf/hnpcc/hnpcc.pdf
1 

Executive Summary 

Introduction 

 Individuals with a familial predisposition to cancer pose an increasing challenge for 

healthcare systems hoping to provide state-of-the-art care. Optimal strategies for recognizing 

them, performing (and interpreting) genetic testing, and preventing cancers in at-risk family 

members have not been well established when considering overall benefits, harms, and costs. 

The challenges involved will likely become increasingly complicated with advances in 

understanding of the molecular genetics underlying cancer risk. In this report, we attempt to 

clarify many of these issues for one form of hereditary cancer, Hereditary Nonpolyposis 

Colorectal Cancer (HNPCC). HNPCC is associated with a substantially increased risk for several 

forms of malignancy but particularly colorectal and endometrial cancer. 

 HNPCC has been defined clinically and genetically. As a genetic disease, it is inherited as an 

autosomal dominant disorder (with variable penetrance) caused by mutations in DNA mismatch 

repair (MMR) genes. As a clinical disorder, it can be defined as a clustering of related cancers 

across generations in a kindred. The disorder has also been referred to as the “Lynch syndrome” 

in recognition of Henry Lynch, who in 1966 described familial aggregation of colorectal cancer 

with gastric and endometrial cancer in two large kindreds (although it was first reported by the 

eminent pathologist Aldred Warthin in 1913). 

 These definitions are not entirely distinct since the disorder is typically recognized in patients 

or kindreds with clinical expression of the disease who have an associated genotype. However, 

the ability to perform predictive genetic testing in individuals with cancer and asymptomatic 

family members makes it imperative to fully understand its implications. There remain many 

uncertainties regarding how HNPCC should be identified in patients presenting with associated 

malignancies, and the full spectrum of implications related to screening and management options 

for the patient and their at-risk family members. Does, for example, the identification of MMR 

genotypes in family members of patients with HNPCC-related cancers improve their outcomes 

compared with management approaches that do not involve predictive genetic testing? What is 

the likelihood that a family member with a MMR mutation is destined to develop HNPCCrelated cancers, and does screening for these cancers improve outcomes? 

 There were three main objectives of this report: (1) to assess the sensitivity, specificity and 

reliability of laboratory and genetic tests commonly used in evaluating patients for HNPCC 

(analytic validity); (2) to summarize the accuracy of commonly used clinical and laboratory 

characteristics for predicting the presence of HNPCC in patients with colorectal cancer (clinical 

validity) and to describe the efficiency of various strategies for identifying patients with a 

mismatch repair mutation; and (3) to describe the benefits and harms related to screening and testing 

patients with colorectal cancer (CRC) and their family members for HNPCC. 

 This report is based upon a systematic review of the literature. The Key Questions that it 

addresses were proposed through the Agency for Healthcare Research and Quality (AHRQ) on 

behalf of the Centers for Disease Control and Prevention (CDC) Evaluation of Genomic 

Applications in Practice and Prevention (EGAPP) Project. EGAPP is a three-year model project 

developed by CDC’s Office of Genomics and Disease Prevention to address the increasingly 

urgent need for timely and objective information that will allow health care providers, 
2 

consumers, policy makers and payers to distinguish tests that are safe and useful, and to guide 

their appropriate use in practice. 

The Key Questions Addressed Include the Following: 

Key Question 1: Does risk assessment and HNPCC mutation testing in patients with newly 

diagnosed CRC lead to improved outcomes for the patient or family members, or is it useful 

in medical, personal, or public health decision making? (Over-arching question). 

Key Question 2a: Assuming a clinical definition of the Lynch Syndrome, what proportion of 

patients has a mismatch repair mutation? 

2b: Assuming a clinical definition of the Lynch Syndrome, what proportion of patients has 

MSI? 

2c: Assuming a clinical definition of the Lynch Syndrome, what proportion of patients has 

abnormal protein expression by immunohistochemistry? 

2: How accurate are various predictors, assuming a genetic definition of the Lynch 

Syndrome? 

 

Key Question 3: What are the harms associated with screening high-risk individuals for 

HNPCC? 

Key Question 4: What is known about the analytic (sensitivity, specificity, reproducibility, 

reliability) and clinical validity of tests that identify HNPCC mutations? 

Key Question 5: What are the harms associated with screening for high-risk individuals? 

Key Question 6a: What are the management options for CRC patients who are HNPCC 

positive? 

6b: Does the identification of HNPCC mutations lead to improved patient outcomes in terms 

of early detection, mortality/morbidity or management decisions (e.g., counseling, 

surveillance, treatment, other decision making) by patients and providers? 

Key Question 7: What are the harms associated with subsequent management options after 

identification of HNPCC mutations in CRC patients? 

Key Question 8a: What is the efficacy of pre-test genetic counseling for informing family 

members of potential risks and benefits of testing? 

8b: What is the accuracy of HNPCC testing in family members in predicting the risk of 

CRC? 

8c: Do other factors, such as race/ethnicity, age, gender, or co-morbidities affect the 

accuracy of the testing?

Key Question 9: What are the harms associated with informing/counseling family members or 

with subsequent testing for HNPCC mutations? 
3 

Key Question 10a: What are the management options for family members of CRC patients who 

have a positive HNPCC test? 

10b1: Does the identification of HNPCC mutations lead to improved outcomes in terms of 

decision making by patients, family members and providers, or public health policy? 

10b2: Does the identification of HNPCC mutations lead to improved outcomes in terms of 

early detection and mortality/morbidity of patients, family members? 

Key Question 11: What are the harms associated with subsequent actions or interventions for 

family members? 

 These questions were based upon an analytic framework that begins with a patient with 

colorectal cancer and examines the full spectrum of implications of identifying HNPCC in the 

patient and their at-risk family members, and the potential benefits and harms of this process 

from the perspectives of the patient, provider, family member and the public health. 

Screening for HNPCC in Newly Diagnosed CRC Patients and 

Their Family Members

Patients

with newly 

diagnosed 

CRC

High risk of

HNPCC

HNPCC testing HNPCC mutation 

negative Patient

Outcomes

Medical

Decision 

making

Family members of HNPCC 

positive CRC patients

Genetic Counseling 

HNPCC testing

Risk Assessment

2,3 Average risk

of HNPCC

1

4,5

Screening

HNPCC

mutation

positive

6,7

Intervention

8,9

Family Outcomes

Increased early 

detection, 

decreased 

mortality/morbidity,

improved decision 

making 

HNPCC

mutation

negative

HNPCC

mutation

positive

10,

11

Methods 

 We performed an electronic search of the literature followed by review of abstracts and then 

full-text review of potentially relevant studies. We also retrieved additional studies based on 

bibliographies of retrieved articles and suggestions from a technical expert panel. We evaluated 

all studies critically based upon pre-specified criteria. 

 We reviewed the full-text of 523 publications. One hundred fifteen papers fulfilled eligibility 

criteria but 11 were duplicate reports and were therefore excluded (or used to provide 

supplementary information). Thus, 104 unique studies were included for this review (40 were 

pertinent to Key Question 2 – clinical validity; 3 were pertinent to Key Question 4 – analytic 
4 

validity; and 61 to the remaining Key Questions – benefits and harms), while 408 did not meet 

the inclusion criteria and were rejected. 

 We reviewed each study for its quality. We assigned an overall quality score (A, B or C) to 

provide a short hand appraisal of the overall validity of the study but also performed analysis of 

specific components of study quality that may have influenced the conclusions. 

 We combined data in studies that used similar methodology and definitions of endpoints in 

similarly selected CRC populations using meta-analysis to provide a point estimate and 95% 

confidence interval, mainly for questions pertaining to clinical validity. 

 We constructed decision trees models to calculate the reported outcomes of various strategies 

for identifying HNPCC among patients with colorectal cancer. The models were based upon 

parameters estimated from data presented in this report. 

Results 

Analytic Validity 

 Key Question 4: What is known about the analytic (sensitivity, specificity, reproducibility, 

reliability) and clinical validity of tests that identify HNPCC mutations? 

The major laboratory tests used in the evaluation of patients suspected of having HNPCC 

include testing of tumor tissue using immunohistochemistry (IHC), microsatellite instability 

(MSI) testing, or germline (generally from peripheral blood mononuclear cells) testing for 

mismatch repair defects. Analytic validity may also apply to the accuracy of the family history. 

Family members generally undergo only germline genetic testing (unless they have also 

developed a relevant cancer), ideally based upon the genotype of the proband. Detection of a 

pathogenic mutation (i.e., one known to be associated with HNPCC) in a proband permits testing 

of at-risk family members for the genotype. Family members with the same genotype have 

HNPCC while HNPCC can be excluded in those who do not. The situation is more complex 

when the probands do not have a detectable DNA alteration associated with HNPCC or when an 

alteration with unclear clinical significance is detected. 

There was little published information on the analytic validity of laboratory testing in 

patients or family members suspected of having HNPCC. Thus, the analytic validity of the tests 

used to evaluate HNPCC is substantially uncertain. However, there was heterogeneity in the type 

of testing offered by commercial laboratories, and one study demonstrated variability in the 

accuracy of protein staining by IHC across facilities. 

Additional information that could shed light upon analytic validity is available but would 

require evaluation of non-published data sources. For example, information has been collected 

through an external proficiency-testing program for MSI conducted by the College of American 

Pathologists and through internal testing performed by individual laboratories. Committees of 

experts could also review the strengths and limitations of specific testing techniques based upon 

clinical experience with these methods in HNPCC and in other genetically based disorders. 

Clinical Validity 

Key Questions 2a, 2b and 2c seek to evaluate the prevalence of MMR mutations, suggestive 

MSI and IHC, respectively, among patients fulfilling the Amsterdam I criteria or the Amsterdam 
5 

II criteria. We considered several variables in making comparisons across studies such as how 

populations were selected, the methods used for genetic testing, whether and how mutations 

were described as being pathogenic, and whether testing for MMR genes other than MLH1 and 

MSH2 was performed. 

 Key Question 2a: Assuming a clinical definition of the Lynch Syndrome, what proportion 

of patients has a mismatch repair mutation? 

 Most eligible studies evaluated only MLH1 and MSH2 genes; only three studies assessed 

other MMR genes. Among CRC fulfilling Amsterdam I criteria, the random effects summary 

prevalence of MLH1 and MSH2 gene mutations was 44% (95% CI: 35, 52; n=19 studies, 464 

patients), with evidence for substantial between-study heterogeneity (p<0.01; I2

=52%). The six 

studies that performed sequencing among all Amsterdam I patients had a summary prevalence of 

51% (95% CI: 35, 66%). 

 For patients fulfilling the Amsterdam II criteria, the corresponding prevalence values were 

39% (95% CI: 30, 49%; 10 studies 279 Amsterdam II patients) and 40% (95% CI: 30, 52%) 

based upon two studies that performed sequencing on all 87 Amsterdam II patients. 

 The three studies examining additional genes (MSH6, PMS1 and PMS2) in Amsterdam I 

patients did not identify any additional MMR mutations. Two additional MSH6 mutations (in 

addition to five MLH1 and MSH2 mutations) were found among 20 Amsterdam II patients in a 

single study. 

 There were limited studies that performed more comprehensive genetic testing for 

pathogenic MMR genotypes associated with HNPCC or evaluated all the MMR genes that have 

been associated with HNPCC. For example, only one study performed sequencing in all samples 

and tested for large genomic mutations/rearrangements for three MMR genes (MLH1, MSH2, 

MSH6). The study included only 22 Amsterdam I patients; the prevalence of MMR mutation 

carriers was 64%. 

 Limited data suggested that approximately one-fourth to one-third of genotypes associated 

with HNPCC are related to deletions or rearrangements that would be missed through 

sequencing alone. As a result, the prevalence of mutations in Amsterdam I or II patients assessed 

from studies that performed sequencing alone are likely to be underestimates. Accounting for 

this effect, one may derive a prevalence of MMR mutations of approximately 63% to 67% in 

Amsterdam I patients. For Amsterdam II patients the corresponding prevalence values would be 

50% to 53%. 

 Furthermore, limited data suggest that approximately 10% of MMR genotypes involve MMR 

genes other than MLH1 and MSH2. Thus, assuming an additional 10% increase in mutations by 

assessing more genes would result in an overall prevalence of up to 70% to 75% for Amsterdam 

I patients and 55 to 59% for Amsterdam II patients. 

 Because of the limited data, these calculations should be considered as being highly 

imprecise. Furthermore, other considerations, such as how patients are selected and how 

genotypes are classified as being pathogenic, may affect these estimates. 

 Key Question 2b: Assuming a clinical definition of the Lynch Syndrome, what proportion 

of patients has MSI? 

 Among patients fulfilling the Amsterdam I criteria, 71% (95% CI: 63, 78%; n=11 studies, 

159 patients) of tumors were MSI-H. Among patients who fulfilled the Amsterdam II criteria, the 

corresponding summary prevalence was 68% (95% CI: 58, 76%; n=4 studies, 102 patients). 
6 

 

 Key Question 2c: Assuming a clinical definition of the Lynch Syndrome, what proportion 

of patients has abnormal protein expression by immunohistochemistry? 

 Among patients fulfilling the Amsterdam I criteria the overall prevalence of tumors with loss 

of protein expression by IHC was 40% (95% CI: 28, 53%; n= 6 studies, 63 patients) with no 

evidence for between-study heterogeneity (p=0.75, I2=0%). 

 Only one eligible study provided relevant data for 20 patients fulfilling the Amsterdam II 

criteria. Eight out of 20 tumors had suggestive IHC for the MLH1, MSH2 or MSH6 genes (40% 

[95% CI: 9, 64%]). 

 MSI and IHC testing often depended upon practical and logistical considerations (e.g., 

patient availability and consent and availability of tumor tissue). Thus, not all patients had all 

tests and it was unclear whether additional bias may have been introduced in selecting patients 

for testing. 

 Key Question 2: How accurate are various predictors assuming a genetic definition of the 

Lynch Syndrome? 

 Five studies provided information on eight clinical predictors and on suggestive MSI testing 

for predicting the presence of MMR mutations among unselected incident CRC. All were limited 

by verification bias. The table below summarizes the sensitivity and specificity of the predictors 

most commonly reported (see Executive Summary Table 1). 

Executive Summary Table 1. Diagnostic ability of various predictors to detect MMR mutations among 

unselected incident CRC 

Predictor Unselected CRC probands 

N Sensitivity [%]

(95% CI) 

Specificity [%] 

(95% CI) 

Amsterdam I criteria 2 45 (29, 63) 99 (74, 100) 

Amsterdam II criteria 2 28 (15, 47) 99 (97, 100) 

Modified Amsterdam criteria 0 ND ND 

Bethesda guidelines 1 73 (39, 94) 82 (80, 84) 

Revised Bethesda guidelines 1 91 (59, 100) 77 (75, 79) 

Age <50 years 3 31 (18, 47) 95 (94, 96) 

1st degree family history of CRC or 

EC 

4 76 (50, 91) 87 (86, 89) 

Multiple CRC or EC tumors in the 

same patient 

3 38 (25, 54) 97 (91, 99) 

Age <50 years, family history of CRC 

or EC, or multiple tumors in same 

patient 

3 88 (60, 97) 77 (74, 81) 

Suggestive MSIa

 2 100 (88, 100) 90 (88, 92) 

Suggestive IHC 0 ND ND 

CRC: colorectal cancer; EC: endometrial cancer; N: Number of studies; ND: no data. a Estimates are the same for combined MSI-H and MSI-L versus MSS and for MSI-H versus MSS 

Decision Tree Modeling of Genetic Testing Strategies. We evaluated nine strategies for 

identifying patients with CRC with MMR mutations based upon combinations of clinical 

features, laboratory testing of tumor tissue (i.e., IHC and MSI testing). These can be 

conceptually organized into four general strategies: 

− Group 1: Perform genetic test on everyone 

− Group 2: Screen with a set of clinical criteria 
7 

− Group 3: Screen tumor tissue with a laboratory test 

− Group 4: Screen using two serial tests: a set of clinical criteria first, and then a laboratory 

test of tumor tissue 

 Of these, Group 4 strategies selected relatively fewer patients for genetic testing (one out of 

twenty-five or fewer) and missed at most in approximately 27% of patients with HNPCC. 

Mismatch repair mutation testing would be performed in less than 6% of CRC patients using 

these strategies. In contrast, for strategies in groups 1 to 3, more than 13% and up to 100% of 

newly diagnosed CRC patients would be genetically tested, and approximately 5% to 16% of 

patients with HNPCC would be missed. Similar results were obtained for both the low (0.90%) 

and the higher (2.75%) estimate for the prevalence of mutation carriers among incident 

unselected CRC (see Executive Summary Table 2). 

Executive Summary Table 2. Expected number of MMR, MSI or IHC tests and expected MMR testing results 

with the nine strategies, assuming a population of 100,000 incident cases of CRC 

Strategy Received tests Number of MMR tests that were 

MMR MSI IHC Positive True 

positive 

Inconclusive 

Unidentified 

MMR 

mutation 

carriers 

Low prevalence estimate for MMR mutation carriers (0.90%) 

MMR-All 100,000 0 0 1,351 855 252 45 

BethR-All 23,612 0 0 892 778 61 122 

3Clinical-All 23,585 0 0 866 752 61 148 

MSI-All 13,738 100,000 0 877 812 37 88 

IHC-All 13,702 0 100,000 843 778 37 122 

BethR-MSI 3,741 23,612 0 754 739 12 161 

3Clinical-MSI 3,716 23,585 0 730 715 11 185 

BethR-IHC 1,828 0 23,612 659 654 6 246 

3Clinical-IHC 3,684 0 23,585 700 685 11 215 

Higher prevalence estimate for MMR mutation carriers (2.75%) 

MMR-All 100,000 0 0 3,098 2,612 257 138 

BethR-All 24,870 0 0 2,489 2,377 69 373 

3Clinical-All 24,787 0 0 2,410 2,299 69 451 

MSI-All 15,255 100,000 0 2,545 2,481 46 269 

IHC-All 15,145 0 100,000 2,440 2,377 45 373 

BethR-MSI 5,285 24,870 0 2,273 2,258 20 492 

3Clinical-MSI 5,206 24,787 0 2,198 2,184 20 566 

BethR-IHC 3,220 0 24,870 2,002 1,997 14 753 

3Clinical-IHC 5,110 0 24,787 2,106 2,092 20 658 

In the hypothetical population, for the low prevalence estimate 900/100,000 patients are assumed to carry MMR 

mutations; for the high prevalence estimate 2750 people are assumed to carry MMR mutations. Strategies are 

presented with respect to the group (1 to 4) to which they belong. MMR-All: test all for mismatch repair mutations; 

BethR-All: test all using the revised Bethesda criteria; 3 Clinical-all: Perform MMR testing only among those fulfilling 

at least one of the three simple clinical criteria (age <50y at diagnosis, family history of CRC or endometrial cancer, or 

multiple tumors, synchronous or metachronous, in the same patient); MSI-All: Perform MSI testing on all patients, 

followed by MMR testing only among those with suggestive MSI test; IHC-all: Perform immunohistochemistry (IHC) 

testing on all patients followed by MMR testing only among those with suggestive IHC test; BethR-MSI: Perform MSI 

testing on patients fulfilling the revised Bethesda guidelines, perform MMR only among those with suggestive MSI 

test;3 Clinical-MSI: Perform MSI testing on patients fulfilling at least one of the three simple clinical criteria (age <50y 

at diagnosis, family history of CRC or endometrial cancer or multiple tumors, synchronous or metachronous, in the 

same patient); perform MMR only among those with suggestive MSI test; BethR-IHC: Perform IHC testing on patients 

fulfilling the revised Bethesda guidelines; perform MMR only among those with suggestive IHC test; 3 Clinical-IHC: 

Perform IHC testing on patients fulfilling at least one of the three simple clinical criteria (age <50y at diagnosis, family 

history of CRC or endometrial cancer or multiple tumors, synchronous or metachronous, in the same patient; perform 

MMR only among those with suggestive IHC test. 
8 

 

 

The overall (strategy-level) sensitivity and specificity are shown in Executive Summary 

Table 3. The overall specificity was high in all strategies. Group 4 strategies had the lowest 

overall sensitivity (ranging between 73% and 82%). Overall sensitivity and specificity estimates 

were similar for both the low (0.90%) and the higher (2.75%) prevalence estimates. 

Executive Summary Table 3. Overall sensitivity and specificity for each of the nine strategies 

Strategy Sensitivity of strategy 

(%) 

Specificity of 

strategy (%) 

MMR-All 95.0 99.3 

BethR-All 86.5 99.8 

3Clinical-All 83.6 99.8 

MSI-All 90.3 99.9 

IHC-All 86.5 99.9 

BethR-MSI 82.1 100.0 

3Clinical-MSI 79.4 100.0 

BethR-IHC 72.6 100.0 

3Clinical-IHC 76.1 100.0 

Strategies are presented with respect to the group (1 to 4) to which they belong. In the above estimates the 

sensitivity and the specificity of the whole strategy was calculated. Inconclusive MMR tests were assumed to be false 

negative (for the calculation of overall sensitivity for each strategy) or false positive (for the calculation of overall 

specificity for each strategy). 

 Thus, of the nine clinical strategies, the presence of at least one of three clinical predictors: 

(i) age less than 50 years old at diagnosis, or ii) a history of colorectal or endometrial cancer in a 

first degree family member, or iii) the presence of multiple, synchronous or metachronous 

colorectal or endometrial cancers in the proband, combined with testing of tumor tissue for either 

IHC or MSI, identified a similar number of patients with colorectal cancer who had mismatch 

repair mutations associated with HNPCC (and failed to identify a similar number) compared 

with other, more complex approaches. There were relatively more studies demonstrating test 

characteristics of MSI testing compared with IHC and thus greater precision in the estimates of 

sensitivity and specificity of MSI testing. 

Benefits and Harms 

 Key Question 1: Does risk assessment and HNPCC mutation testing in patients with newly 

diagnosed CRC lead to improved outcomes for the patient or family members, or is it useful in 

medical, personal, or public health decision making? (Over-arching question) 

 No study directly addressed Key Question 1. 

 Key Question 3: What are the harms associated with screening high-risk individuals for 

HNPCC?

 Studies were considered eligible for Key Question 3 if they reported harms of a risk 

assessment process (e.g., Amsterdam, Bethesda and/or MSI, IHC) used to identify CRC patients 

at increased risk for HNPCC. 
9 

 No study described harms of the risk assessment process in CRC patients at increased risk for 

HNPCC. 

 Key Question 5: What are the harms associated with screening for high-risk individuals? 

 Studies were considered eligible for Key Question 5 if they reported the harms associated 

with testing CRC patients for MMR mutations. Common harms that are thought to be associated 

with genetic testing are labeling, discrimination in health coverage, and emotional distress. 

 Three quantitative, comparative studies of quality A and B, and one qualitative study of B 

quality reported harms associated with MMR mutation testing in CRC patients. One 1-year 

prospective study (Grade A) compared the psychological impact of MMR mutation testing 

between mutation carriers and non-carriers. Subjects in this study were CRC probands or 

relatives from HNPCC families with a prior diagnosis of any cancer (excluding non-melanoma 

skin cancer). Anxiety, depression, and quality of life measures did not change over time, and 

there were no differences in these measures between mutation carriers and non-carriers. Distress 

levels were significantly decreased 2 weeks and 6 months after revealing the genetic testing 

results but were not significantly different from the baseline at 1-year follow-up. There was no 

difference in the distress levels between mutation carriers and non-carriers. 

 Another 1-month prospective study (Grade B) found that three of the 27 probands (11%) had 

minor depression at 1 month after revealing the genetic testing results, but the prevalence of 

minor depression was not significantly different compared to the prevalence at baseline or 

between mutation carriers and non-carriers. Of the six probands who received a positive result, 

two (33%) felt severe guilt regarding their children. 

 One prospective study (Grade B) reported changes in the psychological outcomes of CRC 

patients from self-completed questionnaires pre- and 4-6 weeks post-genetic counseling. There 

was no genetic testing performed in this study. There was a trend toward greater anticipated 

ability to cope with a positive gene test after counseling, as reflected by a decrease in anxiety and 

cancer-specific distress. 

 The qualitative study of 111 newly diagnosed CRC patients reported a high acceptance and 

understanding about information on HNPCC. Nineteen percent of participants rated their current 

level of worry caused by the genetics information at or above the midpoint of 4 on a 1 (not at all) 

to 7 (all the time) scale. 

 Key Question 6a: What are the management options for CRC patients who are HNPCC 

positive? 

 6b: Does the identification of HNPCC mutations lead to improved patient outcomes in terms 

of early detection, mortality/morbidity or management decisions (e.g., counseling, surveillance, 

treatment, other decision making) by patients and providers? 

 There are three aspects to Key Question 6: 1) Are management options for patients with CRC 

with a MMR mutation different from those without a MMR mutation? 2) Does the knowledge of 

MMR mutation status change management decisions by patients and providers? 3) Does 

changing management options for MMR positive patients with CRC improve outcomes (e.g., 

prognosis and survival) compared to standard approaches for patients with CRC? 

 We encountered a variety of surgical and medical management options in patients with CRC 

who were MMR positive but there were no comparative studies. 
10 

 Indirect evidence from one study suggested that identification of HNPCC mutations was 

associated with better prognosis of CRC. However, there were no data on whether management 

options for CRC differed based on MMR mutation status. 

 Indirect evidence from one study showed no difference in survival of patients with 

endometrial cancer, comparing those who were mutation positive to those who were mutation 

negative. 

 In five studies with indirect evidence, there was no evidence in favor or against differences in 

survival, when comparing CRC patients who fulfilled different clinical criteria for HNPCC or 

screened positive for HNPCC by suggestive laboratory testing with those who did not. 

 Key Question 7: What are the harms associated with subsequent management options after 

identification of HNPCC mutations in CRC patients?

 No study described harms associated with subsequent management options after 

identification of HNPCC mutations in patients with CRC or other forms of HNPCC-related 

cancers. 

 One study (involving two centers) described the types of colorectal surgery performed on 

CRC patients who were part of an Amsterdam criteria-positive family, and compared rates of 

metachronous cancers that followed each type of index operation. The overall rate of second 

surgeries for metachronous cancer were 23% in patients who underwent right colectomy, 17% in 

patients who underwent left/sigmoid colectomy or proctosigmoidectomy, 0% in patients who 

underwent total/subtotal colectomy, and 44% in patients who underwent segmental colectomy. 

The two centers had significantly different second resection rates for metachronous cancer. 

 One study described the survival rate of 45 patients with gastric cancer from HNPCC 

families with MMR mutations. Many of these patients had already had treatments for other 

HNPCC-related cancers, including CRC. The 5-year survival was higher in patients in whom 

radical surgery was performed (48%) than in patients in whom radical palliative surgery or 

explorative laparotomy alone was performed (15%). 

 Key Question 8b: What is the accuracy of HNPCC testing in family members in predicting 

the risk of CRC? 

8c: Do other factors, such as race/ethnicity, age, gender, or co-morbidities affect the 

accuracy of the testing? 

 Only two studies of B quality reported the risk of CRC in family members of probands with 

positive MMR mutations (the proposed framework). The lifetime risk of CRC was 68.7% for 

men and 52.2% for women with MMR mutations in one study, and it was 74% and 30% 

respectively in the other study. Men had higher lifetime risk of CRC than women in both studies. 

 In a study that reported the risk of CRC in family members of probands with HNPCC based 

on clinical criteria, the cumulative risk of CRC by age 75 years old was 57% and 41% in families 

that fulfilled the Amsterdam I and II criteria, respectively. The cumulative risk of CRC by age 75 

years old was 42% and 23% for men and women, respectively, from families that fulfilled 

Bethesda criteria. In another study, family members of CRC probands who were younger than 50 

years old at cancer diagnosis had a higher risk of CRC, compared to family members of CRC 

probands who were 50 years old and older. The risk of CRC was increased three-fold, comparing 
11 

first-degree relatives of CRC probands who developed a second primary in the HNPCC spectrum 

with the single primary group in a third report. 

 In a study that reported the risk of CRC in kindreds with HNPCC based on genetic criteria, 

the cumulative risk of CRC by age 70 yr was 82% in MLH1/MSH2 mutation carriers. The 

cumulative incidence of CRC was 100% in men and 54% in women. There was overall a higher 

risk of CRC in men, but the sex difference was not consistent among HNPCC kindreds with 

different MMR mutations. 

 Key Question 9: What are the harms associated with informing/counseling family members 

or with subsequent testing for HNPCC mutations? 

 Key Question 8a: What is the efficacy of pre-test genetic counseling for informing family 

members of potential risks and benefits of testing? 

Studies were considered eligible for Key Question 8a if they summarized the efficacy of pretest genetic counseling immediately after counseling and before performing genetic testing. 

Studies addressing Key Question 9 were considered together with those addressing Key Question 

8a because there was substantial overlap in the studies addressing these questions. The studies 

addressed the long-term efficacy of pre-test genetic counseling or harms associated with 

screening high-risk individuals (such as by using clinical criteria, MSI, or IHC), genetic testing, 

and informing/counseling family members or with subsequent testing for HNPCC mutations. 

 Four studies addressed the efficacy of pre-test genetic counseling immediately after 

counseling and before performing MMR genetic testing. Of these, three were of B quality and 

one was of C quality. 

 Eight comparative and four qualitative studies addressed the harms associated with genetic 

testing for HNPCC mutation or with informing/counseling family members or with subsequent 

testing for HNPCC mutations. Of these, one study was of A quality, six of B quality, and five of 

C quality. 

 Overall, pre-test genetic counseling had good efficacy in improving knowledge about 

HNPCC, and resulted in high likelihood of proceeding with genetic testing, satisfaction in the 

decision to undergo genetic testing, and decreasing depression and distress levels among family 

members of HNPCC probands or among asymptomatic individuals from HNPCC families. 

Family members of HNPCC who received positive MMR mutation test results had higher 

psychological distress levels and anxiety compared to those who received negative test results, 

but this difference generally disappeared with time. However, all of these psychological 

measures were within the normal ranges for the general populations. There were no differences 

in quality of life, comparing mutation carriers to non-carriers or the general population.

 Key Question 10a: What are the management options for family members of CRC patients 

who have a positive HNPCC test? 

b1: Does the identification of HNPCC mutations lead to improved outcomes in terms of 

decision making by patients, family members and providers, or public health policy? 

b2: Does the identification of HNPCC mutations lead to improved outcomes in terms of 

early detection and mortality/morbidity of patients, family members? 

 We included studies evaluating all forms of cancer related to HNPCC since family members 

of CRC probands are potentially at risk for all such cancers. 
12 

 Six studies examined the impact of mutation testing on the decision to undergo specific 

management recommendations among family members of CRC patients or asymptomatic 

individuals from HNPCC families. Of these, four were of B quality and two were of C quality. 

No study directly examined the impact of HNPCC mutation testing on public health policy or 

decision making by insurance providers. 

 Two studies (in three publications) of B and C quality indirectly addressed outcomes of early 

detection and mortality/morbidity in relation to the identification of MMR mutations in family 

members of CRC probands or asymptomatic individuals from HNPCC families. These studies 

were limited by potential selection bias, and/or unclear effects from treatments or subsequent 

interventions. 

 Identification of HNPCC mutations was associated with improved outcomes in terms of 

decision making to undergo screening for cancers in family members of HNPCC. HNPCC 

family members who took subsequent actions or interventions had a lower risk of developing 

HNPCC-related cancers and lower mortality rates, compared to those who did not take actions. 

Most data pertained to screening for CRC with colonoscopy while there was less information 

about screening for other forms of HNPCC-related cancer. 

 Key Question 11: What are the harms associated with subsequent actions or interventions 

for family members? 

 

 We included studies that reported any outcome relating to subsequent management options or 

interventions in HNPCC family members. 

 Nine studies reported outcomes related to subsequent actions or interventions among family 

members. Of these, six were of B quality and three were of C quality. Four of these nine studies 

reported harms or adverse events associated with subsequent actions or interventions for family 

members. In addition, one study of C quality examined the psychological impacts associated 

with colonoscopies. Some of these studies did not have a control group of subjects who declined 

to undergo surveillance and most results did not adjust for potential confounders such as age, 

personal history of cancer, and educational levels. 

 Less than 0.5 percent of family members experienced harms associated with screening or 

surveillance examination or surgical procedures in the studies we evaluated. However, 

complication rates associated with these interventions in the non-HNPCC setting are probably 

applicable. There was some negative psychological impact associated with colonoscopies. 

Implications for Future Research 

 Our report identified several areas for future research; we considered the following to be 

particularly important priorities: 

• There is very little information regarding the analytic validity of tests used in the diagnosis of 

HNPCC. Studies specifically addressing sensitivity, specificity and reliability of all of the 

laboratory and genetic testing methods in HNPCC are needed. Such studies should focus on 

contemporary testing methods and compare them against well-defined reference standards in 

tissue samples representative of the spectrum of genotypes associated with HNPCC. 

Unpublished information regarding analytic validity is also available; it may be feasible to 
13 

obtain information from commercial or private laboratories performing such testing. It may 

also be possible to obtain data from the College of American Pathologists regarding their 

MSI proficiency program once experience has accumulated. Experience with genetic testing 

methods in other conditions is likely to be relevant to HNPCC; a review of such information 

could be conducted by groups of experts on genetic testing techniques. 

• Additional studies are needed to clarify the validity of specific clinical and laboratory 

predictors of HNPCC in patients with CRC who are representative of the general population 

of patients with CRC. 

• Future studies should consider all forms of genetically based CRC cancer predisposition to 

fully understand the effectiveness of various diagnostic strategies. Such studies should 

consider all known genetic causes of cancer predisposition and the accuracy of clinical and 

laboratory testing in identifying these disorders in individuals who are representative of the 

general population. 

• Additional studies are needed to establish the availability of genetic testing centers that can 

provide adequate counseling and whether there are barriers to access them. Such studies may 

involve electronic, mail, or telephone surveys. 

• More studies are needed to understand what forms of surveillance should be offered to MMR 

mutation carriers for HNPCC-related cancers other than CRC. Well-designed controlled 

trials comparing various surveillance (or other management) strategies could be helpful. 

• More studies are needed to clarify the risk of cancer in family members of probands with an 

HNPCC-related cancer who are found to carry MMR mutations. Such studies would ideally 

be prospective, fully account for interventions (such as cancer screening procedures) in those 

at-risk, and have a well-defined control population of individuals at average risk for cancer. 

• Standards for reporting studies of genetically based diseases (including those addressing all 

aspects of the ACCE model) should be developed. A consensus development process with 

publication of a guideline(s) could be helpful. 

Evidence Report 

17

Chapter 1. Introduction 

Background 

 Individuals with a familial predisposition to cancer pose an increasing challenge for 

healthcare systems hoping to provide state-of-the-art care. Optimal strategies for recognizing 

them, performing (and interpreting) genetic testing, and preventing cancers in at-risk family 

members have not been well established when considering overall benefits, harms, and costs. 

The challenges involved will likely become increasingly complicated with advances in 

understanding of the molecular genetics underlying cancer risk. In this report, we attempt to 

clarify many of these issues for one form of hereditary cancer (hereditary nonpolyposis 

colorectal cancer). 

 This evidence review is based upon a systematic review of the literature. The Key Questions 

that it addresses were proposed by the Agency for Healthcare Research and Quality (AHRQ) on 

behalf of the Centers for Disease Control and Prevention (CDC) Evaluation of Genomic 

Applications in Practice and Prevention (EGAPP) Project. EGAPP is a three-year model project 

developed by CDC’s Office of Genomics and Disease Prevention to address the increasingly 

urgent need for timely and objective information that will allow health care providers, 

consumers, policy makers, and payers to distinguish tests that are safe and useful, and to guide 

their appropriate use in practice. 

 Hereditary Nonpolyposis Colorectal Cancer (HNPCC) can be defined clinically or 

genetically as will be described below. As a genetic disease it is inherited as an autosomal 

dominant disorder (with variable penetrance) and is caused by mutations in DNA mismatch 

repair genes. HNPCC is associated with a substantially increased risk for several forms of 

malignancy but particularly colorectal and endometrial cancer1-3 (see Table 1). 

Table 1. Lifetime cancer risk in HNPCC 

Cancer HNPCC 

% 

General 

Population 

% 

Colorectal 80-82 5-6 

Endometrial 50-60 2-3 

Gastric 13 1 

Ovarian 12 1-2 

Small bowel 1-4 0.01 

Bladder 4 1-3 

Brain 4 0.6 

Kidney, renal pelvis 3 1 

Biliary tract 2 0.5 

From Chung, DC. Ann Intern Med 2003; 138:560; HNPCC: Refers to individuals with Lynch Syndrome. 

 The disorder has also been referred to as the “Lynch syndrome” in recognition of Henry 

Lynch, who in 1966 first described familial aggregation of colorectal cancer with gastric and 
18

endometrial cancer in two large kindreds (although it was first reported by the eminent 

pathologist Aldred Warthin in 1913).4

 Lynch I syndrome (also referred to as HNPCC I) refers to 

kindreds in which colorectal cancer predominates, while Lynch II syndrome (HNPCC II) refers 

to kindreds who also have extracolonic tumors. The Muir-Torre syndrome (sebaceous gland 

tumors with or without keratoacanthomas associated with visceral malignancy) and Turcot 

syndrome (HNPCC-related tumors associated with glioblastoma multiforme) describe additional 

subsets of HNPCC with tumor types that appear to cluster in affected families. These 

distinctions have become less clear as more families have been studied, and as a result, these 

clinical classifications are being supplanted by genetic classification.5-7 The most common 

mismatch repair mutations associated with HNPCC are MLH1 and MSH2 (which together are 

believed to account for about 80 percent of cases) while MSH6 and PMS2 are less common.7

The precise cancer burden due to HNPCC has not been well defined. However, a germline 

mismatch repair mutation associated with HNPCC has been described in 1-5% of patients 

diagnosed with colorectal cancer in various reports.8-12 Thus, HNPCC accounted for 

approximately 1400 to 7300 cases of colorectal cancer in 2005 in the United States based upon 

the overall estimate of 145,290 new cases of colorectal cancer.13 Similarly, approximately 0.5-

2% of patients with endometrial cancer has a history compatible with HNPCC.14,15 More than 1 

in 3100 people between the ages of 15 and 74 are estimated to carry a defective DNA mismatch 

repair gene associated with HNPCC and thus are at risk for developing an HNPCC-related 

cancer.16 

 The recognition of a heightened cancer risk in carriers of mismatch repair mutations provides 

hope for offering screening, intensive surveillance or other measures (such as colectomy or 

hysterectomy) aimed at reducing the risk that cancer will develop. Furthermore, identification of 

a specific gene defect allows for testing of family members potentially sparing them the worry, 

bother, and expense associated with lifelong cancer surveillance if they do not carry the 

mutation. Notably, HNPCC has been excluded in 97 living members of the original family 

described by Henry Lynch based upon genetic testing.17

However, many uncertainties remain on the sequence of events that should occur in selecting 

patients with cancer to undergo testing for HNPCC and the spectrum of implications that 

screening, surveillance, and other management strategies have on affected patients and their 

families. The following summarizes many of these issues, while outlining those that are the 

subject of this report. 

The framework for this report begins with patients with colorectal cancer, and explores the 

issues of screening and testing patients with colorectal cancer, identifying, counseling and testing 

at-risk family members, and the benefits and harms of subsequent management options for the 

probands and family members. We evaluate these issues from the perspectives of the patient, 

caregiver, family member, and policy-maker. The analytic framework is described in further 

detail in Chapters 2 and 3. 

Who Should be Screened? 

 Screening for HNPCC has been most widely advocated in patients with colorectal cancer 

while there is much less information about screening in patients with other forms of HNPCCrelated cancers. This report focuses only on colorectal cancer. However, recognition of HNPCC 

may also be possible in patients presenting with other HNPCC-related cancers. In one study, for 

example, one-half of women with HNPCC (defined by the Amsterdam criteria) presented with 
19

endometrial cancer.18 It may also be feasible to identify an HNPCC kindred from individuals 

without a personal family history of an HNPCC-related cancer by obtaining a detailed family 

history. 

 Several studies have described estimates of the proportion of patients with colorectal cancer 

who have a mismatch repair mutation, fulfill clinical criteria for a familial cancer predisposition, 

or have clinical (e.g., tumor location or histology) or laboratory (e.g., abnormal staining for 

mismatch repair proteins [IHC] or microsatellite instability [MSI]) characteristics of their tumor 

tissue that suggest the diagnosis. In this report, we evaluate such studies in detail to produce 

estimates of these parameters and attempt to explain variability across studies to an extent 

possible. These parameters are important for defining cost-effective pathways for evaluating 

patients with colorectal cancer for HNPCC and caring for family members.19 However, this 

report does not include a formal cost-effectiveness analysis. 

How Should HNPCC be Defined? 

 The definition of HNPCC continues to evolve. Some authorities consider HNPCC to 

represent a superset of individuals with a mismatch repair mutation of whom a subset is 

considered to have the familial cancer clustering described by Henry Lynch. Such an approach 

recognizes the uncertainty that remains regarding the penetrance of cancer in carriers of 

mismatch repair mutations; the risk of cancer in a family with Lynch Syndrome may be 

substantially different compared with the risk associated with a mismatch repair mutation alone 

without such a history.20 

 A different view is that Lynch Syndrome represents a description of familial cancer 

clustering, a subset of which is caused by mismatch repair mutations. Patients with familial 

clustering of HNPCC-related cancers but without a mutation may have a different form of 

hereditary cancer (e.g., cancers caused by mutations in the MYH gene)21 while others may have 

HNPCC caused by a mismatch repair mutation that was not sought, or from a false negative 

result of testing. These distinctions are important because they identify subgroups of patients 

with variable cancer risk, and they may influence how family members are screened and 

subsequent surveillance strategies. For example, at least one report suggested that families who 

fulfill the Amsterdam criteria for Lynch syndrome (described below), but do not have an 

identifiable mismatch repair mutation, might be at lower cancer risk compared with those in 

whom a mutation is identified.22 

 An advantage of this view is that it establishes a reference standard for Lynch syndrome that 

is relatively more objective than one based upon a clinical definition. For example, it may be 

possible to establish the diagnosis of HNPCC in a proband with colorectal cancer who does not 

fulfill classical criteria for Lynch syndrome. In addition, a genetically based reference standard 

provides a framework for calculating sensitivity, specificity and predictive values of various 

predictors of mismatch repair mutation that might be useful in selecting patients for genetic 

studies (see Table 2). 
20

Table 2. 2x2 table for test characteristics when considering the presence of a mismatch repair mutation as 

the reference standard 

Patients with Colorectal Cancer Pathogenic Mismatch Repair 

Mutation 

Test Detected Not detected 

Positive A B 

Microsatellite instability 

Abnormal immunohistochemistry 

Amsterdam Criteria I 

Amsterdam Criteria II 

Bethesda Guidelines 

Revised Bethesda Guidelines 

Other 

Negative C D 

 As will be described further in the methods section, in this report we take both views on 

HNPCC depending upon which Key Question is being addressed. For questions related to the 

test characteristics of predictor tests (such as clinical criteria or laboratory tests of tumor tissue 

described below), HNPCC can be defined as the presence of a pathogenic mismatch repair 

mutation in a patient with an HNPCC-related mutation (see Table 2). In this model, test 

characteristics depend upon how comprehensively mutation testing (e.g., the number of 

mismatch repair mutations tested and methods of testing, and the accuracy of the specific 

laboratory methods used), and predictor tests on tumor tissue (e.g., the quality of IHC analysis 

and the specific methods used to determine MSI status) were carried out and whether a mutation 

was known to be pathogenic. We describe the type of testing performed in all studies included in 

this report and provide a general description of how they are used in Chapters 2 and 3. 

 Not all mismatch repair mutations that can be identified using modern genetic testing are 

known to be pathogenic, leaving some uncertainty as to whether a mismatch repair mutation 

found in a patient with an HNPCC-related cancer is responsible for the increased cancer risk. 

The strongest evidence that a mutation is pathogenic is when its presence correlates strongly with 

the clinical expression of the disease. A mutation may also be considered pathogenic when its 

predicted protein sequence is expected to lead to a dysfunctional protein. In this report, the 

methods by which the authors attempted to define pathogenic mutations (if at all) were recorded 

for all eligible studies. 

 However, in some cases, the observed genotype has an unclear relationship to the clinical 

expression of the disease. Thus, even under the best of circumstances, genetic testing may 

produce an ambiguous result, making it unclear how the patient and their family should be 

counseled.2

 

 Test characteristics are also vulnerable to several other features of study design, particularly 

selection bias, spectrum effects and verification bias, potentially helping to explain the variable 

results that have been described in the literature. In this report, we attempt to define these issues 

clearly to permit valid comparisons among studies. For example, a study applying clinical 

criteria to an unselected, consecutive population of patients with colorectal cancer may produce 

substantially different estimates of the accuracy of the Amsterdam criteria compared with a study 

enrolling patients who were referred to a tertiary care medical center because of multiple 

occurrences of cancer in the family. The latter group would be expected to have a higher 

prevalence of HNPCC and correspondingly better predictive values for the Amsterdam criteria. 

 In considering a clinical diagnosis of Lynch syndrome, sensitivity can also be defined as the 

proportion of patients with Lynch syndrome (defined by the Amsterdam criteria) with a specific 
21

predictor (see Table 3). Specificity would be the proportion of unselected patients with colorectal 

cancer in whom these predictor variables are present. 

Table 3. 2x2 table for test characteristics when considering Amsterdam criteria I as the reference standard 

Patients with Colorectal Cancer Lynch syndrome 

(Amsterdam criteria I) 

Test Present Absent 

Positive A B 

Microsatellite instability 

Abnormal 

immunohistochemistry 

Negative C D 

 However, this view permits only a limited understanding of the sensitivity or specificity of 

clinical criteria used to screen patients for HNPCC since many such criteria are included in the 

Amsterdam criteria. Nevertheless, the proportions determined using a clinical or genetic 

reference standard for HNPCC are complementary since they all describe relationships among 

predictor test, mutations, and the clinical syndrome of familial cancer clustering. 

 

Identifying HNPCC in Patients With Colorectal Cancer

 Most patients with colorectal cancer do not have a mismatch repair mutation making it 

impractical to consider universal genetic testing, especially when considering the cost (about 

$3,000 for comprehensive mutation testing).23 As a result, several strategies have been proposed 

to identify patients who should undergo additional testing. As a general rule, these have been 

based either upon clinical criteria, predictive laboratory testing of tumor tissue, or a combination 

of both. Statistical models incorporating these approaches have also been proposed.11 These 

approaches can be characterized quantitatively by determining their sensitivity, specificity and 

predictive values as described above. 

 In this report, we attempt to understand test characteristics of various approaches to 

identifying HNPCC in patients with colorectal cancer. We used these parameters to develop 

models (based upon decision-analysis) that explore different strategies for recognizing patients 

who carry the mutations. 

Clinical Criteria That Suggest the Diagnosis 

 The recognition that certain types of cancers cluster in families with HNPCC and that cancer 

develops at relatively early ages compared with the general population provided the rationale for 

development of criteria that could be used to aid in the diagnosis. Two sets of criteria (the 

Amsterdam criteria and Bethesda guidelines) developed by a consensus of experts, have been 

most widely accepted and best studied, although many similar criteria have been proposed.24,25

Both have been revised since their initial development.26,27 These criteria have applied by 

healthcare providers and genetic counselors during interviews with the patient and/or their family 

and/or with assistance of written documents such as a survey. 
22

 The Amsterdam criteria (see Table 4) were designed to establish the diagnosis of HNPCC 

based upon familial clustering of HNPCC-related tumors. As described above, some authorities 

consider the Amsterdam criteria as the formal description of Lynch syndrome. 

Table 4. Original and revised Amsterdam criteria 

Original (Amsterdam I) Revised (Amsterdam II) 

• At least 3 relatives with colorectal cancer, one of 

whom must be a first degree relative of the other 

two 

• Involvement of 2 or more generations 

• At least 1 case diagnosed before age 50 

• Familial adenomatous polyposis has been 

excluded 

• At least 3 relatives with HNPCC-associated cancer 

• One should be 1st degree relative of other two 

• At least 2 successive generations affected 

• At least 1 diagnosed before age 50 

• Familial adenomatous polyposis excluded 

• Tumors should be verified by pathologic examination 

 

 By contrast, the Bethesda guidelines (see Table 5) were designed to help predict which 

patients with colorectal cancer are likely to have a mismatch-repair mutation and should thus 

undergo further testing. However, both the Amsterdam criteria and Bethesda guidelines have 

been studied for predicting the presence of mismatch repair mutations. The Amsterdam criteria 

are much stricter than the Bethesda guidelines and thus have lower sensitivity but higher 

specificity. The Bethesda guidelines are also more applicable in small families. 

Table 5. Original and revised Bethesda guidelines 

Original Revised 

• Individuals with cancer in families that meet the 

Amsterdam criteria 

• Patients with two HNPCC-related cancers, including 

synchronous and metachronous colorectal cancer or 

associated extracolonic cancers (endometrial, ovarian, 

gastric, hepatobiliary, small bowel, or transitional cell 

carcinoma of the renal pelvis or ureter). 

• Patients with colorectal cancer and a first-degree 

relative with colorectal cancer and/or HNPCC-related 

extracolonic cancer and/or a colorectal adenoma with 

one of the cancers diagnosed before age 45 years, and 

the adenoma diagnosed before age 40 years. 

• Patients with right-sided colorectal cancer having an 

undifferentiated pattern (solid/cribriform) on 

histopathologic diagnosis before age 45 years. 

• Patients with signet-ring cell type colorectal cancer 

diagnosed before age 45. 

• Patients with adenomas diagnosed before age 40. 

• Colorectal cancer (CRC) diagnosed in a 

patient <50 

• Presence of synchronous, metachronous 

colorectal or other HNPCC-associated 

tumors regardless of age 

• CRC with the MSI-H-like histology 

diagnosed in a patient less than 60 

• CRC diagnosed in a patient with one or 

more 1st degree relatives with an HNPCCrelated tumor, with one of the cancers 

being diagnosed under age 50 

• CRC in a patient with two or more 1st or 

2nd degree relatives with HNPCC-related 

tumors, regardless of age

 Although the Bethesda guidelines and Amsterdam criteria continue to be used widely, 

several studies evaluating them (both the original and revised) have underscored the limitations 

of their accuracy in predicting the presence of mismatch repair mutations.11,12,20,28,29 A 2006 

review of the literature reported that the sensitivity of the original Amsterdam criteria ranged 

from 54 to 91%.7

 Such a wide range of estimates leaves substantial uncertainty as to the role of 
23

the Amsterdam criteria as a screening test for mismatch repair mutations. As described, above, 

there are many potential explanations for the variability across studies. In this report, we attempt 

to clarify important differences across studies that may help explain the variability. 

 In addition to the limitations regarding their predictive accuracy, there are practical problems 

with policies based on the implementation of these clinical criteria. Patients’ report of the family 

history may not be accurate, particularly for cancers other than colorectal that are potentially 

related to HNPCC.30 Issues of uncertain paternity may also be relevant in some families while 

some families may be too small (or have insufficient contact among family members) to obtain a 

clinically meaningful family history. In addition, the criteria are not always remembered or 

practical to obtain; as a result many caregivers (including oncologists) fail to obtain a detailed 

family history, and among those who do, many do not act appropriately upon it.30,31 

Testing All Cancers Regardless of the Family History 

 Because of the limitations of relying on clinical criteria to guide testing, some authorities 

have proposed that tumors from patients with colorectal cancer be evaluated for markers of 

HNPCC regardless of the family history.8,32 One of the largest studies evaluating this approach8

included 1066 patients with colorectal cancer whose tumors were tested for MSI. Patients with 

suggestive MSI results were tested for germ-line mutations in the mismatch repair genes (MSH2, 

MLH1, MSH6, and PMS2) by IHC, genomic sequencing, and deletion studies. A mutation 

causing HNPCC was detected in 23 patients (2.2 percent) of whom ten were older than 50 and 

five did not meet the Amsterdam criteria or Bethesda guidelines. 

 These data suggest that the Amsterdam or Bethesda criteria alone may miss as many as 22 

percent of patients with HNPCC. However, only five additional individuals from the cohort of 

1066 subjects (one-half of 1 percent) would have been identified by routine molecular analysis of 

all colon cancers fulfilling the Bethesda criteria, making such an approach impractically 

expensive for routine clinical use. Furthermore, the detailed laboratory analysis the authors 

performed on tumor tissue is not widely available. Despite these considerations, we include a 

strategy of testing all patients with colorectal cancer for mismatch repair mutations for 

comparison against alternative strategies. 

Combinations of Family History and Laboratory Testing 

 Most expert guidelines on HNPCC suggest a combination of sequential laboratory testing in 

patients who fulfill the Amsterdam criteria or Bethesda guidelines to minimize costs and 

maximize test accuracy.1,33 Approaches based on such a strategy have been considered to be 

cost-effective.19,34 However, the exact methods and order of testing are unsettled. Proposed 

strategies include initial testing of tumors for MSI with or without IHC for loss or expression of 

mismatch repair proteins, with germline gene sequencing reserved for patients with suggestive 

results. Certain histologic features of HNPCC-related tumors may also raise clinical suspicion, 

but none is sufficiently specific to establish the diagnosis.35,36 One report identified a specific 

oral manifestation (Fordyce granules) as highly predictive of mismatch repair mutations, but this 

observation has not yet been confirmed.37 Several other strategies for selecting patients for 

genetic testing have been described, but none has been widely adopted. Thus, this report focuses 

mainly on the predictive accuracy of testing tumor tissue for MSI and IHC. 
24

 Microsatellite instability occurs as a result of “slippage” of DNA polymerase during DNA 

replication of microsatellite DNA sequences (short dinucleotide or mononucleotide repeats).1

These are normally repaired by DNA repair mechanisms. In the presence of deficient mismatch 

repair functions (such as in HNPCC) these errors are not corrected, leading to a state that is 

referred to as microsatellite instability. The United States National Cancer Institute (NCI) defines 

the MSI-high (MSI-H) when two of five microsatellite markers from a standard panel display 

instability and the MSI-low phenotype when only one marker is unstable. Tumors without 

instability are labeled as microsatellite stable (MSS). 

 The NCI panel has been widely adopted in recent years, although some centers use additional 

or different markers. MSI-H tumors are generally more predictive of mismatch repair mutations 

than MSI-low tumors. However, approximately 10 to 20 percent of spontaneous colorectal 

cancer test positive for MSI-H, not all laboratories test for the full panel of microsatellite markers 

suggested by the National Cancer Institute, MSI-testing is not widely available, and archived 

tissue may not be readily available to perform such testing. 

 In some studies, MSI-H and MSI-L tumors are combined and compared to MSS, while in 

others each category has been considered separately. In this report, we record the specific 

methods used for determining MSI status and how tumors were categorized to permit valid 

comparisons among studies. 

 IHC techniques can identify the expression of mismatch repair proteins.38,39 Testing for other 

mismatch repair proteins has not been performed routinely, although it may be important in some 

families. This approach is less costly than MSI testing and is technically much easier. Mutations 

associated with HNPCC generally lead to the absence of a detectable gene product, although 

some may lead to a dysfunctional protein that can still be detected (and hence cause a false 

negative result). 

 Some studies have suggested that almost all tumors in which MSH2 or MLH2 is absent by 

IHC demonstrate MSI-H, while approximately 8 percent of MSI-H tumors will demonstrate 

retained immunostaining.39 However, the extent to which IHC and MSI correlate with one 

another is not known precisely. Nevertheless, some authorities have proposed that IHC may be a 

suitable alternative to MSI testing while others consider the two to be complementary. In this 

report we attempt to clarify these issues by providing test characteristics of each approach used 

alone or in combination. 

Analytic Validity 

 As noted above, there are several laboratory methods used for predictive testing for mismatch 

repair mutations and for genetic testing itself. The accuracy of these methods can be influenced 

by several factors such as the definition of the reference standard, how tissue was collected and 

processed, and the specific method by which it was analyzed. Laboratory errors (e.g., 

mislabeling of a specimen, contamination, incorrect interpretation of results) all weigh into 

overall accuracy. These considerations have been collectively referred to as analytic validity. 

 A related issue is the reliability of testing (both within a laboratory and between 

laboratories). In some clinical areas, reliability has been assessed by a method known as 

“proficiency testing” in which samples of known positive and negative biological materials are 

submitted blindly to a laboratory.40 Results can be used to determine sensitivity, specificity, and 

reliability. Proficiency testing for MSI became available in the United States in 2006. 
25

 In this report we attempt to define analytic validity and reliability of the predictive laboratory 

tests (i.e., IHC and MSI) and genetic testing methods. However, there are several limitations to 

attempting to assess these parameters using a literature-based approach: 

• The literature search was based upon HNPCC, not the specific laboratory techniques, thus 

limiting the pool of potentially relevant studies. 

• Many of the techniques described in the literature are experimental or old and thus do not 

reflect contemporary methods. 

• There is likely to be publication bias since information regarding reliability and accuracy of 

the laboratory methods have been evaluated by individual laboratories or by manufacturers of 

the testing methods. 

• Because it can be difficult to establish a clear reference-standard, many studies attempting to 

define analytic validity included the predictor tests in the reference standard, thereby making 

the results of test characteristics uninterpretable. 

 We attempted to clarify these points both by adhering to strict criteria for literature selection 

and in analyzing individual studies. 

 

Benefits and Harms of Screening, Testing, 

and Subsequent Management Strategies 

 Genetic testing for a cancer predisposition has profound implications for the affected patients 

and their families. The genetic test results have the potential to prevent cancer in the affected 

patient and their families, and may influence how patients with cancer are managed, but they can 

also lead to harm from discrimination, the risks associated with surveillance strategies or other 

interventions (such as colectomy or hysterectomy), and the psychological impact of recognizing 

a cancer predisposition. These are issues that are common to all forms of genetically based 

diseases and represent areas of intense study. In this report we critically evaluate the literature 

exploring these issues in patients and their families with HNPCC from the perspective of the 

affected patients, their family and from the point of view of providers and policy-makers. 

 Enthusiasm for genetic testing is based upon the belief that knowledge of the genetic basis 

for a disease will allow for improved treatment and prevention. How these objectives can be best 

achieved in the care of patients and families with HNPCC has not been well established. Few 

high-quality studies have evaluated the effectiveness of screening strategies based upon the 

results of genetic testing for HNPCC. It is generally agreed that patients undergoing genetic 

testing should fully understand the implications of a positive and a negative result and the level 

of certainty of a positive or negative result as a predictor of disease.41 The knowledge-base used 

to counsel patients on these parameters is still evolving. 

 Correlation of genotypes with phenotypes in HNPCC is incompletely understood, as are the 

corresponding implications for screening for HNPCC-related tumors. At least two studies42,43

and a cost-effectiveness analysis 19 suggested a reduction in colorectal cancer and mortality from 

colorectal cancer screening based upon results of genetic testing. However, the primary studies 
26

were not randomized and were vulnerable to selection bias. There is even less information 

regarding the effectives of screening for other forms of HNPCC-related cancers (particularly 

endometrial cancer).44,45 

 The uncertain benefits must be balanced against the potential for harms, which include the 

risks associated with screening procedures, the potential for false-positive results leading to 

further, possibly unnecessary testing, and the psychological, social, and economic implications 

from stigmatization. An observational study that included 16 HNPCC and HNPCC-like families 

illustrated the difficulties that may be encountered when attempting to implement a program of 

genetic screening and counseling.46 Problems encountered included lack of compliance, 

ambiguous results of genetic tests, incomplete documentation of pathologic materials or medical 

history, poor cooperation among family members and/or their physicians, patient fear and 

anxiety and perception of insurance discrimination, and lack of knowledge among referring 

physicians. Thus, the realities of implementing a program for testing patients with colorectal 

cancer for HNPCC must be understood along with the full spectrum of implications of various 

strategies for establishing the diagnosis and testing family members 
27

Chapter 2. Methods 

Key Questions Addressed in This Report 

The following Key Questions are addressed in this evidence report: 

Key Question 1: Does risk assessment and HNPCC mutation testing in patients with newly 

diagnosed CRC lead to improved outcomes for the patient or family members, or is it useful in 

medical, personal, or public health decision making? (Over-arching question). 

Key Question 2a: Assuming a clinical definition of the Lynch Syndrome, what proportion of 

patients has a mismatch repair mutation? 

2b: Assuming a clinical definition of the Lynch Syndrome, what proportion of patients has 

MSI? 

2c: Assuming a clinical definition of the Lynch Syndrome, what proportion of patients has 

abnormal protein expression by immunohistochemistry? 

2: How accurate are various predictors assuming a genetic definition of the Lynch Syndrome? 

 

Key Question 3: What are the harms associated with screening high-risk individuals for 

HNPCC? 

Key Question 4: What is known about the analytic (sensitivity, specificity, reproducibility, 

reliability) and clinical validity of tests that identify HNPCC mutations? 

 

Key Question 5: What are the harms associated with screening for high-risk individuals 

Key Question 6a: What are the management options for CRC patients who are HNPCC 

positive? 

6b: Does the identification of HNPCC mutations lead to improved patient outcomes in terms of 

early detection, mortality/morbidity or management decisions (e.g., counseling, surveillance, 

treatment, other decision making) by patients and providers? 

Key Question 7: What are the harms associated with subsequent management options after 

identification of HNPCC mutations in CRC patients? 

Key Question 8a: What is the efficacy of pre-test genetic counseling for informing family 

members of potential risks and benefits of testing? 

8b: What is the accuracy of HNPCC testing in family members in predicting the risk of CRC? 

8c: Do other factors, such as race/ethnicity, age, gender, or co-morbidities affect the accuracy of 

the testing? 

Key Question 9: What are the harms associated with informing/counseling family members or 

with subsequent testing for HNPCC mutations? 
28

Key Question 10a: What are the management options for family members of CRC patients who 

have a positive HNPCC test? 

10b1: Does the identification of HNPCC mutations lead to improved outcomes in terms of 

decision-making by patients, family members and providers, or public health policy? 

10b2: Does the identification of HNPCC mutations lead to improved outcomes in terms of early 

detection and mortality/morbidity of patients, family members? 

Key Question 11: What are the harms associated with subsequent actions or interventions for 

family members? 

These questions were formulated by EGAPP based upon an analytic model that begins with a 

patient with CRC and proceeds to genetic testing of family members. They broadly reflected the 

conceptual framework proposed by the ACCE Project from the Office of Genomics and Disease 

Prevention at the Centers for Disease Control and Prevention (CDC). The aim of the project is to 

develop a model system for assembling, analyzing, disseminating and updating existing data on 

the safety and effectiveness of DNA-based genetic tests and testing algorithms (See 

http://www.cdc.gov/genomics/gtesting/ACCE.htm for details). ACCE takes its name from its 

four components of evaluation—analytic validity, clinical validity, clinical utility and associated 

ethical, legal and social implications. It is intended to provide a model process for evaluating 

data on emerging genetic tests. The process includes collecting, evaluating, interpreting, and 

reporting data about DNA (and related) testing for disorders with a genetic component in a 

format that allows policy makers to have access to up-to-date and reliable information for 

decision making. The CDC Office of Genomics and Disease Prevention has previously published 

a mini review on HNPCC based upon the ACCE framework, which underscored the need for a 

more comprehensive review (see http://www.cdc.gov/genomics/gtesting/ACCE/fbr.htm). 

Figure 1. Analytic framework that served as the basis of the key questions proposed by the CDC Office of 

Genomics and Disease Prevention 2005 

Screening for HNPCC in Newly Diagnosed CRC Patients and 

Their Family Members

Patients

with newly 

diagnosed 

CRC

High risk of

HNPCC

HNPCC testing HNPCC mutation 

negative Patient

Outcomes

Medical

Decision 

making

Family members of HNPCC 

positive CRC patients

Genetic Counseling 

HNPCC testing

Risk Assessment

2,3 Average risk

of HNPCC

1

4,5

Screening

HNPCC

mutation

positive

6,7

Intervention

8,9

Family Outcomes

Increased early 

detection, 

decreased 

mortality/morbidity,

improved decision 

making 

HNPCC

mutation

negative

HNPCC

mutation

positive

10,

11
29

The Key Questions were refined in several teleconferences with members of EGAPP and the 

Technical Expert Panel (TEP), and following review of draft reports. 

The Key Questions and subset questions were divided conceptually into three general 

domains: clinical validity; analytic validity; and benefits and harms of screening, genetic testing 

and various management strategies from the patient, family member, provider, and public health 

perspectives. (Table 6) These domains were based upon overlapping concepts implied by the 

Key Questions and reflect the core components of the ACCE model. Thus, Key Questions were 

grouped according to the domain, and studies that addressed one of the three domains were often 

relevant to more than one Key Question, but generally not to more than one domain. 

Table 6. Summary of key questions 

Domain Key questions addressed 

Analytic validity 4 

Clinical validity 2, 2a, 2b, 2c 

Benefits and harms (including clinical utility and associated ethical, legal and social 

implications in the ACCE model) 

1, 3, 4, 5, 6a, 6b, 7, 8a, 8b, 8c, 9, 10a, 

10b1, 10b2, 11 

 

We undertook the following steps in conducting this review: 

• Performed an electronic search of the literature followed by review of relevant abstracts and 

then full-text review of potentially relevant studies. 

• Retrieved additional studies from bibliographies of retrieved citations and suggestions of the 

TEP. 

• Included or excluded studies based upon prespecified criteria. 

• Identified duplicate reports of the same patients by comparing authors and study centers. 

Data were included only once, except when duplicate studies reported complementary 

information (e.g., data at one month and then one year). 

• Developed data extraction forms for each domain (i.e., three data extraction forms) and tested 

them until we achieved consensus on the meaning of the data elements in each extraction 

form. 

• Evaluated each study critically according to quality criteria described below. 

• Verified all data with at least two extractors. 

• Summarized data in tables that addressed specific Key Questions (or groups of Key 

Questions) that corresponded to the three domains described above. Because only a few 

studies addressed analytic validity, we described them in the text of the report rather than in 

tables. 
30

• Reviewed drafts of the summary tables with members of the TEP. 

• Pooled data where studies used similar methodology and definition of endpoints using metaanalysis to provide a point estimate and 95% confidence interval, mainly for questions 

pertaining to clinical validity. We performed multiple sensitivity analyses to explore possible 

explanations when studies demonstrated statistically significant heterogeneity. 

• Constructed models depicting various strategies for identifying HNPCC among patients with 

colorectal cancer using decision trees. The models were based upon parameters estimated 

from data presented in this report. 

• Prepared a draft report, which underwent peer review and addressed each comment in a 

revised final report. 

Literature Search Strategy 

We conducted a literature search of MEDLINE® using PubMed on January 10, 2006. We 

used MEDLINE® subject headings and text words to capture relevant English language 

publications of human studies. Additional sources of potentially relevant studies included 

technical experts and hand searching of bibliographic references of reviews. An automatic 

updated search results from PubMed was received on April 1, 2006 after which additional 

studies were included only if the investigators or TEP considered them to provide substantive 

new information that might influence the conclusions (see Appendix A*). 

We reviewed all abstracts for their relevance to the Key Questions and retrieved the full-text 

article of potentially relevant citations. We reviewed bibliographies of studies included in the 

report (as well as previous review articles or meta-analyses, which were not included) to identify 

additional citations, all of which were retrieved for review. We identified duplicate reports of the 

same patients by comparing authors and study centers. Duplicate reports were excluded unless 

they provided complementary information (such as outcomes at different time points) in which 

case they were considered together. 

Inclusion/Exclusion Criteria and Data Extraction 

 We applied prespecified inclusion and exclusion criteria in considering each study. The 

criteria corresponded to the three domains described above. 

Studies Related to Analytic Validity 

We required the following for studies of analytic validity: 

• The study evaluated biological material from patients with colorectal cancer considered to be 

at risk for HNPCC. This criterion was selected to choose studies evaluating laboratory 

techniques that were directly applicable to the spectrum of genotypes associated with 

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm
31

HNPCC. We did not consider it feasible to conduct a systematic review on the individual 

laboratories tests used for other conditions. 

• Reported any of the following (these criteria were selected to choose studies evaluating the 

most common tests performed in HNPCC): 

1. Proportion of tumors that were MSI-H with National Cancer Institute (NCI) panel of 

markers (i.e., BAT-25, BAT-26 D2S123, DS346 and D17S250) versus other markers 

2. Sensitivity or specificity of MSI-H using NCI markers compared with a reference 

standard that the study claimed was better 

3. Sensitivity or specificity of IHC compared with an immunohistochemical standard 

that study claimed was better 

4. Sensitivity or specificity of a genetic technique compared with a reference standard 

(or combination of standards) 

5. Reliability of MSI, IHC or genetic methods across laboratories or within a laboratory. 

• Data were extractable into 2x2 tables. 

We excluded studies that included the index test in the reference standard. For example, 

some studies attempted to calculate sensitivity of a specific NCI MSI marker by comparing it 

with a reference standard that included the marker plus additional markers. Such an approach 

produces an invalid estimate of sensitivity since the calculation includes the index test (i.e., the 

specific NCI marker) in the numerator and the denominator. 

Studies Related to Clinical Validity 

We required all three of the following criteria to be met for studies of clinical validity with 

extractable data: 

• Enrolled patients with colorectal cancer 

• Compared an index test to genetic testing (at least one of the following: suggestive family 

history, MSI, or IHC) 

• Sought mutations using DNA sequencing (or other similar genetic approaches) for a 

minimum of MLH1 and MSH2. 

HNPCC has been defined clinically and genetically as described in Chapter 1. We used both 

approaches depending upon which Key Questions were being addressed. In all cases, the 

definitions used are described explicitly in the tables and figures. 

Key Questions 2a-2c focus on the prevalence of MMR mutations, suggestive MSI or ICH 

among patients with a clinical definition of HNPCC. We defined Lynch Syndrome I and Lynch 

Syndrome II using the Amsterdam I and II criteria, respectively. Studies that reported this 

proportion or provided sufficient data for it to be calculated were eligible. 

We required that studies evaluated all available patients who fulfilled the Amsterdam criteria 

or a representative (random) sample thereof. We excluded studies that did not evaluate all 
32

patients because they selected a non-random patient sample (e.g., patients who fulfilled the 

Amsterdam criteria who were also younger than a certain age). 

Key Question 2 pertains to the predictive accuracy of clinical or laboratory features for 

determining the presence of mismatch repair mutations. We assessed the performance of 

different clinical and laboratory predictors (preliminary tests) to identify carriers of MMR gene 

mutations. We considered all studies that provided relevant data. When the primary study 

reported proportions rather than actual counts, we reconstructed the 2x2 tables using the 

information conveyed by the proportions and their 95% confidence intervals. 

We analyzed the following predictors: 

Laboratory predictors:

1. MSI high versus MSI stable. 

2. MSI high and low versus MSI stable. 

3. Suggestive IHC versus non suggestive IHC. 

Clinical predictors specified a priori: 

1. Amsterdam I criteria fulfilled versus not fulfilled. 

2. Amsterdam II criteria fulfilled versus not fulfilled. 

3. Modified Amsterdam criteria fulfilled versus not fulfilled. 

4. Bethesda guidelines fulfilled versus not fulfilled. 

5. Revised Bethesda guidelines fulfilled versus not fulfilled. 

6. Young age of onset (<50 years) versus later age of onset . 

7. Presence of CRC or HNPCC related cancer in first degree family versus sporadic 

CRC cases. 

8. Presence of CRC or HNPCC related cancer in family (any definition) versus sporadic 

CRC cases (irrespectively of how they were selected). 

Clinical predictors specified a posteriori: 

9. Presence of multiple tumors in a CRC proband versus probands without multiple 

tumors. 

10. Presence of young age of onset (<50 years) or suggestive family history of cancer or 

multiple tumors in a CRC proband (i.e., predictors #6 or #7 or #8) versus absence of 

all three characteristics. 

We focused only on patients with CRC who received at least some form of genetic testing to 

minimize the effects of verification bias. Verification bias occurs when patients with a negative 

test result are not evaluated with the reference test.47 We generally did not accept screening with 

MSI or IHC as a substitute for genetic testing. A single exception pertained to studies assessing 

clinical predictors among newly diagnosed, unselected, non-referral CRC, because this was of 

particular clinical importance and there were few studies available for analysis. For such studies 

we accepted the authors’ assumption that patients who were not tested for mutations based on 

MSI and IHC test results were indeed mutation negative. 
33

Studies Related to Benefits and Harms 

For studies related to benefits and harms, we accepted studies of virtually any design and 

using any definition of HNPCC that reported any outcomes or other findings pertinent to patients 

or families with HNPCC, or provided insights into these issues from a public health perspective. 

We did not confine our inclusion criteria to studies of patients with colorectal cancer even 

though this report focuses on patients with colorectal cancer. Benefits and harms of genetic 

testing, counseling and other management approaches are pertinent to the full spectrum of 

tumors associated with HNPCC. For example, a woman with colorectal cancer who is found to 

have HNPCC is at risk for other forms of HNPCC-related cancer (such as endometrial cancer). 

Thus, the benefits and harms related to genetic testing may not only be relevant to clinical issues 

related to colorectal cancer management (and prevention of metachronous cancers) but also 

management (and prevention) of other HNPCC-related cancer in the patient or her family 

members. We expanded the scope of all pertinent Key Questions to provide as comprehensive a 

view on these issues as possible. 

Although we attempted to be as comprehensive as possible in including studies related to 

benefits and harms, we occasionally encountered studies that did not report any outcomes or 

other findings that appeared to be relevant to the Key Questions and thus excluded them. As 

noted above, the reasons that specific studies were excluded are summarized in the Appendix D*

. 

List of Excluded Studies. 

Evaluation of Study Quality 

We evaluated the quality of each study based upon multiple quality features. Evaluation of 

study quality is a complex process since there is no established method that can comprehensively 

describe all features that are pertinent to the validity of a study. The included studies varied in 

the rigor with which they were designed, conducted, analyzed, and reported. Deficiencies in any 

of these areas can lead to biased reporting and interpretation of results. 

Furthermore, the quality of a study and its applicability are not always related; a study that is 

considered to be of relatively low quality may be more applicable to a specific question (i.e., 

answers it directly) than a study of higher quality. In addition, assessment of quality is based 

upon information reported and not necessarily how the study was conducted since there may be 

omissions or editorial constraints in the published manuscript. 

We hoped to critically assess the quality of each study and to present studies based upon their 

applicability to each Key Question. Thus, we attempted to feature studies that answered Key 

Questions most directly and reported a composite quality score (A, B or C, described below) to 

allow comparison among studies. We based the composite quality score upon an overall 

assessment of the degree to which individual elements describing study quality were fulfilled and 

the implications of those that were not. There was an element of judgment for the final scoring of 

each study but we attempted to be as consistent as possible. 

 The composite quality scores are helpful for giving a shorthand, qualitative appraisal of the 

overall study quality, but they do not necessarily reflect particular deficiencies or strengths that 

might be important for fully understanding the results of the study or interpreting the body of 

knowledge. Thus, strengths and weaknesses of individual studies might be important when 

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm
34

considering their validity and relevance for specific Key Questions. We attempted to highlight 

study features that we considered to be most relevant while making comments as to specific 

deficiencies to allow readers to have a view on this that was transparent and succinct. The 

components of the individual quality scores for each study are available in Appendix C*

. In 

addition, important features of each study are summarized in the tables included in the body of 

the text following each Key Question to allow for easy comparison among them. 

 We used the composite quality score formally for sensitivity analysis, mainly for studies of 

clinical validity to determine whether study quality correlated with test characteristics. For 

example, we determined whether sensitivity and specificity were better for high compared with 

low quality studies. 

 The quality criteria selected for this report (summarized below) were based upon discussions 

with the TEP and quality elements that have been proposed to be relevant for the specific types 

of studies that we included. All investigators discussed details of the meaning of each quality 

element to help assure that they were applied uniformly. Investigators discussed the quality of 

individual studies whenever there were questions related to the overall score that the study 

should receive until consensus was achieved. 

 The Tufts-NEMC EPC has used similar systems for several other evidence reports. 

However, it must be acknowledged that the reliability and validity of quality scoring systems 

used for systematic reviews, including the one used for this report, have not been extensively 

evaluated.48 

Table 7. Interpretation of overall quality grading of individual studies 

Grade Explanation for quality scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those 

that are not. 

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not 

C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by 

multiple omissions in the required criteria for an acceptable study 

 

We used the following approach for grading studies related to clinical validity. These criteria 

were adapted, in part from the Centers for Disease Control and Prevention and the QUADAS 

tool for assessing diagnostic test accuracy.49,50

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm
35

Table 8. Quality criteria used to characterize studies of clinical validity 

Item Criteria Yes No Unclear 

General quality criteria 

1 Were unselected patients with CRC included (i.e., were representative of patients seen in clinical 

practice not selected based upon a suggestive family history or other criteria that may cause 

selection bias)? 

 

2 Inclusion criteria clear? 

3 Did the whole sample or a random selection of the sample (i.e., total population of patients with 

CRC) receive verification using gene sequencing? 

 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of 

sequencing (i.e., was there blinding)? 

 

5 Were the results of sequencing interpreted without knowledge of the results of the index test 

results (i.e., was there blinding)? 

 

6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g., badly 

stained tissues etc)? 

 

7 Were withdrawals from the study explained? 

8 Did the authors report AND analyze results for deleterious MMR mutants? 

Additional relevant quality items 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others 

could replicate it (e.g., either a full description or relevant references)? 

 

10 Did authors describe what specimens were tested (e.g., blood, tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a similar time interval between specimen 

collection and processing for all subjects evaluated? 

 

12 Was there a clear description of which mismatch repair mutations were being tested for? 

13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data 

were obtained across study groups (e.g., the proportion of patients who fulfilled and did not fulfill 

clinical criteria or those who did or did not have MSI who underwent sequencing.... i.e., avoid 

verification bias)? 

 

14 Were quality control methods described for the molecular and genetic tests? 

15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 

16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did 

not fulfill clinical criteria or those who did or did not have MSI) included in each batch analyzed? 

(i.e., this helps minimize the effect of random errors.) 

 

We used the following quality criteria for studies related to analytic validity. These criteria 

were adapted from those proposed by the Centers for Disease Control and Prevention.49
36

Table 9. Quality criteria used to characterize studies of analytic validity 

 Study quality Yes No Unc 

1 Was the description of how MSI or IHC and other genetic techniques described in sufficient detail that 

others could replicate it (e.g., either a full description or relevant references)? 

 

2 Did authors describe what specimens were tested (e.g., blood, tumor tissue etc.)? 

3 Was MSI, IHC, other genetic testing performed at a similar time interval between specimen collection 

and processing for all subjects evaluated? 

 

4 Was there a clear description of which mismatch repair mutations were being tested for? 

5 Were quality control methods described for the molecular and genetic tests? 

6 Did the authors attempt to address the reproducibility of results (reliability of tests)? 

7 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not 

fulfill clinical criteria or those who did or did not have MSI) included in each batch analyzed? (i.e., this 

helps minimize the effect of random errors). 

 

8 Was microdissection (technique for removing only tumor tissue from gross specimen) performed? 

9 Did the study specify whether the biological tissues were from patients known to have HNPCC 

clinically? 

 

10 Did the study include a control group in which biological material was obtained from patients known 

not to have HNPCC clinically 

 

We used the following quality criteria for studies related to benefits and harms. These criteria 

were adapted from the Cochrane Collaboration Handbook for Systematic Reviews of Health 

Promotion and Public Health Interventions for Evaluation of Studies Related to Public health.51
37

Table 10. Quality criteria for studies on benefits and harms 

Domain/question Place an “X” in one Overall rating 

Selection bias A 

(strong)

B 

(moderate)

C 

(weak)

Are individuals selected to 

participate likely to be 

representative of target 

population? 

Very likely Somewhat likely Not 

likely 

What % of selected 

individuals agreed to 

participate? 

80-100 60-79 <60 ND NA 

Allocation bias 

(RCTs only, for quasiexperimental, casecontrol/before/after, 

no control group or other 

skip to “Confounders”)

A 

(strong)

B 

(moderate)

C 

(weak)

Is the method of random 

allocation stated? 

Yes No 

If the method of random 

allocation is stated, is it 

appropriate? 

Yes No 

Was the method of random 

allocation reported as 

concealed? 

Yes No 

Confounders A 

(strong)

B 

(moderate)

C 

(weak)

Prior to the intervention, 

were there between group 

differences for important 

confounders reported in the 

paper? 

Yes No Can’t 

tell 

If there were differences 

between groups for 

important confounders, 

were they adequately 

managed in the analysis? 

Yes No NA 

Were there important 

confounders NOT reported 

in the paper (describe 

above under quality score)? 

Yes No 

Blinding A 

(strong)

B 

(moderate)

C 

(weak)

Was (were) the outcome 

assessor(s) blinded to the 

intervention or exposure 

status of the participants? 

Yes No ND NA 

Data collection methods A 

(strong)

B 

(moderate)

C 

(weak)

Were data collection tools 

shown or are they known to 

be valid? 

Yes No 

Were data collection tools 

shown or are they known to 

be reliable? 

Yes No 
38

Table 10. Quality criteria for studies on benefits and harms (continued) 

Domain/question Place an “X” in one Overall rating

Withdrawals and 

dropouts 

A 

(strong)

B 

(moderate)

C 

(weak)

Indicate the % of 

participants completing the 

study. (If the % differs by 

groups, record the lowest). 

80-100 60-79 <60 ND NA 

Analysis A 

(strong)

B 

(moderate)

C 

(weak)

Is there a sample size 

calculation or power 

calculation? 

Yes Partially No 

Is there a statistically 

significant difference 

between groups? 

Yes No ND 

Are the statistical methods 

appropriate? 

Yes No ND 

Indicate the unit of 

allocation 

Community Organization/group Provider Client Institution 

Indicate the unit of analysis Community Organization/group Provider Client Institution 

If the unit of allocation and 

analysis differed, was the 

cluster analysis done? 

Yes No NA 

Is the analysis performed 

by intervention allocation 

status (i.e., intention to 

treat) rather than the actual 

intervention received? 

Yes No Can’t 

tell 

Intervention integrity A 

(strong) 

B 

(moderate) 

C 

(weak) 

What % of participants 

received the allocated 

intervention or exposure of 

interest? 

80-100 60-79 <60 ND NA 

Was the consistency of the 

intervention measured (i.e., 

intervention was provided to 

all participants in the same 

way)? 

Yes No ND NA 

Is it likely that subjects 

received an unintended 

intervention (contamination 

or cointervention) that may 

influence the results? 

Yes No Can’t 

tell 
39

Data Synthesis, Analysis, and Reporting 

Questions Related to Analytic Validity 

 As will be discussed in Chapter 3, we encountered only a few studies of analytic validity, and 

thus these are described in the text rather than in tables. There were insufficient data for a pooled 

analysis. 

Questions Related to Clinical Validity 

 Studies relevant to each Key Question were grouped together, organized according to their 

applicability to the specific Key Question and their overall quality score. In some cases, Key 

Questions that had substantive overlap in the type of findings reported were grouped together to 

provide a comprehensive yet succinct overview of the literature. 

Questions related to clinical validity: For questions related to clinical validity, sensitivity and 

specificity were based upon the definitions summarized in Chapter 1. We presented studies by 

categorizing them for important features (such as use of similar definitions and selection criteria) 

so that similarities and differences would be as transparent as possible. 

Prevalence of MMR Mutations, MSI or Suggestive IHC Among Patients Fulfilling 

Amsterdam I and II Criteria. For each study we estimated the proportion (and exact binomial 

95% confidence interval) of Amsterdam I and II patients with MMR mutations, MSI or 

suggestive IHC. We estimated the corresponding summary prevalence values with random 

effects meta-analyses.52,53 For the quantitative syntheses, proportions from each study were logit

transformed to stabilize variances, and then back-transformed to their natural scale. Analyses 

using the more drastic Tuckey-Freeman arcsin transform yielded largely similar estimates, and 

thus are not reported. 

Diagnostic Ability of Predictors (Preliminary Tests) To Identify MMR Mutation Carriers. 

As mentioned above, the preliminary tests that identify MMR mutation carriers are either sets of 

clinical criteria, or laboratory tests (namely MSI and IHC). Different considerations are 

applicable to the analysis of clinical and laboratory predictors across a selection of studies. 

Considerations on study populations for clinical predictors (preliminary tests). In HNPCC the 

clinical preliminary tests are highly susceptible to spectrum effects across populations that have 

been selected with eligibility criteria of a clinical nature. Although the detailed analysis of this 

statement is cumbersome, it can be intuitively evaluated through an illustrative example. 

Assume that the preliminary test of interest is the clinical criterion of “age less than 50 years at 

diagnosis of CRC”. It is expected that the sensitivity and specificity of this criterion will be 

different among unselected people with CRC compared to CRC patients who were less than 55 

years at diagnosis. The second population has already been “pre-selected” based on a clinical 

criterion (less than 55 years at CRC diagnosis) that is quite similar to the evaluated “preliminary 

test” (less than 50 years at CRC diagnosis). This pre-selection alters the composition of the 

population with respect to the clinical predictor in a systematic and non-random way, both 

among MMR mutation carriers and among non-carriers. The net effect is a change in the 
40

apparent sensitivity and specificity across populations with increasing prevalence of MMR 

mutation carriers (spectrum effects). 

 Because of the aforementioned consideration we decided a priori not to synthesize the 

sensitivity and specificity of clinical predictors across populations that had been defined using 

different clinical eligibility criteria. Identifying such homogeneous populations is challenging, 

especially given the incomplete descriptions of the studied populations in many of the assessed 

studies. 

 Thus we decided to focus on populations fulfilling sets of criteria that are most frequently 

used for HNPCC, are well known, and presumably assessed identically by different research 

teams. In order of increasing prevalence of MMR mutation carriers these homogeneous 

populations were: unselected, incident CRC, patients selected by the Bethesda guidelines and 

revised Bethesda guidelines, modified Amsterdam criteria, and Amsterdam II and I criteria. We 

also created separate estimates for the sensitivity and specificity for patients who were preselected based on suggestive MSI and/or IHC results. 

Considerations on study populations for laboratory predictors (preliminary tests). The above 

consideration may not be as important for laboratory predictors (e.g., IHC) because there is no 

strong reason to assume that pre-selection with a clinical eligibility criterion (e.g., “age less than 

55 at CRC diagnosis”, as above) in a study would be correlated with IHC results both in MMR 

carriers and in those who are not MMR mutation carriers. However, it is possible that some 

laboratory predictor tests (e.g., MSI analysis) may be vulnerable to spectrum effects.54 Thus, for 

laboratory predictors, we did not require the strict similarity of populations across studies as 

above, and considered all studies together. 

Separate analyses of sensitivity and specificity. For each study we estimated the sensitivity and 

specificity (and exact binomial 95% confidence intervals thereof) for the clinical and laboratory 

predictors (preliminary tests) of interest. As described before, we derived summary sensitivity 

and specificity estimates with random effects syntheses using logit-transformations of 

proportions for the meta-analyses. As a general rule, this approach tends to underestimate the 

diagnostic performance, because it ignores the correlation between the sensitivity and the 

corresponding specificity from the same study. However, it provides a pooled estimate (and 95% 

confidence interval) of sensitivity and specificity that can be useful for providing overall 

appraisal of test performance. 

Summary receiver operating characteristic curve analyses. For laboratory predictors only, we 

summarize test performance graphically using summary receiver operating characteristic curve 

(SROC) analyses. SROC curve analysis can be used to graphically describe the tradeoff between 

sensitivity and specificity across studies. The sensitivity and specificity of each study are 

represented in the graph, thereby depicting the operating characteristics of a group of studies.

The tradeoff between sensitivity and specificity generally reflects the threshold for calling a 

test positive or negative. Such a tradeoff can be understood easily when considering a single 

study that assessed various cutoff values of a diagnostic test. By contrast, the threshold implied 

in an SROC curve analysis is not always apparent. Different estimates of sensitivity and 

specificity across studies may be due to several considerations such as variations in how the tests 

were implemented (e.g., differences in the number of microsatellite markers used) or in 

interpretation of results. With regard to the latter, the same specimen (e.g., a tumor stained for 

IHC) may be interpreted differently when the interpreter believes the test is being used for 
41

screening versus confirmation of a high-risk patient. Thus, by grouping studies according to 

specific study features, SROC analysis can be used to help explain differences in test 

characteristics across studies that seemingly used the same diagnostic test. However, it does not 

necessarily define an explicit threshold effect. 

The area under a receiver operating curve has been used to describe the overall diagnostic 

ability of a test; the greater the area, the better the test. However, calculating the area under a 

SROC curve requires extrapolation outside the range of the sensitivity and specificity values of 

the analyzed studies and thus may not produce a valid appraisal of the test’s accuracy. As a 

result, we did not attempt to calculate the area under the SROC curve. 

Subgroup and Sensitivity Analyses. We defined a priori factors that might explain 

heterogeneity of pooled estimates. These included: 

Overall quality. We compared results from high and low quality studies (as defined by the A, B 

or C classification described above). 

Study size. For the questions relating to the proportion of patients with the Lynch Syndrome 

with mutations or abnormal MSI or IHC results, we used a cutoff of 20 patients (≥20 versus <20) 

to distinguish larger and smaller studies. Although this was a largely arbitrary cutoff, a single 

misclassification in smaller studies would result in more than a 5% misclassification rate, which 

we considered clinically important. Similarly, we used a cutoff of at least 40 people in the 2 by 2 

table for studies that were used to assess sensitivity and specificity. 

 

Characteristics of how genetic testing was performed. Studies were categorized in groups 

according to the comprehensiveness of the genetic testing strategy they used. As least 

comprehensive, we classified studies that used only gene screening methods to detect MMR 

mutations, and did not perform sequencing on any sample; performed sequencing on samples 

with suggestive gene screening analyses; or performed sequencing on all available samples. As 

most comprehensive we considered studies that performed sequencing and analyses for large 

genomic deletions/rearrangements in all samples. The application of more advanced techniques 

such as conversion analysis or mono allelic mutation analysis (MAMA) was only sporadic or in 

the context of demonstrating their feasibility in only a few samples, and thus did not comprise a 

separate category. Examples of genetic screening strategies are single-stranded conformation 

polymorphism (SSCP), conformation sensitive gel electrophoresis (CSGE), denaturing gradient 

gel electrophoresis (DGGE), and denaturing high-pressure liquid chromatography (DHPLC). 

Examples of methods to detect large genomic deletions are: southern blotting and multiplex 

ligation-dependent probe amplification (MLPA). These methods are described in more detail on 

Chapter 3 in the section on Analytic Validity. 

Whether and how the study defined pathogenic mutations. We determined if and how studies 

defined mutations as being pathogenic (i.e., known to be associated with HNPCC versus a 

variant of unknown significance). It is possible that the same mutations might have been defined 

as pathogenic in one study and non pathogenic in another.55 Furthermore, not all definitions are 

equally valid and the methods used to define pathogenicity may not have been conducted with 

equal rigor. For example evidence from functional studies (i.e., in which the function of the 

mutated gene was assessed) may provide strong support that the mutation is pathogenic, whereas 

absence of the mutation in a small sample of healthy controls is not as strong. 
42

Characteristics of MSI testing. We assessed whether studies that used the panel of markers 

recommended by the NCI and whether they performed microdissection. Microdissection helps 

assure that the sample analyzed was from malignant tissue and did not contain DNA from 

surrounding, healthy colonic tissue. Microdissection is not pertinent to germline MMR mutation 

testing or IHC. Germline mutations are typically assessed in blood samples (non-malignant 

tissue). For IHC, a pathologist studies a tissue section to evaluate MMR protein expression in 

regions of malignancy (microdissection it typically not needed). 

The sample selection process. We attempted to identify studies that had a biased sample 

selection process. Studies with transparent sample selection process clearly stated that they 

selected their samples among all available patients using a set of eligibility criteria. Studies with 

non-transparent sample selection process did not report that they applied the same criteria to all 

available patients. For example, they may have used a convenience sample of cases from various 

sources. 

 Despite our efforts, we caution that studies that applied seemingly transparent eligibility 

criteria may still have a highly biased selection process. For example, studies from a referral 

center may have recruited patients with a relatively higher prevalence of familial cancer 

compared with studies that recruited consecutive patients with colorectal cancer in the 

community. 

Whether the study used consecutive, unselected patients with CRC. We identified studies that 

used consecutive CRC (including retrospective studies that assessed all unselected patients that 

were diagnosed during a specific time period) that were otherwise nonselective or representative 

of the general population. Populations evaluated in studies from specialized centers or studies 

that imposed clinical or other criteria to select their populations were not considered to be 

representative of the general population. 

Decision Tree Model Methods. We performed analyses using decision trees to model the 

expected outcomes with different testing strategies from the payers’/third party perspective. The 

outcomes were the number of incident CRC with positive diagnosis for HNPCC, and the number 

of tests (MMR, MSI, or IHC) needed to detect them. We also assessed how many patients found 

to be mutation carriers with each strategy would be truly positive. 

 The decision trees pertain to a cross-section in time. We used a hypothetical population of 

100,000 incident cases of CRC. This number is in the order of incident cases expected annually 

in the US (approximately 150,000, given an annual incidence of 50/100,000 and a population of 

300 million) and is a number convenient for calculations. 

 We assessed nine different strategies, which were most commonly represented in the studies 

summarized in this review. The strategies used clinical criteria, MSI, IHC or a combination of 

clinical criteria with MSI or IHC to select patients for MMR mutation testing. The probabilities 

that were used in the decision trees were derived from the eligible studies. See Chapter 3 for a 

complete description of the modeled strategies and the probabilities that were used. 

Questions Related to Benefits and Harms 

For studies related to benefits and harms, we grouped studies according to their relevance to 

the Key Questions, presenting comparative trials and higher quality studies first followed by 
43

qualitative and other types of descriptive studies. Statistical comparisons made within each study 

are presented; we did not attempt to recalculate these comparisons; however, specific comments 

about study methods are included in footnotes. 

The majority of studies related to benefits and harms were qualitative. Among the 

comparative trials, there were insufficient studies of similar design to allow for meta-analysis. 

We present major findings that were considered to be clinically important or addressed 

(directly or indirectly) the Key Questions and subset questions. We described together studies 

that used similar endpoints (e.g., quality of life instruments or depression scales) and 

interventions (e.g., surveillance colonoscopy) whenever possible. 

Evidence and Summary Tables 

 Evidence tables offer a detailed description of the studies that addressed each of the Key 

Questions. Each study appears once regardless of how many interventions or outcomes were 

reported. We did not attempt to construct evidence tables for all the extracted studies but all data 

extracted for each study are included in data extraction forms that are available in Appendix C*. 

 Summary tables succinctly report summary measures of the main study features and 

outcomes evaluated. They are designed to facilitate comparisons and synthesis across studies. 

Individual studies may appear more than once if the data they contain are relevant to more than 

one Key Question. The summary tables are featured in sections corresponding to each Key 

Question in Chapter 3. 

 

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm

45

Chapter 3. Results 

Overview 

 The MEDLINE® literature search produced 3,018 abstracts, which were screened for their 

relevance. The full text article was retrieved for all studies that appeared to be relevant to the 

Key Questions. With the addition of titles from technical experts and reviews of bibliographies, 

523 publications were retrieved for review. One hundred fifteen papers fulfilled eligibility 

criteria but 11 were duplicate reports and were therefore excluded (or used to provide 

supplementary information as described in Chapter 2). Thus, 104 unique studies were included 

for this review (40 for clinical validity, 3 for analytic validity and 61 for benefits and harms) 

while 410 did not meet the inclusion criteria and were rejected (see Figure 2; see also 

Appendix D*). 

Figure 2. Literature search results 

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm

PubMed

3,018 titles

Technical experts/

bibliographic references

>67 unique titlesa

Database

3,084 titles

523 Articles retrieved

&

reviewed

408 articles

rejected

115 studies

accepted

Analytic validity

3 studies

Clinical validity

40 studies

Benefits & harms

61 studies

a Additional titles overlapping with PubMed not counted.

11 duplicates 
46

 In the following sections, we present results for Key Questions as described in Chapter 2. The 

Key Questions pertaining to each domain (i.e., clinical validity, analytic validity, and benefits 

and harms) are presented within each section. 

Analytic Validity 

Key Question 4: What is Known About the Analytic (Sensitivity, 

Specificity, Reproducibility, Reliability) and Clinical Validity of Tests 

That Identify HNPCC Mutations? 

High risk of

HNPCC

HNPCC testing HNPCC mutation 

negative

Average risk

of HNPCC

4,5

Screening

HNPCC

mutation

positive

Definition 

The analytic validity of a laboratory test refers to its accuracy and reliability for identifying a 

finding of interest (such as a genotype). There are four general elements of analytic validity (see 

http://www.cdc.gov/genomics/gtesting/ACCE.htm for further details): 

1) Sensitivity (i.e., the ability to detect a finding when it is present) 

2) Specificity (i.e., the ability to exclude a finding when it is absent) 

3) Laboratory quality control (i.e., the procedures used in a testing laboratory to ensure that 

results fall within specified limits) 

4) Assay robustness (i.e., how resistant the assay is to changes in pre-analytic and analytic 

variables). 

Laboratory Tests Used in HNPCC 

The major laboratory tests used in the evaluation of patients suspected of having HNPCC 

include testing of tumor tissue using IHC, MSI testing, or germline (generally from peripheral 

blood mononuclear cells) testing for mismatch repair defects. Family members generally 

undergo only germline genetic testing (unless they have also developed a relevant cancer), 

ideally based upon the genotype of the proband. Detection of a pathogenic mutation (i.e., one 

known to be associated with HNPCC) in a proband permits testing of at-risk family members for 

the genotype. Family members with the same genotype have HNPCC while HNPCC can be 

excluded in those who do not. The situation is more complex when the proband does not have a 

detectable DNA alteration associated with HNPCC or when an alteration with unclear clinical 
47

significance is detected. In such cases, it may not be possible to exclude HNPCC in family 

members; as a result, they may be empirically offered enhanced cancer surveillance. 

The laboratory tests used in HNPCC have been described in several reviews, including a 

comprehensive summary published in 2006 to which we refer readers seeking a detailed review.7

The specific tests and order of testing used in the studies included in this review are summarized 

in Table 11. 

Table 11. Genetic testing strategies used by studies included in quantitative analyses 

Study, year Brief description of genetic 

strategy 

MMR genes 

other than 

MLH1, MSH2 

Sequencing Gene 

screening 

Deletion 

analysis 

Syngal, 2000 

Wahlberg, 2002 

Rossi, 2002 

Wolf, 2005 

Lee, 2005 

Moslein, 1996 

Debniak, 2000 

Peel, 1999 

PCR Æ Sequencing No All samples X X 

Farrington, 

1998 

PCR Æ IVSP Æ Sequencing 

PCR Æ Sequencing 

No All samples √ X 

Barnetson, 

2006 

PCR Æ Sequencing (MSH6, some 

MLH1 and MSH2 exons) 

PCR Æ DHPLC Æ Sequencing 

MSH6 All samples √ X 

Wang, 1999 PCR Æ IVSP Æ Sequencing 

PCR Æ HD Æ Sequencing 

in vivo MLH1 expression analysis 

PCR Æ Sequencing (of all other 

samples) 

MSH6 

PMS1 

PMS2 

Practically 

all samplesa √ √

Durno, 2005 [used PTA and Sequencing] No Some 

samples?b √ X 

Aaltonen, 1998 MSI Æ PCR Æ DGGE (some 

samples) Æ Sequencing; 

 MSI Æ PCR Æ Sequencing (all 

 other samples) 

PCR for founder mutations in 

MLH1 (all samples) 

No Some 

samples 

√ √

Salovaara, 

2000 

MSI Æ PCR Æ Sequencing 

PCR for founder mutations in 

MLH1 gene 

No Some 

samples 

X √

Liu, 2004 

Colombino, 

2005 Yuan, 

2004 

De Abajo, 2005 

PCR Æ DHPLC Æ Sequencing No Some 

samples 

√ X 

Southey, 2005 PCR Æ DHPLC Æ Sequencing 

MLPA (in only 10 tumors)c MSH6 

PMS2 

Some 

samples 

X √

Curia, 1999 

de Leon, 1999 

Dieumegard, 

2000 Yuan, 

1998 

PCR Æ SSCP Æ Sequencing No Some 

samples 

√ X 

Katballe, 2002 

Christensen, 

2002 

PCR Æ SSCP & HD ÆSequencing No Some 

samples 

√ X 

Luce, 1995 PCR Æ IVTT Æ Sequencing No Some 

samples 

√ X 
48

Table 11. Genetic testing strategies used by studies included in quantitative analyses (continued) 

Study, year Brief description of genetic 

strategy 

MMR genes 

other than 

MLH1, MSH2 

Sequencing Gene 

screening 

Deletion 

analysis 

Zhu, 2005 MLPA Æ Sequencing No Some 

samples 

X √

Park, 1999 PCR Æ SSCP 

Southern blotting 

PMS1 

PMS2d None √ √

Callistri, 2000 PCR Æ SSCP No None √ X 

Lamberti, 1999 PCR Æ SSCP 

RT-PCR, PTA 

No None √ X 

Samowitz 2001 MSI Æ PCR Æ Sequencing 

MSI Æ PCR for founder mutations 

in MLH1 

No Some 

samples 

X √

Raedle, 2001 

Pistorius, 2000 

MSI Æ PCR Æ Sequencing No Some 

samples 

X X 

Terdiman, 2001 MSI Æ PCR Æ DGGE Æ

Sequencing 

No Some 

samples 

√ X 

Nakahara, 1997 MSI Æ PCRÆ SSCP Æ

Sequencing 

No Some 

samples 

√ X 

Casey, 2005 MSI, IHC Æ PCR Æ Sequencing 

MSI, IHC Æ Conversion analysis 

MSI, IHC Æ Deletion analysis 

No All 

availablee

 

X √

Pinol, 2005 MSI, IHC Æ PCR Æ Sequencing 

MSI, IHC Æ MLPA 

No Some 

samples 

X √

Stormorken, 

2001 

Not described MSH6 Unclear Unclear Unclear 

DHPLC: denaturating high performance liquid chromatography; DGGE: Denaturating gradient gel electrophoresis; HD: 

heteroduplex formation; IHC: immunohistochemistry; IVSP: in vitro synthesized protein test; IVTT: in vitro transcription 

translation; MLPA: multiplex ligation-dependent probe amplification; MSI: Microsatellite instability testing; PCR: polymerase 

chain reaction; SSCP: Single-stranded conformation polymorphism. 

Shown are the genetic strategies used in the various studies that were assessed in the quantitative analyses of this report. All 

studies assessed MLH1 and MSH2 at minimum. a In Wang 1999 essentially all patients were sequenced, but not completely; however, complete sequencing was performed for 

those testing negative with other methods. b Durno 1999 does not describe the used strategy in detail. c The 10 tumors were MSI positive, IHC negative and MMR negative with the usual strategy. d In Park 1999 PMS1 was assessed in 27 samples and PMS2 on 24 samples out of 123 samples for MLH1 and MSH2 (all were 

negative, unclear how the 27 and 24 were selected). e All available patients were tested with full sequencing. However, the sample was assembled mainly on the basis of suggestive 

MSI testing (85 out the 89 patients had suggestive MSI testing). 

Immunohistochemistry. Pathogenic mutations in mismatch repair proteins usually lead to the 

absence of a detectable gene product providing the rationale for IHC techniques used to detect 

underexpression. Tumors from patients suspected of having HNPCC (based upon clinical or 

pathologic findings) can be stained for mismatch repair proteins. The surrounding normal 

colonic tissue can be used as a positive control. 

As noted in the table below, testing is available commercially for MLH1, MSH2, MSH6 and 

PMS2, although the extent to which various laboratories stain for all of these proteins is unclear. 

Furthermore, such staining is relatively easy to perform and available in kits. As a result, local 

pathologists, who may select to stain for some or all of these proteins, can perform it. 

Knowledge of how the mismatch repair proteins interact during DNA repair can help 

interpret the results of such testing and be useful for guiding germline genetic testing. For 

example, MSH2 forms a heterodimer with MHS6; MLH1 complexes with PMS2 and binds to 

the MSH2-MSH6 heterodimer. When MSH2 is not expressed in a tumor, MSH6 is also not 
49

expressed. Because MSH2 and MLH1 are the most common mismatch repair proteins implicated 

in HNPCC, a patient who has a tumor that does not stain for MSH2 (and whose normal 

surrounding colonic mucosal demonstrates preserved staining) is most likely to have a mutation 

in MSH2 (but could also possibly have a mutation in MHS6). Similarly, MSH6 may be the likely 

gene involved in a patient with a tumor that expresses MLH1 and not MSH6 (again with normal 

staining patterns in surrounding colonic mucosal). The situation is more complex with lack of 

expression of MLH1; promoter hypermethylation of MLH1 is common with sporadic colorectal 

cancer and may lead to its underexpression. Some laboratories offer methylation analysis to help 

determine whether lack of expression is due to promoter hypermethylation. Such an approach 

has been suggested in various reviews of HNPCC. 

Immunohistochemistry has an advantage over other techniques (particularly MSI testing) 

since it is much easier to perform and is less expensive. However, the technique is vulnerable to 

the quality of tissue preparation, staining, and interpretation. This concern is not merely, 

hypothetical; our literature search revealed that sensitivity to detect loss of MSH2 expression 

ranged from 84 to 100 percent in a study of 18 participating centers performing such testing.56

Variability in specificity was even greater (see below). 

Microsatellite Instability. Microsatellite instability (MSI) refers to a variety of patterns of 

microsatellite repeats observed when DNA is amplified from a tumor with defective mismatch 

repair compared with DNA amplified from surrounding normal colonic tissue. Repetitive monoor dinucleotide DNA regions are particularly vulnerable to defective mismatch repair. For 

example, the mononucleotide sequence “AAAAAAAAAAAAAAAAAAAAAAAAAA” is 

located on chromosome 2P, near the MSH2 gene locus. This sequence is referred to as the “Big 

A Tract-26” (BAT26). As a result, a tumor suspected to result from mismatch repair defects can 

be tested for the presence of these repeats.

MSI testing involves amplification of a standardized panel of DNA markers; five markers 

were agreed upon by a consensus panel convened by the National Institutes of Health in 1997 

(BAT25, BAT26, D2S123, D5S345, and D17S250) as described in Chapter 1. Three categories 

of MSI have been recognized based upon these panels: MSI-high (instability of two or more 

markers), MSI-low (instability of one marker), and MS-stable (no instability). More recently, 

some laboratories have begun using ten or more markers. In such cases MSI is defined as 

“stable” when fewer than 10% of markers are unstable, “low” when 10 to 30% of markers are 

unstable, and “high” when greater than 30% of markers are unstable (some laboratories use 

40%). We recorded the markers used in each study included in this review (see section on 

clinical validity and accompanying tables). 

There are several pitfalls of MSI testing. First, it is labor intensive, relatively costly 

(compared with IHC), and requires expert pathologic services. In addition, tissue to be amplified 

should ideally be microdissected to avoid amplifying DNA from normal colonic mucosa. We 

systematically recorded whether microdissection was performed for all studies related to clinical 

validity. As a practical consideration, tissue may not always be available since the diagnosis of 

HNPCC may not be suspected when the cancer was first diagnosed. 

Genetic Testing. Multiple methods have been used for genetic testing in HNPCC. The methods 

used should ideally be able to detect the many potential genotypes associated with HNPCC (e.g., 

nonsense, missense, and frameshift mutations, genomic deletions, duplications, and 

rearrangements). We recorded the specific methods and order of testing used in all studies 

included in this report and, in the case of clinical validity, attempted to discern whether the 
50

specific testing methods were associated with the accuracy of HNPCC testing (see sections and 

corresponding tables on clinical validity). 

A detailed review of these methods is beyond the scope of this report. However, the 

following summarizes major categories of testing that are used currently, and that were reported 

in the studies included in this report. 

High Output Screening Techniques. High output screening techniques include single stranded 

conformation polymorphisms (SSCP), conformation sensitive gel electrophoresis (CSGE), 

denaturing gradient gel electrophoresis (DGGE) and denaturing high-pressure liquid 

chromatography (DHPLC). These methods all take advantage of the observation that alteration 

of DNA (due to a polymorphisms or mutation) confers chemical properties that allow it to be 

differentiated from normal DNA. These approaches can be performed relatively rapidly and 

allow more detailed studies (such as DNA sequencing) to be targeted to specific regions of DNA. 

 

DNA Sequencing. DNA sequencing can be used following a high output screening technique or 

as a primary approach (particularly when IHC patterns allow for targeting of a specific mismatch 

repair gene). It is considered the method of choice for detecting most mismatch repair gene 

mutation. However, it does not reliably allow for detection of deletions or rearrangements, which 

are important in HNPCC. DNA sequencing has become automated in recent years, greatly 

reducing the required time, costs, and expertise. 

Conversion analysis. Conversion analysis involves converting diploid cells to haploid cells so 

that only a single allele is analyzed at a time. The rationale is based upon the observation that a 

wild-type allele can mask the presence of a mutant allele when performing DNA sequencing 

(thereby obscuring the presence of a mutation). Conversion testing can increase the yield of 

genetic testing in HNPCC but is technically complicated, expensive, and, as a result, not widely 

available. We recorded whether conversion analysis was performed in all studies included in this 

report. 

Methods To Detect Large Structural DNA Abnormalities. Large structural DNA 

abnormalities (such as large genomic deletions, rearrangements) are potentially important in 

HNPCC but are not detected by the high output screening techniques or DNA sequencing. There 

are several methods for detecting these defects. We recorded the specific methods used for all 

studies included in this report. 

Southern blotting involves digestion of genomic DNA (which breaks it into pieces), 

separation of fragments using electrophoresis, transfer of the fragments to a membrane, and 

hybridization using probes to recognize deletions, duplications or rearrangements. Southern 

blotting has not yet been automated to the extent of DNA sequencing and is time-consuming. 

Multiplex ligation-dependent probe amplification (MLPA) is a newer technique. It involves 

measurement of the relative copy number of DNA sequences. MLPA has evolved to become a 

standard approach for analyzing mismatch repair genes for deletions. 

 

Family History. The family history (and related risk assessment tools such as the Amsterdam 

criteria) can also be considered as a type of laboratory testing, which can be applied to both the 

probands and their family members. The accuracy of the family history in predicting the 

presence of germline mismatch repair mutations is described in the sections on clinical validity. 
51

By contrast, the analytic validity of the family history can be considered the accuracy with which 

individuals are able to report their family history. 

We did not identify any studies that assessed the analytic validity of the family history in 

patients or families with HNPCC. However, we confined our literature search specifically to 

HNPCC, while there are several studies that have assessed the validity of the family history for a 

variety of tumor types, including those associated with HNPCC such as colorectal or endometrial 

cancer.57-60 A systematic review of these data found that (in individuals without a personal 

history of cancer) the positive and negative likelihood ratios of a family history for colon cancer 

in a 1st degree relative were 23.0 (95% CI 6.4-81.0) and 0.25 (95% CI 0.1-0.63), respectively.59

These values were 14.0 (95% CI 2.2-83.4) and 0.68 (95% CI 0.31-1.52), respectively for 

endometrial cancer. In another report, the degree of relationship to the probands, type of cancer, 

age at diagnosis of the probands, and source of ascertainment of probands were all statistically 

significant predictors of the accuracy of reporting.60 The extent to which these data can be 

generalized to patients and families with HNPCC is unclear. 

Limitations of a Literature-Based Approach to Analytic Validity 

We encountered only three studies that were eligible based upon our inclusion criteria, and 

these are summarized below. This was disappointing but expected, since a literature-based 

approach to understanding analytic validity has severe limitations as described in Chapter 1: 

A) Many of the laboratory techniques (e.g., DNA sequencing) are used not only in 

testing for HNPCC but also in testing for a variety of genetic disorders. As a result, a 

comprehensive search of issues related to analytic validity should consider the entire 

literature evaluating such testing, an undertaking that is neither practical nor feasible. 

A search in MEDLINE® on DNA sequencing, for example, will yield more than 

416,000 citations. 

As noted in the methods section, we confined our search to studies that used 

biological materials from patients or family members suspected of having HNPCC to 

make the results as applicable to the population of interest as possible. The accuracy 

of these methods used for other disorders may not be generalizable to HNPCC. The 

extent to which such sources of indirect evidence may be applicable to HNPCC was 

beyond the scope of this review. 

B) The methods used in laboratory testing evolve rapidly. The specific methods used are 

commercialized by companies or individuals who developed them. As a result, the 

published literature may not (and often does not) reflect the actual testing performed 

in clinical laboratories. It is customary for commercial laboratories to assess at least 

some components of analytic validity for these contemporary tests, but laboratories 

are under no obligation to publish the results. 

A partial list of laboratories offering testing services for HNPCC is available through 

“Genetests” an organization funded through the National Institutes of Health (see 

http://www.genetests.org/servlet/access?id=8888891&key=AmmF6UdFjuxHB&fcn=

y&fw=vDlR&filename=/). A summary of the tests listed on this Website is presented 
52

in Table 12 below. We attempted to contact one of the laboratories (chosen at 

random) to determine whether they would share information related to analytic 

validity. Our attempt had limited success. Such a review of “gray” literature may be 

helpful for understanding the analytic validity of these tests but would require 

appropriate resources, including incentives, for participating laboratories. 

Some commercial laboratories participate in proficiency testing, but the results are 

generally not published. Proficiency testing involves distributing samples of tissue 

that are known to be positive or negative for findings of interest. Laboratories 

participating in such programs can obtain a benchmark for how they are performing 

in detecting (or excluding) such findings (e.g., genotypes of interest). Proficiency 

testing also helps understand issues related to reliability. 

A major limitation of proficiency testing is that it does not consider the pre-analytic 

components of testing; for example, the accuracy of a test may be greatly affected by 

the methods used to acquire tissue. In addition, proficiency testing does not allow for 

a detailed understanding of the spectrum of abnormalities (e.g., various genotypes) 

that may be clinically relevant. 

Proficiency testing for MSI was introduced in 2006 and is being conducted by the 

College of American Pathologists. Further information is available at their Website 

(see http://www.cap.org/apps/cap.portal) but the initial results of their program have 

not been made public. 

C) Studies evaluating analytic validity often suffer from serious methodologic 

limitations that potentially invalidate the results. Particularly problematic was the 

inappropriate use of reference standards or lack of clear definitions of references 

standards altogether. For example, a common pitfall was the inclusion of the test 

under evaluation as part of the reference standard. 

We chose inclusion and exclusion criteria carefully to select for high quality studies 

while excluding studies whose results are misleading or clinically irrelevant. There 

were remarkably few high-quality studies directly addressing analytic validity using 

biological materials from probands or family members proven or suspected to have 

HNPCC.

Eligible Studies. We identified only three studies that fulfilled eligibility criteria for analytic 

validity.

• One study compared conversion analysis (in which alleleles are separated into hybrids prior 

to mutation screening) with DNA sequencing alone to detect heterogeneous germline 

mutations in MLH1, MSH2 and MSH6 in patients with colorectal cancer.61 The authors 

estimated that conversion analysis provided an increased yield of 56% (35/63 cases) 

compared with DNA sequencing alone. The study was rated a methodologic quality B 

because the investigators were not blinded to the method used. 
53

• Another study included 20 patients with CRC with known mutations in MLH1 or MSH2.56 A 

set of two unstained slides from each case were sent to participating medical centers with 

capability of performing immunoperoxidase assays for MLH1 and MSH2. 

 Of 18 participating centers 2 were excluded: one because slides were damaged in transit 

and the other because of insufficient staining. Sensitivity for detecting loss of MSH2 

expression ranged from 84 to 100%; 10 centers identified all six. Five out of six false 

positive results were in the same case suggesting that staining or interpretation were not 

random. Fourteen out of 16 laboratories showed 100% specificity (one laboratory had 93% 

specificity due to staining failure on one slide and one lab demonstrated 45% specificity due 

to weak or absent staining in most cases.) 

 Re-review of returned MSH2 slides showed lack of internal positive control staining in at 

least 2 of the 6 MSH2-negative cases from 8 of 16 centers. The other 8 centers had 100% 

sensitivity and 93-100% specificity on re-review. The slides that lacked internal positive 

control staining were largely accounted for by two cases. 

 The study was rated a methodologic quality B because of its small sample size, and 

because it did not describe quality control methods or whether microdissection had been 

performed when preparing the specimens. 

• A third study evaluated the sensitivity and specificity of DHPLC analysis compared with 

DNA sequencing in 46 patients with colorectal cancer from families with HNPCC.62 DHPLC 

analysis identified 19 changes previously identified by DNA sequencing and 16 new 

alterations not previously described. DHPLC was considered to be highly sensitive, detecting 

a mutation in all patients with no false negative results. The study was rated methodologic 

quality C because of several deficiencies in reporting. 

Summary. In summary, the analytic validity of the tests used to evaluate HNPCC is substantially 

uncertain. However, there is heterogeneity in the type of testing offered by commercial 

laboratories and the available data suggest that there may be variability across testing facilities. 

Additional information that could shed light upon analytic validity is available but would require 

evaluation of non-published data sources. Committees of experts could also review the strengths 

and limitations of specific testing techniques based upon clinical experience, with particular 

reference given to experience with these methods in other genetically-based disorders.

 Table 12 presents information from North American laboratories offering genetic testing for 

HNPCC. Most of the data was retrieved through the website, GeneTests.org. When available, 

links to the clinical laboratory websites offering genetic testing service were accessed to gather 

additional detail on type of tests offered. The information presented is not comprehensive as 

reporting to GeneTests.org is voluntary.
54

Table 12. North American laboratories offering clinical testing for Hereditary Nonpolyposis Colon Cancer (Lynch Syndrome)* Clinical laboratories Location 

Analysis of 

entire coding 

region: 

Sequence 

analysis 

Sequence 

analysis of 

select 

exons 

Analysis of 

entire coding 

region: 

Mutation 

scanning 

Targeted 

mutation 

analysis 

Linkage 

analysis 

Microsatellite 

instability testing 

(MSI) 

Deletion/

duplication 

analysis 

Mutation 

scanning of 

select 

exons 

h

ARUP Laboratories, Inc. Salt Lake City, UT 

--- --- --- --- --- PCR: 

BAT25, BAT26, 

D2S123, D5S346, 

D17S250 in MSI low 

(instability<30%) 

samples, additional 

markers available: 

BAT40, MYCL1, 

TGS-beta-R2, 

D10S197, D18S58 

--- --- 

Baylor College of Medicine Medical Genetics Laboratories Houston, TX 

PCR-based 

assay: MLH1, MSH2, MSH6 

aMLH1 

promoter 

methylation 

assay (in 

combination 

with MSI 

analysis): 

MLH1, MSH2, 

MSH6 

--- --- --- --- Multiplex PCR-based 

assay (in combination 

with MSI analysis): 

BAT25, BAT26, 

NR21, NR22, 

D2S123, D17S250, 

D5S346, D18S35, 

DIS2883 

PCRbased 

assay: 

MLH1, 

MSH2 

--- 

Boston University School of Medicine Center for Human Genetics Boston, MA 

MLH1, MSH2, 

MSH6 

MLH1, 

MSH2, 

MSH6 

--- --- --- --- MLPA --- 
55

Table 12. North American laboratories offering clinical testing for Hereditary Nonpolyposis Colon Cancer (Lynch Syndrome)* (Clinical laboratories Location 

Analysis of 

entire coding 

region: 

Sequence 

analysis 

Sequence 

analysis of 

select 

exons 

Analysis of 

entire coding 

region: 

Mutation 

scanning 

Targeted 

mutation 

analysis 

Linkage 

analysis 

Microsatellite 

instability testing 

(MSI) 

Deletion/

duplication 

analysis 

Mutation 

scanning of 

select 

exons 

h

Children's Hospital of Eastern Ontario Molecular Genetics Diagnostic Laboratory Ottawa, Ontario, Canada 

MLH1, MSH2 --- --- --- --- --- MLH1, 

MSH2 

--- 

City of Hope Clinical Molecular Diagnostic Laboratory Duarte, CA 

√ --- Fluorescent 

sequencer: 

MLH1, MSH2, 

MSH6 

√ --- Amplification ≥5 

markers by 

denaturing 

polyacrylamide gel 

electrophoresis: MSI 

≥2 markers, low MSI 

= 1 marker 

--- --- 

Creighton University Medical Center Creighton Medical Laboratories Omaha, NE 

--- --- --- --- --- √ --- --- 

Fox Chase Cancer Center Clinical Molecular Genetics Laboratory Philadelphia, PA 

--- --- --- --- --- √ --- √ 
56

Table 12. North American laboratories offering clinical testing for Hereditary Nonpolyposis Colon Cancer (Lynch Syndrome)* (c

Clinical laboratories Location 

Analysis of 

entire coding 

region: 

Sequence 

analysis 

Sequence 

analysis of 

select 

exons 

Analysis of 

entire coding 

region: 

Mutation 

scanning 

Targeted 

mutation 

analysis 

Linkage 

analysis 

Microsatellite 

instability testing 

(MSI) 

Deletion/

duplication 

analysis 

Mutation 

scanning of 

select 

exons 

h

Huntington Medical Research Institutes Molecular Oncology & Cancer Genetics Laboratory Pasadena, CA 

MSH2, MLH1, 

MSH6 

MSH2, 

MLH1 

--- --- MSH2, 

MLH1 

PCR: MSH2, MLH1 √ --- 

London Health Sciences Centre Molecular Diagnostic Laboratory London, Ontario, Canada 

MSH2, MLH1 --- --- --- --- √ √ --- M

w

Mayo Clinic Molecular Genetics Laboratory Rochester, MN 

MLH1, MSH2, 

MSH6 

--- --- --- --- --- MLPA: 

MLH1, 

MSH2 

--- 

Memorial University of Newfoundland Molecular Genetics Laboratory St. John's, Newfoundland, Canada 

--- --- --- MSH2, Del 

50 

CODONS 

--- --- --- --- 
57

Table 12. North American laborato ries offering clinical testing for Hereditary Nonpolyposis Colon Cancer (Lynch Syndrome)* (Clinical laboratories Location 

Analysis of 

entire coding 

region: 

Sequence 

analysis 

Sequence 

analysis of 

select 

exons 

Analysis of 

entire coding 

region: 

Mutation 

scanning 

Targeted 

mutation 

analysis 

Linkage 

analysis 

Microsatellite 

instability testing 

(MSI) 

Deletion/

duplication 

analysis 

Mutation 

scanning of 

select 

exons 

h

Myriad Genetics Laboratory Salt Lake City, UT 

MLH1, MSH2, 

MSH6 

(Southern Blot 

available for 

MLH1 and 

MSH2 

--- --- √ --- --- √ --- 

North York General Hospital Molecular Genetics Laboratory North York , Ontario, Canada 

--- --- --- --- --- √ --- --- 

Ohio State University Molecular Pathology Laboratory Columbus, OH 

--- --- --- --- --- BAT 25, 

BAT 26 

--- --- 

Quest Diagnostics, Inc. Molecular Genetics Laboratory San Juan Capistrano, CA 

MLH1, MSH2, 

MSH6 

--- --- --- --- Multiplex polymerase 

chain reaction (PCR): 

BAT 25, BAT 26, 

D2S123, D5S346, 

D17S250 

√ --- 

Saint Louis University Health Science Center DNA Diagnostic Laboratory Saint Louis, MO 

--- --- --- --- --- √ --- --- 
58

Table 12. North American laborato ries offering clinical testing for Hereditary Nonpolyposis Colon Cancer (Lynch Syndrome)* (Clinical laboratories Location 

Analysis of 

entire coding 

region: 

Sequence 

analysis 

Sequence 

analysis of 

select 

exons 

Analysis of 

entire coding 

region: 

Mutation 

scanning 

Targeted 

mutation 

analysis 

Linkage 

analysis 

Microsatellite 

instability testing 

(MSI) 

Deletion/

duplication 

analysis 

Mutation 

scanning of 

select 

exons 

h

UCLA Medical Center Diagnostic Molecular Pathology Laboratory Los Angeles, CA 

--- --- --- --- --- √ --- --- 

University of Alberta Molecular Diagnostic Laboratory Edmonton, Alberta, Canada 

--- --- MSH2, 

MLH1 

--- --- --- √ --- 

University of Pennsylvania School of Medicine The Genetic Diagnostic Laboratory Philadelphia, PA 

MSH2, 

MLH1 

(If negative, 

Southern blot 

optional) 

--- √ --- √ --- √ --- 

*May include Muir-Torre Syndrome, Turcot Syndrome. √ = Genetests.org indicates offered by laboratory, but no additional information given. Abbreviations: PCR, Polymerase Chain Reaction/Fragment Analysis. a due to space limitations promoter methylation assay was reported in this column.
59

Clinical Validity 

Key Question 2 and Pertinent Subquestions 

Patients

with newly 

diagnosed 

CRC

High risk of

HNPCC

Risk Assessment

2,3 Average risk

of HNPCC

Screening

 

 

 HNPCC has been defined clinically and genetically as described in Chapter 1. The following 

sections present analyses using either the clinical or the genetic definition of the condition, 

depending on which Key Question was being addressed. 

 Key Questions 2a to 2c seek to evaluate the prevalence of MMR mutations and suggestive 

MSI and IHC among patients with the Lynch Syndrome. Thus, they assume a clinical definition 

of the condition. 

 Key Question 2 pertains to the ability of clinical and laboratory predictors to identify the 

presence of MMR mutations. Thus, it assumes a genetic definition of the condition. 

 We first present analyses based on a clinical definition of HNPCC. In these analyses we 

estimated the frequency of mismatch repair gene mutations and tumors with MSI and IHC 

among CRC patients fulfilling the Amsterdam I and II criteria. 

 We present subsequent analyses based on the genetic definition of the Lynch syndrome. 

These help define test characteristics of various strategies (such as the combination of a clinical 

history with laboratory testing of tumor tissue) for predicting the presence of mismatch repair 

mutations. 

 Finally, we used a decision tree model to calculate the expected number of patients with 

MMR mutations among unselected patients presenting with CRC using various predictive 

strategies. The parameters used in the calculations are based on our best estimates derived from 

this systematic review. 

Key Question 2a: Assuming a Clinical Definition of the Lynch 

Syndrome, What Proportion of Patients has a Mismatch Repair 

Mutation? 

 Among CRC fulfilling Amsterdam I criteria, the random effects summary prevalence of 

MLH1 and MSH2 gene mutations was 44% (95% CI: 35, 52; n=19 studies, 464 CRC patients), 

with evidence for substantial between-study heterogeneity (p<0.01; I2

=52%). The six studies that 

performed genetic testing among all Amsterdam I patients had a summary prevalence of 51% 

(95% CI: 35, 66%; 84 CRC patients). 
60

 For patients fulfilling the Amsterdam II criteria, the corresponding prevalence values were 

39% (95% CI: 30, 49%; 10 studies, 279 CRC patients) and 40% (95% CI: 30, 52%) based upon 

2 studies that performed sequencing on all 87 Amsterdam II patients. 

 Only three studies examined other MMR genes (MSH6, PMS1 and PMS2) in Amsterdam I 

patients, without identifying any additional mutations. Two additional MSH6 mutations were 

found among 20 Amsterdam II patients in a single study. 

Frequency of MMR Gene Mutations Among Patients Fulfilling Amsterdam Criteria I. 

Description of studies. Nineteen studies (described in 21 papers 63-83) provided data for the 

prevalence of MMR mutations among CRC probands who fulfilled the Amsterdam I criteria. The 

median number of patients with relevant data in each study was only 13 (interquartile range: 10, 

22), and the actual number ranged between 3 and 154 patients. However, study sizes (all 

included patients) ranged between 19 and 509. Only five studies included at least 20 probands 

fulfilling the Amsterdam I criteria.67,72,76,79-81 Nine studies were rated grade B for their overall 

methodologic quality63,65-67,70,71,74,77,79-81 while the rest were rated grade C (Appendix F-1*). 

 Six studies performed bidirectional sequencing on all patients who fulfilled the Amsterdam I 

criteria.69,75,77,79,81,83 (Colombino 200583 performed full sequencing only among familial CRC 

cases, which included Amsterdam I patients). Ten studies performed full sequencing only on 

patients who were selected by gene screening methods such as DHPLC or SSCP.63,66-

68,70,71,73,74,76,82 The remaining three studies did not describe the presence of any mutation with 

bidirectional sequencing in any patient64,72 or did not provide any details on the genetic testing 

strategy they used.78 None of these 19 studies used conversion analysis to detect mutations. 

Assessment of mutations in MMR genes other than MLH1 and MSH2. All reports assessed 

mutations in the MLH1 and MSH2 genes. Additional MMR genes were assessed in three 

studies.76,78,81 Park 199976 assessed the PMS1 and PMS2 genes in a minority of their Amsterdam 

I patients (24 and 27 patients respectively, less than 20% of their sample), without identifying 

any additional mutations(Appendix F-1*). Wang 199981 also tested for MSH6, PMS1 and PMS2, 

but found no additional mutations among the subgroup of patients fulfilling the Amsterdam I 

criteria. Stormorken 200178 also tested for MSH6 mutations, but found none among Amsterdam I 

patients.

Analyses. Overall, the random effects summary estimate from the 19 studies was 44% (95% CI: 

35, 52%), with evidence for substantial between-study heterogeneity (p<0.01; I2

=52%) (Table 

13, Figure 3). The six studies that performed full sequencing in all available samples identified 

mutations in 51% (95% CI: 35, 66%) of 84 patients fulfilling the Amsterdam I criteria (Table 

13). The corresponding prevalence estimate was 44% when sequencing was performed after 

suggestive SSCP, DHPLC or other gene screening methods, and only 33% among studies that 

used screening methods without further confirmation by sequencing (Table 13). The prevalence 

of MMR mutations did not differ beyond chance with respect to overall study quality score, 

whether the study was small (i.e., analyzed fewer than 20 patients), or whether the authors 

described how they classified mutations as being pathogenic. 

 Finally, three studies (n=28 eligible patients) that sampled unselected, nonreferral patients 

with CRC found a higher prevalence of MMR mutations (66%) among patients who fulfilled the 

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm
61

Amsterdam I criteria, compared to studies that sampled among referral or otherwise selected 

cases (prevalence 41%; Table 13). The precision of these estimates is uncertain because of the 

very small number of eligible patients. 

Figure 3. Prevalence of mismatch repair gene mutations among colorectal cancer patients fulfilling the 

Amsterdam I criteria. 

Three panels are shown, representing three subgroups of studies. Top: Studies that performed sequencing on all Amsterdam I 

patients. Middle: Studies that performed sequencing only after a suggestive gene screening method. Bottom: studies that did not

perform sequencing on any patient. Within each subgroup, studies are ordered by decreasing number of patients. The bottom 

diamond represents the overall random effects estimate. MMR: mismatch repair. 
62

Table 13. Summary estimates of the prevalence of MMR mutations among CRC fulfilling Amsterdam I criteria 

Summary Number of 

studies (CRC 

patients 

fulfilling AM1) 

% with 

mutations 

(95% CI) 

Heterogeneity 

P-value, (I2

[%]) 

Betweensubgroup 

heterogeneity, 

P-value 

Overall 19 (464) 44 (35, 52) <0.01 (52) NA 

Overall quality scale 

B a 9 (162) 49 (37, 61) 0.09 (41) 

C 10 (302) 39 (28, 51) 0.01 (58) 

0.12 

Sequencing 

All samples 6 (84) 51 (36, 66) 0.17 (35) 

Some b

 10 (298) 44 (33, 55) 0.04 (50) 

None 3 (82) 33 (19, 51) 0.16 (46) 

0.02 

Deletion analysis and sequencing in all samples 

Yes 1 (22) 64 (41, 83) NA 

No 18 (442) 42 (34, 51) 0.03 (51) 

0.09 

Assessment of additional MMR genes (other than MLH1 and MSH2) 

Yes 3 (188) 49 (34, 65) 0.12 (54) 

No 16 (276) 42 (32, 52) 0.01 (51) 

0.12 

Any definition for pathogenic mutations 

Yes 11 (233) 47 (34, 60) <0.01 (63) 

No 8 (231) 40 (30, 51) 0.13 (33) 

0.90 

Total number of patients fulfilling Amsterdam I criteria 

≥20 5 (317) 47 (35, 59) <0.01 (73) 

<20 14 (147) 41 (30, 53) 0.06 (40) 

0.28 

Sampling among unselected, non-referral CRC 

Yes 3 (28) 66 (35, 88) 0.17 (44) 

No 16 (436) 41 (31, 50) 0.01 (52) 

0.09 

AM1: Amsterdam I criteria; CI: confidence interval; CRC: colorectal cancer patients. 

None of the studies was rated A in the overall quality scale. 

Gene sequencing was performed only to those selected by gene screening methods. 

Frequency of MMR Gene Mutations Among Patients Fulfilling Amsterdam II Criteria. 

Description of studies. Ten studies assessed the prevalence of MMR mutations among 271 CRC 

probands who fulfilled the Amsterdam II criteria (Appendix F-2*). They were described in 12 

papers.65,67,71,72,77-80,84-87 

 The total number of patients in the various study populations ranged from 45 to 535 patients 

with CRC. However, the median number of patients fulfilling Amsterdam II criteria was 19.5 

(interquartile range: 5, 35). Only five studies assessed more than 20 probands from families 

fulfilling the Amsterdam II criteria.67,72,79,80,86,87 Eight out of ten studies received grade B in the 

overall quality rating while two72,78 received grade C. 

 Four studies performed bidirectional sequencing in all patients fulfilling Amsterdam II 

criteria.77,79,84,86 Four studies performed full sequencing only to patients who were selected by 

gene screening methods.65,67,71,85,87 The remaining two studies did not describe the presence of 

any mutation with bidirectional sequencing in any patient72 or did not provide any details on the 

genetic testing strategy they used.78 None of these 10 studies used conversion analysis. 

Assessment of mutations in MMR genes other than MLH1 and MSH2. All studies tested for 

MLH1 and MSH2 mutations. Only Stormorken 200178 also tested for MSH6 mutations. In this 

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm
63

study five out of 20 Amsterdam II patients had deleterious MLH1 and MSH2 mutations (25% 

[95% CI: 9, 49%]) and another two had deleterious MSH6 mutations (10% [95% CI: 1, 32%]).

Analyses. As expected, the overall prevalence of MMR gene mutations was lower compared 

with patients who fulfilled the stricter Amsterdam I criteria (39% [95% CI: 30, 49%]; Table 14, 

Figure 4). Between-study heterogeneity was statistically significant. 

 Studies that performed sequencing in all Amsterdam II patients estimated similar prevalence 

of MMR mutations compared to studies that performed sequencing only after suggestive gene 

screening with SSCP, DHPLC, or other methods (40% versus 45%, respectively; Table 14). 

 Studies that were rated C for their overall quality estimated significantly lower prevalence rates 

(27%; Table 14) compared to studies that received B for their overall quality. In the latter, the 

summary estimate was 43% (95% CI: 34, 54%) with no evidence for heterogeneity. 

 Studies that mentioned how pathogenic mutations were characterized as such, contributed 

fewer than 20 patients, or sampled unselected, non-referral patients with CRC, tended to provide 

higher estimates for the prevalence for MMR mutations. However, all of these studies were small 

and thus it is possible that the observed variability could have been due to chance (Table 14). 

Figure 4. Prevalence of mismatch repair gene mutations among colorectal cancer patients fulfilling the 

Amsterdam II criteria

Three panels are shown, representing three subgroups of studies. Top: Studies that performed sequencing in all Amsterdam I 

patients. Middle: Studies that performed sequencing only after a suggestive gene screening method. Bottom: studies that did not

perform sequencing to any patient. Within each subgroup, studies are ordered by decreasing number of patients. The bottom 

diamond represents the overall random effects synthesis. MMR: mismatch repair.
64

Table 14. Summary estimates of the prevalence of MMR mutations among CRC fulfilling 

Amsterdam II criteria 

 Summary Number of 

studies 

(patients 

fulfilling AM2) 

% with 

mutations 

(95% CI) 

Heterogeneity 

P-value, (I2

[%]) 

Betweensubgroup 

heterogeneity, 

P-value 

Overall 10 (279) 39 (30, 49) 0.03 (53) NA 

Overall quality scale 

B a 8 (190) 45 (38, 52) 0.16 (34) 

C 2 (89) 27 (19, 37) 0.36 (0) 

0.01 

Sequencing 

All samples 2 (87) 40 (30, 52) 0.82 (0) 

Some b 4 (111) 45 (26, 65) 0.03 (66) 

None 2 (89) 27 (19, 37) 0.36 (0) 

0.02 

Deletion analysis and sequencing in all samples 

Yes 0 (0) NA NA 

No 10 (279) 39 (30, 49) 0.03 (53) 

NA 

Assessment of additional MMR genes (other than MLH1 and MSH2) 

Yes 1 (20) 35 (15, 59) NA 

No 9 (259) 40 (30, 51) 0.02 (57) 

0.68 

Any definition for pathogenic mutations 

Yes 7 (233) 43 (31, 55) 0.02 (60) 

No 3 (46) 29 (18, 44) 0.45 (0) 

0.14 

Total number of patients fulfilling Amsterdam criteria II 

≥20 5 (226) 36 (24, 49) 0.01 (73) 

<20 5 (53) 44 (31, 58) 0.51 (0) 

0.41 

Sampling among unselected, non-referral CRC 

Yes 2 (23) 57 (23, 85) 0.19 (43) 

No 8 (256) 37 (28, 48) 0.03 (56) 

0.28 

AM2: Amsterdam II criteria; CI: confidence interval; CRC: colorectal cancer patients. a None of the studies was rated A in the overall quality scale. b Gene sequencing was performed only to those selected by gene screening methods. 

Interpretation of the MMR Prevalence Eestimates. The summary prevalence was 44% 

among all studies on Amsterdam I patients, and 51% for studies that sequenced all patients. For 

Amsterdam II the corresponding numbers were 39% and 40%. These estimates pertain mainly to 

MLH1 and MSH2 mutations, because most studies tested only these two genes. The true 

prevalence of MMR mutations among Amsterdam I (or Amsterdam II) patients may be different 

(possibly higher rather than lower) than these estimates for several reasons: 

• Genes other than MLH1 and MSH2: Only a minority of studies tested for mutations in 

other MMR genes (especially MSH6, the gene most commonly implicated in patients 

without mutations in MLH1 or MSH288).

– Stormorken 2001 and Wang 1999 did not find any deleterious MSH6 mutations among 

34 Amsterdam I patients in total. The upper boundary of the exact binomial 95% CI for 

the frequency of MSH6 mutations in these two studies is approximately 10%. 

– Stormorken 2001 found two deleterious MSH6 mutations in addition to five MLH1 and 

MSH2 mutations out of 20 Amsterdam II patients. Thus, MSH6 mutations resulted in an 

increment of 10% (95% CI: 1, 32%). 

Presumably, if other studies had assessed MSH6 routinely, the total prevalence of MMR 

mutations would be higher. However, there were insufficient data to provide robust 

estimates. 
65

• Comprehensiveness of genetic testing – sequencing of all samples and testing for large 

deletions/rearrangements: Studies that performed sequencing in all samples estimated 

higher frequency of MMR mutations among Amsterdam I patients compared to other studies. 

This was not true among studies that focused on Amsterdam II patients. Thus, more 

comprehensive testing resulted in more identified MMR mutations (at least in Amsterdam I 

studies), as expected. 

• Role of founder mutations: The degree to which a search for mutations other than MLH1 

and MSH2 will identify mutations in other MMR genes depends upon the population being 

studied. The spectrum of MMR mutations is variable and some are specific to certain 

populations. An example of the latter is the presence of founder mutations in the MLH1 

genes that are common among patients of Finnish origin, but are not prevalent in patients of 

different descent. The Finnish study63 included only 4 patients fulfilling the Amsterdam 

criteria I, all of whom were found to have MMR mutations. An American founder mutation 

in the MSH2 gene has been described in one kindred, probably originating from German 

immigrants, but it is unlikely to have a large impact in the general population.90,91 The 

Finnish founder mutations are probably not applicable to the US population. Nevertheless, 

the summary estimates are practically identical if the Finnish study63 is excluded. 

• Identifying deleterious mutations: As mentioned in the methods, only mutations that were 

characterized by the primary studies as deleterious or pathogenic were analyzed. 

Misclassifications may have occurred in both directions: 

– Some deleterious missense mutations may have been erroneously misclassified as nonpathogenic. 

– The opposite is also likely, especially when pathogenicity is inferred on the basis that the 

mutation was absent from cancer-free controls. This was done in five studies.63,66,67,82,83

However, most of the studies included only small numbers of cancer-free controls (only 

around 100 patients were used as controls in the five studies). Absence of a mutation in 

such a small population of cancer-free controls does not necessarily prove that the 

mutation is not pathogenic. 

There were insufficient data to provide precise estimates of how many missense mutations 

were found among the subgroup of Amsterdam I (or Amsterdam II) patients in the studies 

that performed comprehensive genetic testing. 

• Sampling of tested Amsterdam I and Amsterdam II cases: The strategy used for 

selecting patients with CRC can have a substantial impact on the prevalence estimates (see 

comments on additional studies that were suggested for consideration by the TEP, below). 

Only three studies on Amsterdam I patients studied unselected patients with CRC, but they 

identified a total of only 28 patients. By contrast, studies that identified cases that had been 

included in cancer databases (e.g., the ICG-HNPCC database that was used in the Park 1999 

study) represent a highly selected population,88 or may pertain almost entirely to individuals 

with a high probability of having mutations (e.g., because of suggestive MSI testing prior to 

their registration in the database).61 Biases resulting from sampling are not only a theoretical 

concern (see below comments on additional studies that were suggested for consideration by 

the TEP). 
66

Additional studies suggested for consideration by the TEP. Several additional studies were 

considered by the TEP during the peer review process as important and potentially relevant that 

had not been included in the draft report. Although none of these studies was eligible for these 

analyses according to the eligibility criteria, they provide insight and complementary data, 

directly supporting the analyses and their interpretation. Most importantly, the findings from 

these studies are in accordance with the notion that sampling of Amsterdam I or II patients from 

different sources may result in very different prevalence estimates, even when the genetic testing 

strategies are comprehensive. 

• Balmana 200692 reported the prevalence of MLH1 and MSH2 gene mutations among 1914 

unrelated people who had been tested for MMR mutations in the Myriad Genetic 

Laboratories. Genetic analysis was performed using a combination of sequencing and 

southern blotting. The cohort included 534 unrelated people fulfilling the Amsterdam II 

criteria (although it was unclear if all of them had CRC). Amsterdam I patients were not 

reported separately. Only half of the Amsterdam II patients (274/534=51%) were tested for 

large deletions or rearrangements. 

 In total, 180 Amsterdam II participants were found to be carriers of MLH1 or MSH2 

mutations (34% [9%% CI: 30, 38%]). This estimate includes large deletions or 

rearrangements and pathogenic and non-pathogenic mutations. About one fourth (27% [95% 

CI: 20, 35%] of the mutations were due to large deletions/rearrangements in a subgroup of 

the 1016 participants who underwent such testing. Extrapolating the proportion of large 

deletions to all Amsterdam II patients would result in a total prevalence of approximately 

38%, very close to the summary estimate provided above. This study was excluded from the 

analyses because it was unclear whether all Amsterdam II participants had CRC. 

• Wagner 200393 evaluated 49 individuals from the Henry Lynch Cohort fulfilling the 

Amsterdam criteria (presumably Amsterdam I criteria), at least 9 of whom were not CRC 

patients. They used DGGE to screen for MMR mutations in the MLH1, MSH2 and MSH6 

genes, and performed sequencing on exons with altered migration patterns. They tested for 

large deletions, and performed mono-allelic mutation analysis (MAMA) in very few samples, 

mainly to demonstrate the feasibility of the technique. 

 Overall, 25 out of 49 had deleterious MLH1 or MSH2 mutations, 1 had deleterious 

MSH6 mutations, and 14 had genomic rearrangements. Thus, the overall proportion was 

82% (95% CI: 68, 91%). In addition, 5 missense mutations were found; were they to be 

considered pathogenic, the overall proportion would be 92% (95% CI: 80, 98%). 

The detection of large deletions/rearrangements resulted in 29% increase in the estimated 

prevalence of MMR mutations. The degree to which the Henry Lynch Cohort is 

representative of the whole Amsterdam I population is unclear. 

• Liu 199694 assessed the MLH1, MSH2 and PMS2 genes 48 families of Amsterdam I patients 

who had suggestive MSI (note that more than one person per family was tested for mutations 

–i.e, reporting was at the family not the individual level). They found a prevalence of 

“drastic” (deleterious) mutations of 54%. Counting all identified mutations, drastic or not, 

71% (95% CI: 56, 83%) of the families carried a mutation. Fifteen families had large 

genomic deletions/rearrangements (31%), but many families had more than one type of 

mutations. 
67

 It is unclear how many unrelated people had only large deletions or rearrangements in 

this study. There might be some overlap with the Wanger 200393 families for patients from 

North America. The authors note that their sample was not representative of the general 

Amsterdam I population. 

Caveat on true overall prevalence of MMR mutation carriers. Limited data suggested that 

approximately one-fourth to one-third of genotypes associated with HNPCC are related to 

deletions or rearrangements that would be missed through sequencing alone. As a result, the 

prevalence of mutations in Amsterdam I (or II patients) assessed from studies that performed 

sequencing alone are likely to be underestimates. 

 Accounting for this effect, one may derive a prevalence of MMR mutations of approximately 

63% to 67% in Amsterdam I patients. For Amsterdam II patients the corresponding prevalence 

values would be 50 to 53%. 

 Furthermore, limited data suggest that approximately 10% of MMR genotypes involve MMR 

genes other than MLH1 and MSH2. Thus, assuming an additional 10% increase in mutations by 

assessing more genes would result in an overall prevalence of up to 70% to 75% for Amsterdam 

I patients, and 55 to 59% for Amsterdam II patients. 

Key Question 2b: Assuming a Clinical Definition of the Lynch 

Syndrome, What Proportion of Patients has MSI? 

 Among patients fulfilling the Amsterdam I criteria (data from 11 studies, n=159 patients 

fulfilling the criteria), 71% (95% CI: 63, 78%) of tumors were found to be MSI-H (p=0.51 for 

heterogeneity; I2=0%). Among patients who fulfilled the Amsterdam II criteria (data from four 

studies, n=102 patients fulfilling the criteria), the corresponding summary prevalence was 68% 

(95% CI: 58, 76%) without evidence for between-study heterogeneity (p=0.91, I2=0%). 

Patients Fulfilling Amsterdam I Criteria. Eleven studies with 159 patients fulfilling the 

Amsterdam I were criteria were included in the analysis (Figure 5). These were described in 13 

papers.63-66,68-72,75,79,80,95 Although the total number of patients with CRC included in the primary 

studies ranged from 11 to 509, the median number of CRC patients eligible for this analysis was 

10 (interquartile range: 9, 18), and only one study assessed more than 20 CRC patients. 

(Lamberti 1999,72 n=57). Five studies 63,65,66,70,71,79,80 received a grade B in the overall quality 

rating while the others were rated a grade C. 
68

Figure 5. Prevalence of MSI in colorectal cancer tumors from patients fulfilling the Amsterdam I criteria 

MSI-H was considered as defined in the primary studies. Two subgroups are shown. The upper subgroup shows studies that used 

the NCI recommended 5 marker set, the lower subgroup shows studies that used other marker sets. Within each subgroup studies 

are ordered by decreasing number of patients. The bottom diamond represents the overall random effects estimate. 

 We considered specific study features as described in Chapter 2 (i.e., whether the study 

reported use of microdissection and used the microsatellite marker set recommended by the 

National Cancer Institute). Only six studies reported that they used microdissection (which helps 

assure that the tissue sample that was tested for MSI consisted mainly of malignant cells).64-

66,68,71,79,80,95 Only two studies (reported in three papers64,79,80) used the marker set recommended 

by the National Cancer Institute (Appendix F-3*). 

 Overall, 71% (95% CI: 63, 78%) of tumors were MSI-H (p=0.51 for heterogeneity; I2=0%). 

The estimate was consistent among the various subgroups that we assessed (Table 15). The 

prevalence rates were generally higher among studies that used the marker sets recommended by 

the National Cancer Institute (Table 15). 

 The prevalence of combined MSI-H and MSI-low was similar to that of MSI-H (Appendix 

F-3*

). 

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm
69

Table 15. Summary estimates of the prevalence of MSI-H in colorectal cancer tumors from patients fulfilling 

Amsterdam I criteria 

 Summary Number of 

studies (CRC 

patients 

fulfilling AM1) 

% with MSI-H 

(95% CI) 

Heterogeneity 

P-value, (I2

[%]) 

Betweensubgroup 

heterogeneity, 

P-value 

Overall 11 (159) 71 (63, 78) 0.51 (0) NA 

Overall quality scale 

B a 5 (58) 79 (67, 88) 0.81 (0) 

C 6 (101) 66 (56, 75) 0.44 (0) 

0.10 

Microdissection 

Yes 6 (84) 70 (58, 80) 0.32 (15) 

No 5 (75) 71 (59, 81) 0.50 (0) 

0.89 

Use of the NCI-recommended marker sets 

Yes 2 (32) 81 (64, 91) 0.69 (0) 

No 9 (127) 68 (59, 76) 0. 52 (0) 

0.17 

Total number of patients fulfilling Amsterdam criteria I 

≥20 1 (49) 71 (57, 83) NA 

<20 10 (110) 70 (60, 79) 0.42 (2) 

0.89 

Sampling among unselected, non-referral CRC 

Yes 2 (14) 70 (62, 78) 0.39 (5) 

No 9 (145) 74 (44, 91) 0.39 (0) 

0.81 

AM1: Amsterdam I criteria; CI: confidence interval; CRC: colorectal cancer patients. a

 None of the studies was rated A in the overall quality scale. 

Patients Fulfilling Amsterdam II Criteria. Only four studies including 102 patients fulfilling 

the Amsterdam II criteria were identified (Appendix F-4*). These were described in five 

papers.65,71,72,85,86 The largest study (Lamberti 199972) analyzed 59 available eligible tumors, and 

the smallest study (Salovaara 200085) had only 4 eligible tumors. The total number of patients 

with CRC included in these studies ranged from 45 to 535. All studies except for the one by 

Lamberti 199972 received grade B for overall quality. None of the studies used the National 

Cancer Institute recommended marker sets, and only two 79,80,86 used microdissection (Appendix 

F-4). Overall, the summary prevalence was 68% (95% CI: 58, 76%) without any evidence for 

between-study heterogeneity (p=0.91, I2=0%) (Figure 6). 

 A pooled estimated for the three fair quality (B overall quality) studies was 66% (95% CI: 

51, 79%) with no evidence for heterogeneity. Estimates from the two studies with more than 20 

patients that fulfilled the Amsterdam II were similar to the overall estimate. The results were also 

similar for studies that used the markers sets recommended by the National Cancer Institute. 

 Finally, results were consistent when the MSI-L and MSI-H tumors were combined in two 

studies (described in three papers 65,71,72, Appendix F-4*

). The overall prevalence was 71% 

(95%CI: 60, 81%) with no statistically significant heterogeneity. 

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm
70

Figure 6. Prevalence of MSI-H in colorectal cancer tumors from patients fulfilling the Amsterdam II criteria 

MSI-H was considered as defined in the primary studies. Studies are ordered by decreasing number of patients. MSI-H: MSI-H. 

Interpretation of the MSI Prevalence Estimates. Some of the biases that may affect the 

calculated prevalence for MMR mutations are also applicable in this section, specifically biases 

that pertain to the selection/sampling of Amsterdam I and Amsterdam II patients who were 

studied. In addition, even among the selected Amsterdam I or Amsterdam II patients in each 

study, MSI testing often depended upon practical and logistical considerations (e.g., patient 

availability and consent and availability of tumor tissue). Thus, not all patients had all tests and it 

was unclear whether additional bias may have been introduced in selecting patients for testing. 

Key Question 2c: Assuming a Clinical Definition of the Lynch 

Syndrome, What Proportion of Patients has Abnormal Protein 

Expression by Immunohistochemistry? 

 Among patients fulfilling the Amsterdam I criteria, the overall prevalence of tumors with loss 

of protein expression was 40% (95% CI: 28, 53%; n= 6 studies, 63 patients) with no evidence for 

between-study heterogeneity (p=0.75, I2=0%). 

 Only one eligible study provided relevant data for 20 patients fulfilling the Amsterdam II 

criteria. Eight out of 20 tumors had suggestive IHC for the MLH1, MSH2 or MSH6 genes (40% 

[95% CI: 9, 64%]). 

Patients Fulfilling Amsterdam I Criteria. Only six studies that included a total of 63 eligible 

patients (median 12 tumors in each study) provided relevant data; no study contributed more than 

15 tumors in this analysis. The total number of patients included in the pertinent studies ranged 

from 30 to 168. These were described in eight publications.65,66,69-71,78-80. 

 Five out of six studies were characterized as grade B quality while two69,78 were rated grade 

C. Only Stormorken 200178 assessed the expression of the MSH6 genes (in addition to MLH1 

and MSH2) (Appendix F-5*

). However, none of the samples was positive for only MSH6. 

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm
71

 The overall prevalence of tumors with loss of protein expression was 43% (95% CI: 30, 

57%) with no evidence for between-study heterogeneity (p=0.85, I2

=0%) (Figure 7). The 

summary estimate was similar (42% [95% CI: 29, 56%] without any heterogeneity) after 

excluding the report by Debniak 2000,69 which was the only one graded C for overall quality. 

Figure 7. Prevalence of suggestive IHC in colorectal cancer tumors from patients fulfilling the 

Amsterdam I criteria 

Studies are ordered by decreasing number of patients. 

Patients Fulfilling Amsterdam II Criteria. Only Stormorken 200178 assessed the prevalence 

of suggestive IHC among 20 patients fulfilling Amsterdam criteria II. This study was rated C for 

its overall methodologic quality. The expression of three MMR genes was sought, namely 

MLH1, MSH2 and MSH6. Eight out of 20 Amsterdam II patients had suggestive IHC (40% 

[95% CI: 19, 64%]). 

 Tumors that have suggestive IHC for MSH2 may also have suggestive IHC for MSH6, 

because in the absence of functional MSH2 protein the MSH2/MSH6 heterodimer is not formed 

correctly. Two out of 20 Amsterdam II patients had deleterious mutations in the MSH6 gene in 

Stormorken 2001.78 None had suggestive IHC only for the MSH6 antibody. 

Interpretation of the IHC Prevalence Estimates. In contrast to MSI testing that detects 

replication errors, and is generally not MMR-gene specific, IHC uses antibodies that target 

specific MMR genes. Only one of the eligible studies used antibodies against MSH6.78 The 

MSH6 gene is the third most common gene with pathogenic mutations in HNPCC patients 

(approximately 6% of the mutations in the ICG-HNPCC database are in MSH688). Thus, the 

prevalence of suggestive IHC would probably be higher if anti-MSH6 or additional antibodies 

were used. 

 Some of the biases that may affect the calculated prevalence for MMR mutations are also 

applicable in this section, specifically biases that pertain to the selection/sampling of Amsterdam 

I and Amsterdam II patients who were studied. In addition, even among the selected Amsterdam 

I or Amsterdam II patients in each study, IHC testing often depended upon practical and 
72

logistical considerations (e.g., patient availability and consent and availability of tumor tissue). 

Thus, not all patients had all tests and it was unclear whether additional bias may have been 

introduced in selecting patients for testing. 

Key Question 2: How Accurate Are Various Predictors Assuming a 

Genetic Definition of the Lynch Syndrome? 

 We examined a variety of clinical and laboratory predictors as described in Chapters 1 and 2. 

A central question of this report relates to the accuracy of various predictors in unselected 

patients with CRC. Because testing for MMR mutations is expensive, most studies performed 

testing only in patients who had been pre-selected based on clinical or laboratory features. Thus, 

it is critically important to describe the specific CRC population when attempting to make 

comparisons among studies. 

 The various clinical predictors defined populations at different risk for HNPCC. Figure 8 

illustrates the relationship among patient populations defined by Amsterdam I criteria, 

Amsterdam criteria II, Bethesda guidelines, and unselected CRC patients. 

Figure 8. Relationship between the CRC populations defined by different clinical predictors 

AM 1

AM 2

Beth

Unselected

CRC

The whole pyramid represents all existing unselected patients with CRC. Each of the different clinical predictors (AM1: 

Amsterdam I criteria; AM2: Amsterdam II criteria; Beth: Bethesda guidelines) defines populations that are a subset of the 

immediately broader definition, as shown in the figure. The precise areas of the populations are only illustrative and not drawn to 

scale; thus they do not represent the actual relative proportions of these different populations.

 Table 16 presents the different populations that underwent MMR testing in all studies 

included in this analysis. Moving to the right, columns represent increasingly selected patient 

populations (by analogy, moving to the top of the pyramid in Figure 8). Table 16 provides a 

roadmap for the analyses presented in the following sections. 
73

Table 16. Overview of available evidence on the sensitivity and specificity of various predictors for MMR 

mutation rates 

Predictor All Specific CRC populations defined by increasingly selective criteria 

studies Unselected 

CRC 

probands 

Revised 

Bethesda 

guidelines 

Bethesda 

guidelines 

Modified 

Amsterdam 

criteria 

Amsterdam 

II criteria 

Amsterdam I 

criteria 

Amsterdam I criteria F-6 

(n=19) 

F-7 

(n=2) 

ND F-8 

(n=2) 

ND F-9 

(n=5) 

Amsterdam II 

criteria 

F-10 

(n=12) 

F-11 

(n=2) 

F-12 

(n=1) 

F-13 

(n=3) 

ND 

Modified 

Amsterdam criteria 

F-14 

(n=2) 

ND ND ND 

Bethesda guidelines F-15 

(n=5) 

F-16 

(n=1) 

ND 

Revised Bethesda 

guidelines 

F-17 

(n=1) 

F-17 

(n=1) 

Young age of onset F-18 

(n=5) 

F-19 

(n=4) 

ND ND ND ND ND 

Family history F-20 

(n=9) 

F-21 

(n=4) 

ND ND ND 

Multiple tumors in 

the same patient 

F-22 

(n=3) 

F-22 

(n=3) 

ND ND ND ND ND 

Age <50 years, 

family history, or 

multiple tumors in 

same patient 

F-23 

(n=3) 

F-23 

(n=3) 

ND ND ND ND ND 

Suggestive MSI 

(MSI-H; MSI-H and 

MSI-L) 

F-24 

(n=16) 

F-25 

(n=2) 

F-26 

(n=1) 

ND ND ND F-27 

(n=3) 

Suggestive IHC F-28 

(n=9) 

ND ND ND ND ND F-29 

(n=2) 

Table rows represent different predictors; the table columns describe the populations addressed in the summary tables. Each cell 

in the table refers to a corresponding Summary Table of the appendix. For example, the second cell of the second row shows that

there were 19 studies that described the ability of Amsterdam I criteria to predict MMR status. The studies are described in 

Appendix F-6. The third cell of the second row shows that of these 19 studies, only two performed MMR testing in unselected 

patients; these studies are described in Appendix F-7, and so on. 

ND: No data 

 Table 17 summarizes the overall sensitivity and specificity of the various predictors among 

several CRC populations. These are described in detail in the sections below. 
74

Table 17. Sensitivity and specificity of various predictors for detecting MMR gene mutations 

Unselected CRC probands Revised Bethesda 

guidelines 

Predictor Bethesda guidelines Amsterdam II criteria Amst

N Sens [%] 

(95% CI) 

Spec [%] 

(95% CI) 

N Sens [%] 

(95% CI) 

Spec [%] 

(95% CI) 

N Sens [%] 

(95% CI) 

Spec [%] 

(95% CI) 

N Sens [%] 

(95% CI) 

Spec [%] 

(95% CI) 

N 

Amsterdam I criteria 2 45 (29, 63) 99 (74, 100) ND 2 57 (46, 68) 48 (35, 62) 5 80 (70, 88) 24 (12, 43) Amsterdam II criteria 2 28 (15, 47) 99 (97, 100) 1 68 (43, 87) 65 (51, 76) 3 84 (74, 91) 62 (53, 69) 

Modified Amsterdam 

criteria 

ND ND ND 

Bethesda guidelines 1 73 (39, 94) 82 (80, 84) ND Revised Bethesda 

guidelines 

1 91 (59, 100) 77 (75, 79) 

Age <50 years 3 31 (18, 47) 95 (94, 96) ND ND ND 1st degree family history 

of CRC or EC 

4 76 (50, 91) 87 (86, 89) ND ND 

Multiple CRC or EC 

tumors in the same patient 

3 38 (25, 54) 97 (91, 99) ND ND ND 

Age <50 years, family 

history of CRC or EC, or multiple tumors in same patient 

3 88 (60, 97) 77 (74, 81) ND ND ND 

Suggestive MSIa 2 100 (88, 100) 90 (88, 92) 1 100 (75, 100) 79 (63, 90) ND ND 3 1

Suggestive IHC ND ND ND ND 2 

CRC: colorectal cancer; EC: endometrial cancer; N: Number of studies; Sens: (summary) sensitivity; Spec: (summary) specificity.a Estimates are the same for combined MSI-H and MSI-L versus MSS and for MSI-H versus MSS. 
75

 We first present an overview of the clinical predictors among unselected CRC patients. 

We then present each one of the clinical predictors separately. 

Overview of Clinical Predictors Among Unselected Patients With CRC 

 In total, five studies provided information on eight clinical predictors among unselected CRC 

probands (Salovaara 2000,85 Aaltonen 1998,63 Colombino 2005,83, Samowitz 2001,10 and Pinol 

200512) (Appendices F-7, F-11, F-16, F-17, F-19, F-21, F-22, F-23, F-25*). All studies received a 

grade B in the overall quality rating. All were limited by verification bias. In the two studies 

from Finland63,85 founder mutations that are unique in the Finnish population were also assessed. 

These specific founder mutations have not been found in the US population. 

 The five studies differed in their testing strategies. Aaltonen 199863 and Salovaara 200085

performed comprehensive genetic testing only among patients whose tumors exhibited MSI. The 

remaining patients were tested only for common founder mutations in the Finnish population 

(large deletions in the MLH1 gene). Samowitz 200110 and Pinol 200512 performed genetic testing 

only among patients with tumors that had suggestive MSI (or suggestive IHC testing for Pinol 

200512). Finally, Colombino 200583 performed comprehensive genetic testing only among 

patients with cancer history in 1st or 2nd degree family, and tested the remaining cases only for 

the mutations that were identified in the former. 

 As noted in the methods, we assumed that all patients who were not selected for genetic 

testing based on the MSI/IHC results were mutation negative. 

 

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm
76

 Figure 9 illustrates the sensitivity and specificity of the clinical criteria among unselected 

CRC patients. 

Figure 9. Sensitivity and specificity of clinical criteria for detecting mismatch repair mutations among 

unselected patients with CRC 

0

50

100

Sensitivity (%)

0 50 100

100 - Specificity (%)

AM1

0

50

100

Sensitivity (%)

0 50 100

100 - Specificity (%)

AM2

0

50

100

Sensitivity (%)

0 50 100

100 - Specificity (%)

Age<50

0

50

100

Sensitivity (%)

0 50 100

100 - Specificity (%)

Many cancers

0

50

100

Sensitivity (%)

0 50 100

100 - Specificity (%)

BG

0

50

100

Sensitivity (%)

0 50 100

100 - Specificity (%)

Fam Hx

0

50

100

Sensitivity (%)

0 50 100

100 - Specificity (%)

3Clinical

0

50

100

Sensitivity (%)

0 50 100

100 - Specificity (%)

RevBG

AM1/AM2: Amsterdam criteria I/II; Age<50: proband age at diagnosis <50 years; Many cancers: Multiple synchronous or 

metachronous colorectal or endometrial cancers in the same proband; BG: Bethesda guidelines; Fam Hx: History of colorectal 

cancer or endometrial cancer in 1st degree family; 3Clinical: Proband age <50y at diagnosis, history of colorectal cancer or 

endometrial cancer in 1st degree family, or multiple synchronous or metachronous tumors in the same proband; RevBG: Revised 

Bethesda guidelines. The study by Samowitz 2001 is not depicted in the graphs because it used slightly different criteria. 

 As shown in Figure 9 and Table 17, the revised Bethesda guidelines had the best sensitivity 

(91%, [95% CI: 59, 100%]). However, clinical predictors that are relatively easier to assess such 

as the family history of colorectal or endometrial cancer, or the fulfillment of at least one of three 

simple clinical criteria (proband age less than 50 years at diagnosis, history of CRC or 

endometrial cancer in 1st degree family, or multiple synchronous or metachronous CRC or 

endometrial cancer in the proband) appeared to have better test performance than the Amsterdam 

I and II criteria, or even the Bethesda guidelines. Specific summary estimates are presented in 

Table 17 and are also mentioned in the following sections. 

 

Amsterdam I Criteria 

 Nineteen studies (described in 21 publications29,61,63-68,71-77,79-83,96) provided information 

needed to calculate sensitivity and specificity for this analysis (Appendix F-6*

). Fifteen assessed 

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm
77

the clinical predictors among all patients, and three (Casey 2005,61 Raedle 2001,29 and Nakahara 

1997,96 Appendix F-6*

) among patients with suggestive MSI. 

 Ten studies were rated grade B for their overall methodologic quality29,61,63,65-67,71,74,77,79-81

and nine received grade C. 

Genetic Testing Irrespective of MSI or IHC Status. Sixteen studies performed some form of 

genetic testing in all people, irrespective of suggestive MSI or IHC (Appendix F-6*

). Only four 

of these studies performed sequencing on all available samples,75,77,79,81 and only Wang 199981

also tested for large deletions/rearrangements. Most studies tested only for mutations in MLH1 

and MSH2. Wang 199981 also assessed the MSH6 gene, the PMS2 and PMS1 genes (no 

deleterious mutations were identified in non-MLH1 and non-MSH2 genes). Park 199976 sought 

mutations in additional MMR genes (PMS1 and PMS2) and in only a minority of patients 

(without finding any additional mutations). Eight out of the sixteen 2x2 tables had at least 40 

patients in total. 

 There was variability in the CRC populations that were examined (Appendix F-6*

). The 

sample selection process was considered to be transparent (based on clearly stated selection 

criteria applied to all available patients) in only eight studies.63,65,67,71,74,77,79-81,83 Of these, three 

sampled from unselected, non-referral CRC populations.63,65,71,83 The remaining five sampled 

from referral or otherwise selected CRC patients67,74,77,79-81 (Appendix F-6*

). 

 Figure 10 graphically depicts the sensitivity and specificity of the 16 studies that performed 

some form of genetic testing in all people irrespective of suggestive MSI/IHC. A study 

suggesting that the Amsterdam I criteria had perfect test characteristics (i.e., sensitivity and 

specificity of 100%) would appear in the upper left corner. The graph shows that the Amsterdam 

I criteria are not highly sensitive for detecting MMR gene mutations. In two studies that enrolled 

unselected patients with CRC, the summary estimate of sensitivity was only 45% (95% CI: 29, 

63%), and the summary specificity was 99% (95% CI: 74, 100%).63,83 However, both studies 

were limited by verification bias, as described below. 

Figure 10. Sensitivity and specificity of the Amsterdam I criteria for detecting mismatch repair mutations in 

patients with CRC 

Aaltonen

Callistri

Colombino

Curia

Katballe

Lamberti

Liu

Luce

Moslein

Park

Rossi

Syngal

Wang

Yuan

de Abajo

de Leon

0

20

40

60

80

100

Sensitivity (%)

0 20 40 60 80 100

100 - Specificity (%)
78

 Overall, the distribution of sensitivity and specificity of the various studies did not follow a 

discernable pattern. The differences may be related to variation in genetic strategies (gene 

screening and deletion analysis), the definition of pathogenic mutations, sample selection 

processes (clearly described versus not), overall study methodologic quality (B versus C), or 

within-study heterogeneity of “baseline risk” for HNPCC (study sample included patients at very 

different risks for HNPCC). However, as the graphs in Figure 11 show, none of these factors 

alone appears to account for the variability. The variability is most likely attributable to the very 

different CRC populations assessed in these studies. 

 

Figure 11. Sensitivity and specificity of the Amsterdam I criteria for detecting mismatch repair mutations: 

study subgroups according to various factors 

0

20

40

60

80

100

Sensitivity (%)

0 20 40 60 80 100

100 - Specificity (%)

Fair quality

0

20

40

60

80

100

Sensitivity (%)

0 20 40 60 80 100

100 - Specificity (%)

>=40 CRC

0

20

40

60

80

100

Sensitivity (%)

0 20 40 60 80 100

100 - Specificity (%)

Pathogenic mutations

0

20

40

60

80

100

Sensitivity (%)

0 20 40 60 80 100

100 - Specificity (%)

Sequencing

0

20

40

60

80

100

Sensitivity (%)

0 20 40 60 80 100

100 - Specificity (%)

Sequenc & deletion

0

20

40

60

80

100

Sensitivity (%)

0 20 40 60 80 100

100 - Specificity (%)

All concecutive CRC

Filled squares versus empty circles imply that the pertinent characteristic is present versus absent. Fair quality: Overall quality B 

versus C; ≥40 CRC: total of ≥40 versus <40 patients in the 2x2 table; Pathogenic mutations: Any versus none definition for 

pathogenic mutations; Sequencing: Sequencing of all available samples; Sequence & Deletion: Sequencing and deletion analysis 

to all samples; All consecutive CRC: Studies were all available consecutive CRC were used versus studies without consecutive 

CRC or studies that included some but not all available consecutive CRC. 

Amsterdam I Criteria Among Different Populations. As discussed in the Methods section, 

clinical predictors are expected to have substantially different sensitivity and specificity to detect 

mismatch repair gene mutations in CRC populations selected using different clinical criteria. We 

assessed the sensitivity and specificity of Amsterdam I criteria among CRC subpopulations that 

were defined consistently across studies (Appendices F-7, F-8 and F-9*

). 

 Figure 12 depicts the change in the sensitivity and specificity of the Amsterdam I criteria 

among CRC populations that had an increasing likelihood of having HNPCC: unselected CRC, 

CRC fulfilling the Bethesda criteria, and CRC fulfilling Amsterdam II criteria. As the likelihood 

for HNPCC increases, the studies shift toward the upper right (i.e., better sensitivity and worse 

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm
79

specificity). Thus, there is evidence for spectrum effects in the diagnostic ability of the 

Amsterdam I set of criteria. 

Figure 12. Sensitivity and specificity of the Amsterdam I criteria for detecting mismatch repair gene 

mutations among populations with an increasing likelihood of having HNPCC 

Aaltonen

Colombino

Syngal

de Abajo

Katballe

Lamberti

Rossi

Syngal

de Abajo

0

20

40

60

80

100

Sensitivity (%)

0 20 40 60 80 100

100 - Specificity (%)

Among CRC patients who

are unselected

fulfill Bethesda guidelines

fulfill AM2

The average patient risk for HNPCC is lowest for unselected colorectal cancer patients populations, higher for patients fulfilling 

the Bethesda guidelines and highest for patients fulfilling Amsterdam II criteria. The same studies (Syngal, de Abajo) have 

information both for the subpopulation of CRC that fulfills the Bethesda guidelines and the subpopulation of CRC that fulfills the 

Amsterdam II criteria. AM2: Amsterdam II criteria; CRC: colorectal cancer. 

Amsterdam I Criteria Among Unselected Patients With CRC. Two studies (Aaltonen 

1998,63 and Colombino 200583) assessed the performance of the Amsterdam I criteria among 

unselected CRC patients (Appendix F-7*

). The first63 assessed all new incident CRC cases from 

nine Finnish hospitals, while the second83 was retrospective and assessed all CRC patients who 

were registered in a tertiary hospital during the 3 years before the study (the authors commented 

that their hospital registered practically all CRC cases diagnosed in Sardinia). 

 Both studies were limited by verification bias. Aaltonen 199863 performed comprehensive 

genetic testing only among 63 patients who had tumors with MSI, and tested the remaining 446 

for a common founder mutation in the MLH1 gene. Colombino 200583 performed detailed 

genetic testing only among patients with family history and assessed patients with apparently 

sporadic CRC only for mutations found in the familial cases. Thus, both studies clearly 

overestimated the sensitivity and the specificity of Amsterdam I criteria to detect mutations. 

Their summary sensitivity was 45% (95% CI: 29, 63%) and their summary specificity was 99% 

(95% CI: 74, 100%). The wide confidence intervals of the sensitivity values reflect that few 

patients fulfilled Amsterdam I criteria (n=4 in Aaltonen 1998,63 and n=21 in Colombino 200583). 

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm
80

Genetic Testing Only Among People Who Have MSI Positive (or IHC Negative) Tumors. 

In the three studies that were pertinent to this category29,61,96 the sensitivities ranged between 

55% and 78% and the specificities between 40% and 100% (Appendix F-6*). 

Amsterdam II Criteria 

 Twelve studies described in 14 publications12,29,65,67,71,77,79,80,84-87,97,98 provided data for 

determining the sensitivity and specificity of the Amsterdam II criteria for predicting the 

presence of mismatch repair mutations (Appendix F-10*). Eight assessed sensitivity and 

specificity of the Amsterdam II criteria among all available patients,65,67,71,77,79,80,84-87 three 

among patients who had tumors with MSI instability,29,97,98 and one among patients whose 

tumors were selected after MSI or IHC testing.12 

Genetic Testing Irrespective of MSI or IHC Status. Eight studies provided data on genetic 

testing irrespective of MSI or IHC status.65,67,71,77,79,80,84-87 The populations evaluated were 

heterogeneous (Appendix F-10*). One study84 received grade C for overall quality rating while 

the rest were rated grade B.65,67,71,77,79,80,85-87 All eight studies tested only for mutations in MLH1 

and MSH2 genes. Seven studies had more than 40 patients in total in the pertinent 2x2 tables (all 

except for Rossi 200277). The sampling process was described in all but one study.87

 Figure 13 provides an overview of the eight studies while Figure 14 shows the various 

studies according to the presence or absence of various characteristics that may affect the 

sensitivity and specificity of the Amsterdam II criteria. Overall, sensitivities varied between 22% 

and 80% and specificities varied between 61% and 100%. The plot suggests that Amsterdam II 

criteria are not a good screening test for HNPCC. The seven studies did not follow any particular 

pattern with respect to the factors addressed in Figure 14. The variability is mostly likely due to 

the very different CRC populations assessed in these studies. 

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm
81

Figure 13. Sensitivity and specificity of the Amsterdam II criteria for detecting mismatch repair 

gene mutations

Salovaara

Katballe

Wolf

Syngal

Lee

Rossi Zhu

de Abajo

0

20

40

60

80

100

Sensitivity

0 20 40 60 80 100

100 - Specificity (%)

Figure 14. Sensitivity and specificity of the Amsterdam II criteria for detecting mismatch repair mutations: 

study subgroups according to various factors 

0

20

40

60

80

100

Sensitivity

0 20 40 60 80 100

100 - Specificity (%)

Fair quality

0

20

40

60

80

100

Sensitivity

0 20 40 60 80 100

100 - Specificity (%)

>=40 CRC

0

20

40

60

80

100

Sensitivity

0 20 40 60 80 100

100 - Specificity (%)

Pathogenic mutations

0

20

40

60

80

100

Sensitivity

0 20 40 60 80 100

100 - Specificity (%)

Sequencing

0

20

40

60

80

100

Sensitivity

0 20 40 60 80 100

100 - Specificity (%)

All concecutive CRC

Filled squares versus empty circles imply that the pertinent characteristic is present versus absent. Fair quality: Overall quality B 

versus C; ≥40 CRC: total of ≥40 versus <40 patients in the 2x2 table; Pathogenic mutations: Any versus none definition for 

pathogenic mutations; Sequencing: Sequencing of all available samples; All consecutive CRC: Studies where all available 

consecutive CRC were used versus studies without consecutive CRC or studies that included some but not all available 

consecutive CRC. 
82

Genetic Testing Only Among People Who Have MSI Positive (or IHC Negative) Tumors. 

Three studies performed genetic testing only among patients who fulfilled the Amsterdam II 

criteria and had MSI high tumors,29,97,98 and one12 among patients selected based on the results of 

MSI and IHC testing (Figure 15). 

Figure 15. Sensitivity and specificity of the Amsterdam II criteria for detecting mismatch repair mutations 

among patients selected after MSI or IHC testing 

Terdiman

Pistorius

Raedle

Pinol

0

20

40

60

80

100

Sensitivity

0 20 40 60 80 100

100 - Specificity (%)

 Pinol 200512 and Raedle 200129 assessed all available consecutive CRC patients. Terdiman 

200198 selected patients who fulfilled the Bethesda guidelines and Pistorius 2000,97 patients in 

whom there was increased clinical suspicion for HNPCC (at least two 1st degree relatives with 

CRC or young age at diagnosis or multiple tumors in the same patient). Sensitivities ranged from 

36% to 80% and specificities ranged from 64% to 99% (Appendix F-10*). 

Amsterdam II Criteria Among Unselected CRC Cases. As noted above, a question of central 

importance in this report is the accuracy of predictors in unselected patients with CRC. Only two 

studies assessed the accuracy of the Amsterdam II criteria in such patients (Pinol 2005,12 and 

Salovaara 200085) (Appendix F-11*). The latter study85 performed comprehensive genetic testing 

in a subset of 66 patients who had tumors with MSI but tested the remaining 469 only for 

founder mutations in the MLH1 gene common in the Finnish population. The study by Pinol 

200512 also performed comprehensive testing in only a subset of patients; however we included it 

by making an assumption that patients who were not selected for genetic testing based on 

MSI/IHC results would test negative for mutations. 

 Two studies that focused on unselected CRC probands (Pinol 2005,12 and Salovaara 200085) 

provided a summary estimate of 28% (95% CI: 15, 47) for sensitivity and 99% (95% CI: 97, 

100) for specificity. However, both studies were limited by verification bias. 

Amsterdam II Criteria Among Different Populations. Figure 16 depicts sensitivity and 

specificity of the Amsterdam II criteria for identifying MMR mutations, among populations at 

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm
83

variable risk for HNPCC. The studies are described in Appendices F-11, F-12 and F-13*

. Studies 

focusing on populations at increasing risk appear to be associated with better sensitivity and 

worse specificity, similar to the findings described above for the Amsterdam I criteria. 

Figure 16. Sensitivity and specificity of the Amsterdam II criteria for detecting mismatch repair mutations 

among populations with an increasing likelihood for HNPCC 

Pinol

Salovaara

Wolf

Syngal

de Abajo

0

20

40

60

80

100

Sensitivity

0 20 40 60 80 100

100 - Specificity (%)

Among CRC patients who

are unselected

fulfill revised Bethesda guidelines

fulfill Bethesda guidelines

The average risk for HNPCC is lowest for unselected colorectal cancer patients and highest for patients fulfilling the revised 

Bethesda guidelines or the Bethesda guidelines. CRC: colorectal cancer patients. 

Modified Amsterdam Criteria 

 The modified Amsterdam criteria are fulfilled when CRC has appeared in more than one 

generation, there are at least two CRC in 1st degree family, and at least one CRC diagnosed at 

age younger than 55 years; or when there are at least two 1st degree relatives affected by CRC 

plus another relative with an unusually early onset neoplasm or endometrial cancer. Two studies 

(described in three papers79,80,86) provided data on the modified Amsterdam criteria (Appendix F14*

). Both studies received grade B for their overall quality. Neither assessed mutations in genes 

other than MLH1 or MSH2. The studies differed in their inclusion criteria and neither included 

unselected patients. Despite these differences, the estimates of sensitivity and specificity were 

similar. Overall, the summary sensitivity was 78% (95% CI: 61, 89%) and the summary 

specificity was 46% (95% CI: 37, 55%). It is likely that the sensitivity of the modified 

Amsterdam criteria in an unselected CRC population would be lower. 

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm
84

Bethesda Guidelines 

 Five studies described in six papers assessed the sensitivity and specificity of the Bethesda 

guidelines to detect MMR mutations: Syngal 2000,79,80 Wolf 2005,86 de Abajo 2005,67 Raedle 

2001,29 and Pinol 2005.12 Three studies performed genetic testing irrespective of tumor MSI 

status67,79,80,86 while the other two performed genetic testing only among patients who were 

selected based on MSI testing29 or MSI and IHC testing.12 All four studies were rated grade B for 

the overall quality. All studies examined only the MLH1 and MSH2 genes. Only Syngal 2000 

and Wolf 2005 performed sequencing to all available samples. 

 Only the study by Pinol 200512 assessed incident, newly diagnosed CRC cases. We included 

it by making an assumption that patients who were not selected for genetic testing based on 

MSI/IHC results would test negative for mutations. Figure 17 depicts the sensitivity and 

specificity of the five studies. 

Figure 17. Sensitivity and specificity of the Bethesda guidelines for detecting mismatch repair gene 

mutations: all available studies 

Wolf

de Abajo Syngal

Pinol

Raedle

0

20

40

60

80

100

Sensitivity (%)

0 20 40 60 80 100

100 - Specificity (%)

Genetic testing

irrespectively of MSI/IHC

based on MSI/IHC

The study by Pinol 2005 included unselected incident CRC probands. See text about the verification bias in this study. 

 Based on the study by Pinol 2005,12 the Bethesda guidelines had a sensitivity of 73% (95% 

CI: 39, 94%) and a specificity of 82% (95% CI: 80, 84%) for detecting MMR mutations among 

unselected patients with CRC (Appendix F-15*

). However, these are probably overestimates 

because of verification bias. 

 Among a referral population (or otherwise selected CRC populations) the Bethesda 

guidelines appeared to have high sensitivity (over 90%) but low specificity (below 50%) in all 

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm
85

three studies (including the study by Raedle 200129 that focused on patients whose tumors 

exhibited MSI-H) (Appendix F-15*

). 

Revised Bethesda Guidelines 

 Only one study (Pinol 200512) provided data on the sensitivity and specificity of the revised 

Bethesda guidelines in a unselected population of patients with CRC (Appendix F-17*). The 

sensitivity and specificity of the clinical criteria were calculated to be 91% (95% CI: 59, 100%) 

and 77% (95% CI: 75, 79%), respectively. Thus, the revised Bethesda guidelines were more 

sensitive and as specific as the original Bethesda guidelines (Appendices F-16 and F-17*

). 

However, these may be overestimates because of the possibility of verification bias as discussed 

above. 

Young Age of Onset 

 Appendix F-18 summarizes five studies that assessed young age of onset as a clinical 

predictor to identify mismatch repair gene mutations (Salovaara 2000,85 Aaltonen 1998,63

Colombino 2005,83 Samowitz 2001,10 and Pinol 200512) (Appendix F-18*). Young age of onset 

was defined as less 50 years in all studies, with the exception of Samowitz 2001,10 where the 

definition was less than 55 years. Colombino 200583 provided data only in patients with familial 

CRC (Appendix F-18*). All studies suffered from verification bias because genetic testing was 

not thorough63,85 or was not performed at all10,12 among patients without MSI (MSI or IHC for 

Pinol 200512). 

 Among the three studies of unselected patients with CRC (Appendix F-19) that defined 

young age of onset as less than 50 years old, a summary estimate of sensitivity was 31% (95% 

CI: 18, 47%) and specificity was 95% (95% CI: 94, 96). Only the specificity estimate was 

heterogeneous (p<0.01, I2=81%) (Appendix F-18 and F-19*). These estimates might be inflated 

because of verification bias. Sensitivity and specificity were similar in the studies by Colombino 

200583 and Samowitz 200110 (Appendix F-19*). 

Familial Cancer History 

 Nine studies provided data eligible for this analysis10,12,63,75,81,84,85,87,99 (Appendix F-20*). Six 

studies were rated with grade B in the overall quality scale,10,12,63,81,85,87 and the remaining three 

studies were rated with grade C. Only one study had fewer than 40 patients in total in the 2x2 

tables.75

 There was variability in the populations that were examined and the definition of familial 

disease. A familial history of cancer was defined as presence of colorectal cancer or endometrial 

cancer in a first-degree family member of the proband in three prospective studies that assessed 

consecutive unselected CRC cases.12,63,85 Another study on incident CRC (Samowitz 200110) 

defined familial cancer as CRC in a first degree family. In these studies, all other CRC cases 

were used as a comparator. 

 In the remaining studies, familial cases were highly selected, based on sets of clinical criteria 

such as the Amsterdam I or II criteria, the Korean HNPCC criteria, the aggregation of HNPCC 

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm
86

related tumors in a family, or combinations thereof. Similarly, in these studies the comparator 

(“sporadic CRC cases”) was defined variably (Appendix F-20*

), and was most often a group of 

“apparently sporadic” CRC that were selected based on their young age of onset or a diagnosis of 

multiple tumors. 

 Figure 18 depicts all studies according to whether they assessed unselected CRC cases. 

Figure 18. Sensitivity and specificity of familial cancer history for detecting mismatch repair gene mutations: 

all available studies 

Salovaara

Aaltonen

Pinol

Samowitz

Zhu

Wang

Yuan

Moslein

Lee

0

20

40

60

80

100

Sensitivity (%)

0 20 40 60 80 100

100 - Specificity (%)

Family Hx assessed among:

unselected consecutive CRC

selected CRC populations

CRC: colorectal cancer; Family Hx: family history (of cancer). Empty circles denote studies that contrasted familial CRC cases 

with increased likelihood for HNPCC against sporadic CRC cases who were also at a higher risk for HNPCC compared to an 

unselected CRC population (i.e., young age of onset or multiple tumors in the same patients). See text and Appendix F-20 for 

more details. 

 Four studies reported estimates in unselected, consecutive patients with CRC.10,12,63,83 Three 

used identical definitions (CRC or endometrial cancer in first degree family),12,63,83 and one 

defined familial cancer as CRC in a first degree family member10 (Appendix F-21*). 

 A summary estimate of sensitivity based on the four studies that used similar definitions was 

76% (95% CI: 50, 91%) with borderline between-study heterogeneity (p=0.13, I2

=50%); the 

summary estimate of specificity was 87% (95% CI: 86, 89%) (without between-study 

heterogeneity: p=0.51, I2

=0%). The estimates were similar when Samowitz 200110 was included 

in the calculations, despite the different definition of familial cases. The other studies assessed 

substantially different populations to allow for a meaningful synthesis (Appendix F-20*

). 

However, they uniformly suggested high sensitivities (above 75%) and variable, but generally 

low, specificities (ranging from 24% to 65%). 

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm
87

Multiple CRC or Endometrial Cancer in the Same Patient 

 Appendix F-22* summarizes three studies that included unselected CRC and assessed the 

sensitivity and specificity of multiple, synchronous or metachronous CRC or endometrial cancer 

in the same proband to predict MMR mutations (Salovaara 2000,85 Aaltonen 1998,63 and Pinol 

200512). Their characteristics and limitations have already been discussed. The respective 

summary estimates of sensitivity and specificity were 38% (95% CI: 25, 54%) with no evidence 

for between-study heterogeneity (p=0.98, I2=0%) and 97% (95% CI: 91, 99%) with significant 

between-study heterogeneity (p<0.01, I2=94%). 

Familial History of Cancer, Age of Onset Less Than 50 Years, or 

Multiple Cancers in the Same Proband 

 Appendix F-23* summarizes three studies that enrolled unselected CRC (Salovaara 2000,85

Aaltonen 1998,63 and Pinol 200512). The sensitivity and specificity of the presence of either of 

the following three simple clinical criteria was assessed: history of colorectal or endometrial 

cancer in first degree family, CRC onset at less than 50 years of age, or multiple tumors in the 

same proband versus all other cases. Their characteristics and limitations have already been 

discussed. Summary estimates of sensitivity and specificity were 88% (95% CI: 60, 97%) with 

little evidence for between-study heterogeneity (p=0.19, I2=39%) and 77% (95% CI: 74, 81%) 

with significant between-study heterogeneity (p=0.03, I2

=73%), respectively. 

MSI 

 The presence of MSI is a marker for replication errors and thus a predictor of MMR 

mutations. We assessed the sensitivity and specificity of MSI-H versus MSS. We also analyzed 

combined MSI-H and MSI-L versus MSS. 

 Studies were considered eligible if they performed both MSI testing and MMR genetic 

testing in CRC patients and each test was applied irrespective of the results of the other test. 

 In these analyses we assumed that any genetic testing was the reference standard. 

Overview of MSI Testing. Overall, MSI testing appeared to have modest to good 

discriminating ability to identify carriers of MLH1 and MSH2 mutations. The sensitivity of MSIH versus MSS ranged between 56% and 100% and the specificity between 17% and 93%. 

MSI-H Versus MSS. Sixteen studies described in 18 publications11,63-66,69-71,75,79,80,84-86,95,100-102

were eligible. One received grade A in the overall quality rating,11 nine received grade 

B,63,65,66,70,71,79,80,85,86,101,102 and six received grade C (Appendix F-24*

).

 Eight studies performed sequencing on all available samples11,69,75,80,84,86,95,101 and one of 

them (Barnetson 200611) also used specific methods to detect large genomic deletions or 

rearrangements. All studies tested for MLH1 and MSH2 gene mutations. One study also assessed 

the presence of MSH6 mutations (Barnetson 200611) and another tested for MSH6 and PMS2 

mutations (Southey 2005102). It was not possible to examine the changes in the sensitivity and 

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm
88

specificity of MSI with and without the non-MLH1/non-MSH2 mutations in these studies. Seven 

out of 16 had information in the 2x2 tables on at least 40 patients (Figure 19, Appendix F-24*

). 

 Two studies analyzed all available incident CRC cases (Aaltonen 199863 and Salovaara 

200085), but had substantial verification bias since individuals who were negative in the MSI 

testing were assessed only for the presence of founder mutations in the MLH1 gene. Two other 

prospective studies analyzed incident CRC patients (Barnetson 2006,11 Southey 2005102) who 

were diagnosed at young age (<55 and <45 years, respectively). 

 The sample selection process was based on clearly stated selection criteria that were applied 

to all available patients in ten studies; in five of them, cases were selected among incident CRC 

patients (Aaltonen 199863, Salovaara 200085, Katballe 200265,71, Southey 2005102 and Barnetson 

200611). Twelve studies used microdissection to help assure that the analyzed tumor tissue 

contained a high proportion of malignant cells,11,64-66,69,71,79,80,84,86,95,100-102 and eight used the 

marker sets proposed by the NCI.11,64,69,79,80,84,100-102 

Figure 19. Sensitivity and specificity of MSI-H to identify mismatch repair gene mutations 

Studies shown according to the total number of patients in the 2x2 tables. Black squares, at least 40; empty circles, 40 or less. 

 Overall, the sensitivity of MSI ranged between 56% and 100% and the specificity between 

17% and 93%. Two small studies found a specificity of only 25% (Durno 2005100 among only 

four patients without a mutation) and 17% (Curia 199966 among 18 patients without a mutation). 

Notably, the study by Curia 199966 found few pathogenic mismatch repair gene mutations among 

CRC cases with HNPCC-related cancers (3/30=10%) and thus its applicability is unclear. 

 We could not explain the heterogeneity among estimates based on the overall study quality, 

the comprehensiveness of the genetic testing, the presence of microdissection, the use of NCIrecommended marker sets, or whether the study used a transparent sample selection process 

(Figure 20). Larger studies (with at least 40 patients in the 2x2 tables) tended to cluster more 

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm

0

20

40

60

80

100

Sensitivity (%)

0 20 40 60 80 100

100 - Specificity (%)
89

around the upper left corner of the plot (Figure 19) than studies that used deletion analysis to test 

for MMR mutations (all four of which had more than 40 patients in the 2x2 tables) (Figure 20). 

Figure 20. Sensitivity and specificity of MSI-H to identify mismatch repair gene mutations based upon 

study characteristic 

0

20

40

60

80

100

Sensitivity (%)

0 20 40 60 80 100

100 - Specificity (%)

Microdissection

0

20

40

60

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100

Sensitivity (%)

0 20 40 60 80 100

100 - Specificity (%)

NCI set

0

20

40

60

80

100

Sensitivity (%)

0 20 40 60 80 100

100 - Specificity (%)

Good/fair quality

0

20

40

60

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Sensitivity (%)

0 20 40 60 80 100

100 - Specificity (%)

Sequencing

0

20

40

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Sensitivity (%)

0 20 40 60 80 100

100 - Specificity (%)

Sequenc & deletion

0

20

40

60

80

100

Sensitivity (%)

0 20 40 60 80 100

100 - Specificity (%)

Transparent sampling

Studies are depicted with filled squares versus empty circles according to the presence or absence of the following characteristics: 

Microdissection: Use versus non-use/not stated use of microdissection; NCI set: Use versus non-use/not stated use of the 

National cancer Institute marker sets; Good/fair (A/B) versus poor (C) overall quality; Sequencing: Sequencing of all available

samples; Sequenc & deletion: Sequencing of all available samples and deletion analysis for large genomic rearrangements; 

Transparent Sampling: Sample selected with clearly reported criteria among all available patients versus not. 

 Among studies with more than 40 patients in the 2x2 tables, a summary estimate of 

sensitivity was 83% (95% CI: 65, 92%), with little evidence for between study heterogeneity 

(p=0.15, I2

=37%). A summary estimate of specificity was 87% (95% CI: 80, 91%) with high 

between-study heterogeneity (p<0.01, I2

=83%). The estimates were similar after the exclusion of 

the two Finnish studies (Salovaara 200085 and Aaltonen 199863) in which genetic testing was less 

rigorous for patients without MSI-H tumors. The latter studies also included the Finnish founder 

mutations in their analyses; these mutations are not found in populations of non-Finnish origin. 

 A summary ROC analysis is shown in Figure 21. 
90

Figure 21. Summary ROC curve for the diagnostic ability of MSI-H and MSI-L to identify mismatch repair 

gene mutations 

0

20

40

60

80

100

Sensitivity (%)

0 20 40 60 80 100

100 - Specificity (%)

Combined MSI-H and MSI-L versus MSS. 

Combined MSI-H and MSI-L Versus MSS. There were seven studies in which allowed the 

comparison of combined MSI-H and MSI-L versus MSS. These were described in eight 

publications.11,63,65,70,71,84,85,102 

 The summary estimate for sensitivity was 94% (95% CI: 86, 97) (no heterogeneity: p=0.85, 

I

2

=0%) and for specificity was 83% (95% CI: 77 to 88%) (with substantial heterogeneity: 

p<0.01, I2=80%). When analyses were limited to the four studies that had at least 40 patients in 

the 2x2 tables,11,63,85,102 the corresponding results were 87% (95% CI: 82, 91%) and 95% (95% 

CI: 86, 98%); both estimates had substantial between-study heterogeneity. 

What is the role of MSI-L in the detection of MMR mutations? We evaluated the sensitivity and 

specificity in the seven studies excluding the MSI-L tumors. 11,63,65,70,71,84,85,102 The summary 

sensitivity became smaller (80% [95% CI: 63, 90%]) and the summary specificity increased 

(88% [95% CI: 83, 91%]). The summary sensitivity estimate was not heterogeneous (p=0.21, 

I

2

=28%) but the summary specificity estimate was heterogeneous (p<0.01, I2=71%). 

MSI-H and Combined MSI-H and MSI-L Versus MSS Among Unselected CRC Probands. 

There were only two relevant studies, both from Finland (Salovaara 200085 and Aaltonen 199863

(Appendix F-25*). MSI was assessed based upon the BAT26 marker set in the former, and on at 

least 30% of at least seven markers being unstable in the latter. Patients without MSI tumors 

were screened only for founder mutations in the MLH1 gene, which are common in Finland, 

whereas patients with MSI tumors received more comprehensive genetic testing (for both MLH1 

and MSH2 genes). Thus, their estimates are likely to be biased. Summary estimates of sensitivity 

and specificity were 100% (95% CI: 88, 100%) and 90% (95% CI: 88, 92%), respectively. 

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm
91

MSI-H Versus MSS Among Patients Fulfilling the Revised Bethesda Guidelines. Only the 

study by Wolf 200586 provided relevant data (Table 17, Appendix F-26*

). Sensitivity was 100% 

(95% CI: 75, 100%) and specificity was 79% (95% CI: 63, 90%). 

MSI-H Versus MSS Among Patients Fulfilling Amsterdam I Criteria. There were very few 

patients for these analyses and thus the precision of the estimates is poor (Table 17, Appendix F27*

). Sensitivity is of MSI-H (and combined MSI-H and MSI-low) versus MSI-stable was 

universally excellent (100% [95% CI: 62, 100%]) in the three studies that provided the relevant 

information,70,75,95 although none contributed more than 10 patients who fulfilled the Amsterdam 

I criteria. Overall specificity was 69% (95% CI: 20, 95%). 

IHC 

 Nine studies (described in 11 publications11,65,66,69-71,79,80,98,100,102) provided information 

needed to calculate the sensitivity and specificity of IHC for predicting MMR mutations 

(Appendix F-28*

). One of these (Terdiman 200198) assessed the ability of IHC only in 

individuals with MSI-H positive tumors. 

Studies That Assessed IHC Irrespective of MSI Testing. Eight studies assessed IHC in 

patients with available tumor tissue; one received grade A in overall quality,11 five grade 

B65,66,70,71,79,80,102 and two grade C.69,100

 Three studies performed sequencing in all available patients,11,69,79 and one of them 

(Barnetson 200611) also used specific methods to detect large genomic deletions or 

rearrangements. 

 All studies tested for MLH1 and MSH2 gene mutations. One study also tested for the 

presence of MSH6 mutations (Barnetson 200611) and another tested for MSH6 and PMS2 

mutations (Southey 2005102). IHC testing was performed for all assessed MMR genes (MLH1, 

MSH2 and the additional MMR genes) in the latter two studies.11,102 It was not possible to 

examine the changes in the sensitivity and specificity of IHC with and without the nonMLH1/non-MSH2 mutations in these studies. 

 Three studies had information in the 2x2 tables on at least 40 patients.11,69,102 Two papers 

described prospective studies that analyzed incident CRC patient (Barnetson 200611 and Southey 

2005102) diagnosed at young age (<55 and <45 years, respectively). 

 Figure 22 shows the distribution of the studies according to the presence or absence of 

several study characteristics. Overall, the sensitivity ranged from 27% to 100% and the 

specificity from 43% to 100%. The six good or fair quality studies (grade A or B respectively) 

had a summary sensitivity of 74% (95% CI: 54, 87%) and specificity of 77% (95% CI: 61, 88%). 

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm
92

Figure 22. Sensitivity and specificity of IHC to identify mismatch repair gene mutations based on study 

characteristics 

0

20

40

60

80

100

Sensitivity (%)

0 20 40 60 80 100

100 - Specificity (%)

Transparent sampling

0

20

40

60

80

100

Sensitivity (%)

0 20 40 60 80 100

100 - Specificity (%)

Good/fair quality

0

20

40

60

80

100

Sensitivity (%)

0 20 40 60 80 100

100 - Specificity (%)

Sequencing

0

20

40

60

80

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Sensitivity (%)

0 20 40 60 80 100

100 - Specificity (%)

Sequenc & deletion

0

20

40

60

80

100

Sensitivity (%)

0 20 40 60 80 100

100 - Specificity (%)

N>=40

Studies are depicted with filled squares versus empty circles according to the presence versus absence of the following 

characteristics: Sample selected with clearly reported criteria among all available patients versus not; Good/fair (A/B) versus 

poor (C) overall methodologic quality; Sequencing: Sequencing performed in all available samples; Sequenc & deletion: 

Sequencing and testing for large genomic deletions/rearrangements performed in all samples; Sample size in the analyses at least 

40 versus less than 40. 
93

 Figure 23 shows the summary ROC curve for the pertinent studies. 

Figure 23. Summary ROC curve for the diagnostic ability of IHC to identify mismatch repair gene mutations 

0

20

40

60

80

100

Sensitivity (%)

0 20 40 60 80 100

100 - Specificity (%)

IHC among people fulfilling Amsterdam I criteria. Only the studies described by Katballe 2000 

(another paper by Christensen 2002 reported the same patients65,71) and the study by Dieumegard 

200070 provided relevant information, and they are described in Appendix F-29*

. Both 

contributed nine or fewer patients and thus estimates of sensitivity and specificity lack precision 

(Table 17). 

Studies That Assessed IHC After Suggestive MSI Testing. A single study by Terdiman 

200198 assessed IHC among individuals who had tumors with MSI-H. The sensitivity of IHC 

was 94% (95% CI: 71, 100%) and the specificity was only 13% (95% CI: 4, 30%).

Expected Outcomes With Different Testing Strategies 

Outline of the Problem 

 The prevalence of MMR mutations among people who are newly diagnosed with CRC is 

low.7,10,12,63,85 Because of practical, economic, and logistical considerations, genetic testing 

would ideally be performed only in patients with high probability of HNPCC. Such patients may 

be selected based on heightened suspicion from the clinical history, suggestive laboratory testing 

of tumor tissue (in particular MSI or IHC), or complex combinations of all of the above. The 

various strategies differ in the number of tests (MMR, MSI or IHC) that need to be performed 

and the accuracy of the diagnosis. 

 We performed analyses using decision trees to model the expected outcomes with different 

testing strategies from the payers’/third party perspective. The outcomes were the number of 

incident CRC with positive diagnosis for HNPCC, and the number of tests (MMR, MSI, or IHC) 

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm
94

needed to detect them. We also assessed how many patients found to be mutation carriers with 

each strategy would be truly positive. 

 This analysis pertains to a cross-section in time. We used a hypothetical population of 

100,000 incident cases of CRC. This number is in the order of incident cases expected annually 

in the US (approximately 150,000, given an annual incidence of 50/100,000 and a population of 

300 million) and is a number convenient for calculations. The interested reader could multiply 

the reported numbers by 1.5 to extrapolate to the whole population in the United States. 

 We assessed nine different strategies, which were most commonly represented in the studies 

summarized in this review. The strategies used clinical criteria, MSI, IHC, or a combination of 

clinical criteria with MSI or IHC to select patients for MMR mutation testing. 

 Among the various clinical criteria that have been proposed, we opted to model the revised 

Bethesda guidelines and a set of three clinical criteria that are more easily ascertained 

(fulfillment of at least one of the following: age less than 50 year old at diagnosis, history of 

CRC or endometrial cancer in 1st degree family, or presence of multiple -synchronous or 

metachronous CRC or endometrial cancer in the proband). The revised Bethesda guidelines were 

selected because they appear to have the best sensitivity and specificity among unselected 

incident patients with CRC (data from Pinol 200512). 

 The set of three clinical criteria is a simple alternative that appears to have comparably high 

sensitivity and specificity, but is much simpler to assess. Interestingly, in the predictive models 

that have been developed in the literature, the aforementioned three simple clinical criteria have 

been the most influential predictors of HNPCC status (based on the coefficients from the logistic 

regression models from Barnetson 2006,11 Wijnen 1998103 and Balmana 200692). 

 The selected strategies are not exhaustive. However, these strategies have been discussed in 

the literature as possible options, and some of them have been evaluated in previous decision 

analyses.19 It would be impractical to provide analyses on a more extensive list of strategies, 

especially given the paucity of relevant data in the literature. 

Brief Description of Strategies and Values Used in the Decision Tree 

Nine strategies were modeled as described briefly below. 

• MMR-All: Perform MMR testing on all patients with CRC. 

• BethR-All: Perform MMR testing only among those fulfilling the revised Bethesda 

guidelines. 

• Clinical-All: Perform MMR testing only among those fulfilling at least one of the three 

simple clinical criteria (age <50y at diagnosis; 1st degree family history of CRC or 

endometrial cancer; or multiple, synchronous or metachronous, CRC or endometrial cancer 

in the same patient). 

• MSI-All: Perform MSI testing on all patients; followed by MMR testing only among those 

with suggestive MSI test. 

• IHC-All: Perform IHC testing on all patients; followed by MMR testing only among those 

with suggestive IHC test. 
95

• BethR-MSI: Perform MSI testing on patients fulfilling the revised Bethesda guidelines; 

perform MMR only among those with suggestive MSI test. 

• 3Clinical-MSI: Perform MSI testing on patients fulfilling at least one of the three simple 

clinical criteria (age <50y at diagnosis; 1st degree family history of CRC or endometrial 

cancer; or multiple, synchronous or metachronous, CRC or endometrial cancer in the same 

patient); perform MMR only among those with suggestive MSI test. 

• BethR-IHC: Perform IHC testing on patients fulfilling the revised Bethesda guidelines; 

perform MMR only among those with suggestive IHC test.

• 3Clinical-IHC: Perform IHC testing on patients fulfilling at least one of the three simple 

clinical criteria (age <50y at diagnosis; 1st degree family history of CRC or endometrial 

cancer; or multiple, synchronous or metachronous, CRC or endometrial cancer in the same 

patient); perform MMR only among those with suggestive IHC test. 

Figure 24 illustrates the general concepts in the architecture of these decision trees. 

The strategies are conceptually organized in similar groups depending upon the pattern they 

follow in selecting who would be sequenced for MMR mutations. 

• Group 1: Test everyone (MMR-All) 

• Group 2: Screen with a set of clinical criteria (BethR-All, 3Clinical-All) 

• Group 3: Screen with a laboratory test (MSI-All, IHC-All) 

• Group 4: Screen using two serial tests: a set of clinical criteria first and then a laboratory test 

(BethR-MSI, BethR-IHC, 3Clinical-MSI, 3Clinical-IHC). 
96

Figure 24. Decision tree model used to calculate the impact of different testing strategies The figure consists of 5 panels arranged horizontally; these correspond to different levels/nesting in the decision tree. The nine branches in the first level stand for the modeled strategies (abbreviations are explained in the text). These may be clustered/organized in 4 co

The second level models the prevalence of HNPCC. The third and fourth levels stand for the screening tests that may be applied. The fifth level repres

mutations. Depending on the strategy, all patients may receive genetic testing (i.e., reach the fifth level) (strategy MMR-All), all may be screened with 

criteria for BethR-All and 3Clinical-All, MSI for MSI-All and IHC for IHC-All), or all may be screened with two serially applied tests (the first based second on MSI or IHC) to select the ones who will reach the fifth level (BethR-MSI, 3Clinical-MSI, BethR-IHC, 3Clinical-IHC). 
97

 Table 18 shows the selected baseline values for the decision tree parameters, as well as the 

ranges of the sensitivity analyses. It should be noted that only some of the conditional 

probabilities that were presented in the previous sections are applicable to the following 

modeling approach; furthermore some conditional probabilities are imputed based on realistic 

assumptions because they were not extractable from the reviewed literature. All probabilities, 

their derivation and the rationale for any assumptions are described in more detail in the sections 

that follow. 

Prevalence of Mutations Among Incident Unselected CRC Patients (Prev). The prevalence 

of mutations among incident unselected CRC is the most influential variable in the analyses (see 

Appendix G*

). Based on the following considerations we present here two sets of results; one 

using a low prevalence estimate (0.9%) and one using a higher prevalence estimate (2.7%). The 

rationale for these values is given below. The prevalence value ranged from 0.6% to 5.1% in the 

sensitivity analyses.

Relevant studies. Among the 40 studies that were assessed for Key Question 2, four (3,332 

analyzed patients in total) evaluated incident unselected CRC patients (Aaltonen 1998,63

Salovaara 2000,85 Pinol 2005,12 and Samowitz 200110). All were limited by verification bias, in 

that comprehensive genetic testing was performed only in patients with suggestive MSI 

results,10,63,85 or MSI and IHC results.12 

 We also considered the study by Hampel 2005,8 although it was not eligible (did not provide 

needed data) for the quantitative analyses described in the previous sections. Patients with 

suggestive MSI were sequenced for MLH1, MSH2, MSH6 and PMS2 mutations; analyses for 

large genomic deletions/rearrangements were also performed. 

Low estimate for mutation prevalence (0.9%). The two Finnish studies63,85 reported founder 

mutations (large genomic deletions) in the MLH1 gene of several CRC patients; however the 

Finnish founder mutations have not been identified in patients of non-Finnish origin.12 Thus, 

they are not applicable to the US population. Excluding the founder mutations from the analyses, 

the proportion of mutation carriers among unselected patients with CRC was estimated at 0.9% 

(95% CI: 0.6 to 1.3%).

 An American Founder Mutation (AFM) in the MSH2 gene has been described in one kindred 

probably originating from German immigrants, but it is unlikely to have a large impact in the 

general population.90,91 AFM is expected to account for very few HNPCC incident cases 

(between 51 and 290 CRC in the US for AMF) annually.90,91 Thus, we did not model the AFM. 

Higher estimate for mutation prevalence (2.7%). In the Hampel 2005 study twenty-three out of 

1066 CRC were identified as carriers (prevalence 2.2% [95% CI: 1.4, 3.2%]), after suggestive 

MSI. Conservative accounting for carriers who did not have suggestive MSI (using the lower 

95% CI boundary for the sensitivity of MSI among unselected CRC) raises the prevalence value 

to 2.7%. 

Range of sensitivity analyses. We used a wide range of prevalence values in the sensitivity 

analyses from 0.6% to 5.1%. 0.6% is the lower 95% CI boundary for the low prevalence 

estimate. 5.1% is 20% more than the upper 95% CI of the higher prevalence estimate and was 

 

* Appendixes cited in this report are provided electronically at http://www.ahrq.gov/clinic/tp/hnpcctp.htm
98

deemed to be high enough to encompass practically all realistic prevalence estimates that have 

been suggested by experts.7

 

Sensitivity and Specificity of Genetic Testing (S_MMR, C_MMR). We assumed:

 

• That complete gene sequencing for MMR testing would be done. 

• Near perfect sensitivity and specificity for genetic testing. 

• The proportion of inconclusive genetic test results (that is mutations of unknown clinical 

significance) was not incorporated in the estimates of sensitivity and specificity of genetic 

testing; it is rather modeled separately (see below). 

 Because the available estimates for the diagnostic performance of all clinical predictors were 

based on MLH1 and MSH2 gene mutation testing in the primary studies, we “penalized” the 

maximum sensitivity of genetic testing to account for HNPCC cases with mutations other than 

MLH1 and MSH2. There were limited data on this proportion in the eligible studies. We 

therefore assumed that 5% of patients with HNPCC would have mutation in MMR genes other 

than MLH1 and MSH2 (thus we set sensitivity to 95% in the base case). In further analyses, we 

examined sensitivity as low as 72% based on data from Southey 2005,102 where 28% of the 

detected mutations among CRC aged less than 45 years of age (a selected population) were due 

to MSH6 and PMS2 mutations. Thus, the modeling accounts for the incremental value of testing 

for additional MMR genes other than MLH1 and MSH2. 

Proportion of Inconclusive Results Among HNPCC With Non-Positive Test Results 

(Pinconcl1), and Proportion of Inconclusive Results Among Patients Without HNPCC 

With Non-Positive Test Results (Pinconcl2). These estimates are associated with considerable 

uncertainty. Syngal 199955 found that 2/18 patients with pathogenic mutations also had 

inconclusive mismatch mutations. Thus we assumed that 11% of HNPCC carriers also have 

inconclusive mutations. 

 The proportion of patients with CRC without HNPCC who carry mismatch mutations is more 

difficult to estimate. We based the estimate on the only study on incident, unselected CRC that 

provided the relevant information.12 This study was published recently and thus the authors 

presumably had access to the latest knowledge on which mutations were pathogenic; the 

corresponding estimate was 0.25%. 

 Both the aforementioned proportions were varied from 0% to their upper 95% CI values in 

sensitivity analyses. 

Sensitivity and Specificity of MSI (S_MSI, C_MSI) and IHC (S_IHC, C_IHC) Among 

Unselected Incident CRC. These values were based on estimates from population-based 

studies that performed some form of genetic testing in all available patients. Because such IHC 

estimates were not available in unselected CRC, we assumed that two studies on patients with 

young age of onset for CRC (Barnetson 200611 and Southey 2005102) provided reliable 

approximations. This is based on the assumption that the diagnostic ability of laboratory 

predictors is relatively independent of clinical criteria that were used to select a population. 

 Similarly, for MSI we used a synthesis from the estimates obtained from four studies on 

incident CRC (Salovaara 200085, Aaltonen 199863, Barnetson 200611 and Southey 2005102). The 
99

values for MSI refer to combined MSI high and MSI low. The range of the sensitivity analyses 

includes the estimates for MSI high only. 

Sensitivity and Specificity of the Revised Bethesda Guidelines Among Unselected CRC 

(S_BethR, C_BethR). These were obtained from the study by Pinol 2005.12

Sensitivity and Specificity of the Three Clinical Criteria (Young Age at Diagnosis, CRC in 

Family or Multiple Tumors in Proband) Among Unselected Patients With CRC 

(S_3Clinical, C_3Clinical). These were obtained from a meta-analysis of pertinent studies 

(Aaltonen 1998,63 Salovaara 2000,85 and Pinol 200512). The estimates for the two Finnish 

studies63,85 were calculated accounting for the presence of founder mutations. 

Sensitivity and Specificity of MSI (S_MSI_3Clinical, C_MSI_3Clinical) and IHC 

(S_IHC_3Clinical, C_IHC_3Clinical) Among People Fulfilling the Three Clinical Criteria 

(Young Age at Diagnosis, CRC in Family or Multiple Tumors in Proband). The values for 

MSI were based on the studies by Aaltonen 1998 and Salovaara 2000.63,85 Values include both 

MSI-H and MSI-low, as discussed above. 

 Similar values for IHC were not available. We assumed that these could be substituted by the 

corresponding estimates for the unselected CRC (which were based on data from Barnetson 2006 

and Southey 2005, two studies11,102 that assessed people at young age of CRC diagnosis). 

Sensitivity and Specificity of MSI (S_MSI_BethR, C_MSI_BethR) and IHC (S_IHC_BethR, 

C_IHC_BethR) Among People Fulfilling the Revised Bethesda Guidelines. A small study on 

81 patients by Wolf 200586 provided the relevant information for the accuracy of MSI. However, 

the study reported perfect sensitivity (100%) for MSI, which was considered improbable. We 

therefore used the same estimates as for patients fulfilling at least one of the three simple 

clinical criteria (<50 years of age at diagnosis, CRC or endometrial cancer in family or multiple 

colorectal or endometrial cancers in proband). 

 There were no available data for the diagnostic accuracy of IHC among people fulfilling the 

revised Bethesda guidelines. Therefore, we assumed that Southey 2005102 (a study that assessed 

incident CRC aged less that 45 at diagnosis) provided suitable corresponding estimates.
100

Table 18. Probabilities and parameters used in the decision trees for different strategies to detect MMR mutations Variable Baseline Range for sensitivity analysis 

Low High Rationale 

R

Prevalence of HNPCC among unselected CRC assuming no founder mutations in non-Finnish populations, (%) Prev 

Low=0.90 

Higher=2.75 

0.6 5.1 Lower limit is the lower 95% CI 

boundary excluding founder mutations in MLH1; Upper limit is more than the upper 95% CI boundary of the higher prevalence estimate The range was intentionally broad. 

AS2H

Sensitivity of genetic testing, (%) S_MMR 

95 72 100 Assumed to be the same in 

every subset of CRC 

AdM

Specificity of genetic testing, (%) C_MMR 

99.5 98 100 Assumed to be the same in 

every subset of CRC 

A

Proportion with inconclusive results among those with HNPCC whose MMR test results were not positive, (%) Pinconcl1 

11 0 35 Upper limit based on 95% CI S

Proportion with inconclusive results among those without HNPCC whose MMR test results were not positive, (%) Pinconcl2

0.25 0 0.7 Upper limit based on 95% CI P

Unselected incident CRC probands 

Sensitivity of MSI testing, (%) S_MSI 

95 81 99 Assuming combined MSI-H and 

MSI-L is “positive”; limits based on 95% CI 

ASBS

Specificity of MSI testing, (%) C_MSI 

87 81 91 Assuming combined MSI-H and 

MSI-L is “positive”; Limits based on 95% CI 

ASBS

Sensitivity of IHC testing, (%) S_IHC 

91 75 97 among <45 and <55y of age; 

Limits based on 95% CI 

SB

Specificity of IHC testing, (%) C_IHC 

87 83 90 among <45 & <55y of age; Limits 

based on 95% CI 

SB

Sensitivity of Revised Bethesda Guidelines, (%) S_BethR 

91 59 100 Limits based on 95% CI P

Specificity of Revised Bethesda Guidelines, (%) C_BethR 

77 75 79 Limits based on 95% CI P
101

Table 18. Probabilities and parameters used in the decision trees for different strategies to detect MMR mutations (continued) Variable Baseline Range for sensitivity analysis 

Low High Rationale 

R

Sensitivity of 3 clinical criteria, (%) S_3Clinical 

88 60 97 Limits based on 95% CI A

S2

Specificity of composite clinical criteria, (%) C_3Clinical 

77 74 81 Limits based on 95% CI A

S2

Among CRC fulfilling the composite clinical criteria 

Sensitivity of MSI testing, (%) S_MSI_3Clinical 

95 65 100 10/10 and 17/18; limits based on 

95% CI 

AS

Specificity of MSI testing, (%) C_MSI_3Clinical

87 73 94 98/118 and 82/99; Limits based 

on 95% CI 

AS

Sensitivity of IHC testing, (%) S_IHC_3Clinical

91 75 97 Assumption, see text [S

B

Specificity of IHC testing, (%) C_IHC_3Clinical

87 83 90 Assumption, see text [S

B

Among CRC fulfilling the Revised Bethesda guidelines 

Sensitivity of MSI testing, (%) S_MSI_BethR

95 65 100 Assumption, see text [A

S

Specificity of MSI testing, (%) C_MSI_BethR

87 73 94 Assumption, see text [A

S

Sensitivity of IHC testing, (%) S_IHC_BethR

84 73 96 Assumption, see text; Limits 

based on 95% CI 

[S

Specificity of IHC testing, (%) C_IHC_BethR 

95 94 97 Assumption, see text; Limits 

based on 95% CI 

[S

In the first column the italicized word is the name of the pertinent variable in the decision trees. For references that are in brackets: The variable value

that the studies referenced within brackets provide suitable estimates. CI: confidence interval; CRC: colorectal cancer; IHC: immunohistochemistry; MSI: MSI.
102

Results With the Different Strategies 

 Testing for MSI (or even IHC) among CRC patients with high clinical suspicion for HNPCC 

may be a reasonable compromise between the number of laboratory tests that would be required 

and an accurate diagnosis in the majority of people with HNPCC (i.e., strategies in group 4). 

 More specifically, group 4 strategies selected relatively fewer patients for genetic testing (6% 

or less) and missed at most 27% of patients with HNPCC. In contrast, for strategies in groups 1 

to 3, more than 13% and up to 100% of newly diagnosed CRC patients would be genetically 

tested, and 5% to 16% of patients with HNPCC would be missed. These descriptives are true for 

both the low (0.9%) and the higher estimate for the prevalence of mutation carriers among 

incident unselected CRC (Table 19). 

 Because the revised Bethesda guidelines are more difficult to ascertain compared to the 

fulfillment of at least one of the three simple clinical criteria, it might be easier to use the 

combination of the three clinical criteria. Although it seemed that MSI was preferable over IHC 

as a screening tool among people with high clinical suspicion, estimates were based on several 

assumptions due to missing information, compromising the validity of this observation. 

 We decided not to highlight the relative ranking of the strategies within each group because 

of the many assumptions in the used probabilities. We therefore focus more on the differences 

across the four groups of strategies. The relative ranking of the 4 strategy groups with respect to 

each outcome was robust in all one-way sensitivity analyses. 

Baseline Analyses. 

Number of patients identified as positive – low estimate for prevalence of mutation carriers. In 

the hypothetical population of 100,000 CRC, 900 individuals had MMR mutations. The number 

of positive diagnoses with each strategy ranged from 1,351 (MMR-All) to 659 (Beth-IHC) 

(Table 19). The number of HNPCC patients who were truly identified was highest in the MMRAll strategy (n=855 out of 900) and lowest in group 4 strategies (ranging between 654 and 739, 

depending on the strategy). Reciprocally, the number of HNPCC patients who were missed with 

each strategy increased for strategies that performed fewer MMR tests. While only 45 patients 

would be missed if all CRC were to be genetically tested, up to one out of five (between 161 and 

246 out of 900) would be missed if MSI or IHC were performed only on people with a high 

clinical suspicion for HNPCC. 

 As shown in Table 19, the number of inconclusive MMR tests increases with the number of 

total MMR tests. In group 4 strategies it ranged between 6 and 12, and in the MMR-All strategy 

it was approximately 250. 

Number of patients identified as positive – higher estimate for prevalence of mutation carriers. 

In the hypothetical population of 100,000 CRC, 2,750 individuals had MMR mutations. The 

number of positive diagnoses with each strategy ranged from 3,098 (MMR-All) to 2,002 (BethIHC) (Table 19). The number of HNPCC patients who were truly identified was highest in the 

MMR-All strategy (n=2,612 out of 2,750) and lowest in group 4 strategies (ranging between 

1,997 and 2,258, depending on the strategy). The number of HNPCC patients who were missed 

with each strategy increased for strategies that performed fewer MMR tests. While only 138 

patients would be missed if all CRC were to be genetically tested, up to one out of four (between 

492 and 753 out of 2,750) would be missed if MSI or IHC were performed only on people with 

high clinical suspicion for HNPCC. 
103

 As shown in Table 19, the number of inconclusive MMR tests increases with the number of 

total MMR tests. In group 4 strategies it ranged between 14 and 20, and in the MMR-All strategy 

it was a bit over 260. 

Number of tests performed for assessments – low estimate for prevalence of mutation carriers. 

The expected percentage of the CRC cohort receiving MMR tests ranged from 100% (MMR-All) 

to 1.83% (BethR-IHC) (Table 19). 

 Generally, strategies that used clinical criteria to select patients to be tested with MSI or IHC 

(BethR-MSI, BethR-IHC, 3Clinical-MSI, 3Clinical-IHC) required fewer MMR genetic tests. 

Fewer than 4% of newly diagnosed patients with CRC would be expected to need MMR genetic 

testing based on this approach. Furthermore, for the strategies in group 4, approximately one out 

of four patients would receive MSI or IHC testing, based upon suggestive clinical criteria. 

 The use of clinical criteria alone to screen cases (BethR-All, 3 Clinical-All) would require 

MMR genetic testing in almost a quarter of the incident cases of CRC. Using laboratory tests as a 

first screening would result in approximately one out of seven patients requiring genetic testing 

(Table 19). By definition, all patients in the strategies that first screen with MSI or IHC would 

receive MSI or IHC testing (MSI-All or IHC-All, respectively), but fewer than one out of four 

patients would need to be screened with MSI or IHC based upon suggestive clinical criteria 

(Table 19). 

Number of tests performed for assessments – higher estimate for prevalence of mutation carriers. 

The expected percentage of the CRC cohort receiving MMR tests ranged from 100% (MMR-All) 

to 3.22% (BethR-IHC) (Table 19). The overall percentages of people receiving genetic testing, 

MSI testing or IHC testing with the various strategies was similar to what was calculated for the 

low prevalence estimates (Table 19). 

 
104

Table 19. Expected number of MMR, MSI or IHC tests and expected MMR testing results with the nine 

strategies, assuming a population of 100,000 incident cases of CRC 

Strategy Received tests Number of MMR tests that were 

MMR MSI IHC Positive True 

positive 

Inconclusive 

Unidentified 

MMR 

mutation 

carriers 

Low prevalence estimate for MMR mutation carriers (0.90%) 

MMR-All 100,000 0 0 1,351 855 252 45 

BethR-All 23,612 0 0 892 778 61 122 

3Clinical-All 23,585 0 0 866 752 61 148 

MSI-All 13,738 100,000 0 877 812 37 88 

IHC-All 13,702 0 100,000 843 778 37 122 

BethR-MSI 3,741 23,612 0 754 739 12 161 

3Clinical-MSI 3,716 23,585 0 730 715 11 185 

BethR-IHC 1,828 0 23,612 659 654 6 246 

3Clinical-IHC 3,684 0 23,585 700 685 11 215 

Higher prevalence estimate for MMR mutation carriers (2.75%) 

MMR-All 100,000 0 0 3,098 2,612 257 138 

BethR-All 24,870 0 0 2,489 2,377 69 373 

3Clinical-All 24,787 0 0 2,410 2,299 69 451 

MSI-All 15,255 100,000 0 2,545 2,481 46 269 

IHC-All 15,145 0 100,000 2,440 2,377 45 373 

BethR-MSI 5,285 24,870 0 2,273 2,258 20 492 

3Clinical-MSI 5,206 24,787 0 2,198 2,184 20 566 

BethR-IHC 3,220 0 24,870 2,002 1,997 14 753 

3Clinical-IHC 5,110 0 24,787 2,106 2,092 20 658 

In the hypothetical population, for the low prevalence estimate 900/100,000 patients are assumed to carry MMR mutations; for 

the high prevalence estimate 2750 people are assumed to carry MMR mutations. Strategies are presented with respect to the 

group (1 to 4) to which they belong. 

 One may consider the nine strategies as nine (composite) diagnostic tests, and thus calculate 

their overall (strategy-level) sensitivity and specificity (Table 20). In these calculations, 

inconclusive tests were assumed to be false negative (for the calculation of overall sensitivity for 

each strategy) or false positive (for the calculation of overall specificity for each strategy). The 

overall specificity was high in all strategies. Group 4 strategies had the lowest overall sensitivity 

(ranging between 73% and 82%). The overall sensitivity and specificity values were similar for 

both the low and the higher estimates for the prevalence of MMR mutation carriers. 

Table 20. Overall sensitivity and specificity for each of the nine strategies 

Strategy Sensitivity of strategy 

(%) 

Specificity of 

strategy (%) 

MMR-All 95.0 99.3 

BethR-All 86.5 99.8 

3Clinical-All 83.6 99.8 

MSI-All 90.3 99.9 

IHC-All 86.5 99.9 

BethR-MSI 82.1 100.0 

3Clinical-MSI 79.4 100.0 

BethR-IHC 72.6 100.0 

3Clinical-IHC 76.1 100.0 

The overall sensitivity and specificity values were very similar for both the low and the higher estimates for the prevalence of 

MMR mutation carriers. 

Strategies are presented with respect to the group (1 to 4) to which they belong. 
105

Benefits and Harms 

Key Question 1: Does Risk Assessment and HNPCC Mutation Testing 

in Patients With Newly Diagnosed CRC Lead to Improved Outcomes 

for the Patient or Family Members, or is it Useful in Medical, Personal, 

or Public Health Decision Making? (Overarching Question) 

 

 Key Question 1 is a general question that includes all other Key Questions. An ideal study 

addressing Key Question 1 would enroll patients with CRC patients and/or their family members 

and randomize them to risk assessment and mutation testing or a control intervention, and follow 

them up prospectively. The study would compare a spectrum of health outcomes among those 

who received screening for HNPCC to those who did not, while considering subsequent 

treatments or interventions in each group. 

Summary of Findings. No study directly addressed Key Question 1 based on the ideal 

framework described above.

Gaps in the Literature. While our literature review did not identify a comprehensive study that 

directly answered Key Question 1, it is unlikely that such an "ideal" study is feasible. Such a 

study would likely be prohibitively complex, require multiple years of follow-up and 

randomization of patients or family members to care that is no longer considered to reflect 

contemporary practices. However, studies addressing specific components of this general 

question (as described in the remaining Key Questions) provide some of necessary pieces to 

better understand this overarching question.

Patients

with newly 

diagnosed 

CRC

High risk of

HNPCC

HNPCC testing HNPCC mutation 

negative Patient

Outcomes

Medical

Decision 

making

Family members of HNPCC 

positive CRC patients

Genetic Counseling 

HNPCC testing

Risk Assessment

2,3 Average risk

of HNPCC

1

4,5

Screening

HNPCC

mutation

positive

6,7

Intervention

8,9

Family Outcomes

Increased early 

detection, 

decreased 

mortality/morbidity,

improved decision 

making 

HNPCC

mutation

negative

HNPCC

mutation

positive

10,

11
106

Key Question 3: What Are the Harms Associated With Screening 

High-Risk Individuals for HNPCC? 

Patients

with newly 

diagnosed 

CRC

High risk of

HNPCC

Risk Assessment

2,3 Average risk

of HNPCC

Screening

 Studies were considered eligible for Key Question 3 if they reported harms of a risk 

assessment process (e.g., Amsterdam, Bethesda and/or MSI, IHC) used to identify CRC patients 

at increased risk for HNPCC. 

Summary of Findings. No study described any harms of the risk assessment process in CRC 

patients at increased risk for HNPCC. 

Gaps in the Literature. The goal of the risk assessment process is to identify individuals 

suspected of having HNPCC who might benefit from subsequent genetic testing. It is possible 

that such a process could lead to harm, such as stigmatization or worry, but no study directly 

assessed such associations.

 The risk assessment tools also have the potential to misclassify patients. The test 

characteristics (i.e., sensitivity, specificity and predictive values) of these methods are 

summarized in the “Clinical Validity” section. We did not consider misclassified patients (i.e., 

false positive or negatives) as having been harmed. However, it is possible that subsequent 

genetic testing and/or management options could harm patients who had been misclassified. The 

harms associated with genetic testing and/or management options are described later in this 

report. 

 Future research is needed to identify the best strategy of the risk assessment process that 

increases the accuracy, feasibility and applicability of diagnostic methodologies to determine 

HNPCC so the potential harms can be minimized. 

Key Question 5: What Are the Harms Associated With Screening for 

High-Risk Individuals? 

High risk of

HNPCC

HNPCC testing HNPCC mutation 

negative

Average risk

of HNPCC

4,5

Screening

HNPCC

mutation

positive
107

 Studies were considered eligible for Key Question 5 if they reported the harms associated 

with testing CRC patients for MMR mutations. Common harms that are thought to be associated 

with genetic testing are labeling, discrimination in health coverage, and emotional distress. 

 Three quantitative, comparative studies of quality A and B, and one qualitative study of B 

quality reported harms associated with MMR mutation testing in CRC patients104-106 (Table 21). 

Summary of Findings. One 1-year prospective study (Grade A) compared the psychological 

impact of MMR mutation testing between mutation carriers and non-carriers.104 Results for noncarriers are summarized below and also in Table 21. Subjects in this study were CRC probands 

or relatives from HNPCC families with a prior diagnosis of any cancer (excluding nonmelanoma skin cancer). Anxiety, depression, and quality of life measures did not change over 

time, and there were no differences in these measures between mutation carriers and noncarriers. Distress levels were significantly decreased 2 weeks and 6 months after revealing the 

genetic testing results, but were not significantly different from the baseline at 1-year follow-up. 

There was no difference in the distress levels between mutation carriers and non-carriers. 

 Another 1-month prospective study (Grade B) found that three of the 27 probands (11%) 

had minor depression at 1 month after revealing the genetic testing results, but the prevalence of 

minor depression was not significantly different compared to the prevalence at baseline or 

between mutation carriers and non-carriers.105 Of the six probands who received a positive result, 

two (33%) felt severe guilt regarding their children. 

 One prospective study (Grade B) reported changes in the psychological outcomes of CRC 

patients from self-completed questionnaires pre- and 4-6 weeks post-genetic counseling.107 There 

was no genetic testing performed in this study. There was a trend toward greater anticipated 

ability to cope with a positive gene test after counseling, as reflected by a decreased in anxiety 

and cancer-specific distress. 

 The qualitative study of 111 newly diagnosed CRC patients reported a high acceptance and 

understanding about information on HNPCC.106 Nineteen percent of participants rated their 

current level of worry caused by the genetics information at or above the midpoint of 4 on a 1 

(not at all) to 7 (all the time) scale. 

Gaps in the Literature. Most research on psychosocial aspects of genetic counseling and 

testing for cancer risk has focused on hereditary breast and ovarian cancer. Genetic testing for 

hereditary colorectal cancer has become available only in the past decade. As a result, only few 

studies with small number of CRC patients have evaluated the acceptability and psychosocial 

sequelae of genetic counseling and testing. Some of the findings from studies of counseling and 

testing in other forms of cancer may be applicable to the CRC setting. 
108

Table 21. Key Question 5. What are the harms associated with genetic testing for HNPCC mutations? 

Observed imp

Author, year Country 

Study 

design 

(follow-up 

duration) 

Target population for 

genetic testing 

(Eligible /Enrolled N) 

N 

evaluated 

% 

Mutation 

positive 

Measures of 

psychological 

impact after 

revealing genetic 

testing results 

Test 

receivers 

vs. normal 

rangesa

Follow-up 

vs. baseline 

(changes 

over time) 

Quantitative, Comparative Studies 

Anxiety (State-Trait) 

⇔ at all f/up 

Depression (CES-D) 

⇔ at all f/up 

Gritz, 2005 Quality of life (SF-36) 

⇔ at all f/up 

US 

Prosp 

(2 wk, 6 

mo & 1 yr) 

Affectedb persons at least 

18 years old with HNPCC 

defined by Amsterdam or 

suggestive family history 

followed by mutation 

testing 

(126/89) 

89 37% 

Distress from 

receiving testing 

results (RIES) 

Æ at 2 wk & 

mo; 

⇔ 1 yr 

Anxiety (HAD) 

Æ

Depression (HAD) 

⇔

Keller, 2002 Germany 

Prosp 

(4-6 wk) 

Patients with HNPCCrelated cancer from 

families at risk for 

HNPCC 

(ND/31) 

31 N/Ac

Cancer-specific 

distress (IES scales) 

Æ

⇓

Minor depression 

(DSM-III-R and DSMIV scales) 

⇔

Murakami, 2004 Japan 

Prosp 

(1 mo) 

Probands whose family 

members had been 

identified as carrying 

the MLH1/MSH2 

mutation, age ≥20 years, 

and opted for genetic 

testing 

(31/27) 

27 22% 

Guilt 

(mutation carriers only) 

Å Statistically increased 

⇑ Increased, but not statistically significant 

⇔ No statistically significant differences Æ Statistically decreased 

⇓ Decreased, but not statistically significant 

a Published population levels or the normal ranges b “Affected” persons included any index patients or relatives with a prior diagnosis of any cancer excluding non-melanoma skin cancer c Patients received only pre-test genetic counseling. Psychological measures were done immediately after counseling without performing genetic testin
109

Table 21. Key Question 5. What are the harms associated with genetic testing for HNPCC mutations? (continued) Author, year Country 

Study 

Design 

(Follow-up 

duration) 

Target population 

for genetic 

testing 

(Eligible /Enrolled 

N) 

N 

evaluated 

% 

Mutation 

positive 

Results 

Qualitative, Non-Comparative Studies Porteous, 2003 UK 

Survey 

(N/A) 

Newly diagnosed 

CRC patients 

under the age of 

55 years old 

(160/111) 

111 N/A 

The majority of participants found it highly acceptable to ha

about HNPCC brought to their attention at a time when 

coping with a new diagnosis of colorectal cancer, despiawareness that the disease could run in families. The vreported high levels of subjective understanding concertesting. 19% of participants (n=21) rated their current lecaused by the genetics information at or above the mid(not at all) to 7 (all the time) scale. 

1 patient (1% ) not opting for genetic testing. 

References for standardized instruments: CES-D: Center for Epidemiologic Studies Depression. DSM-III-R: Association Psychiatric Association. Diagnostic and statistical manual of mental disorders. 3rd edition, revised. Washington, DC: Americ

DSM-IV: Association Psychiatric Association. Diagnostic and statistical manual of mental disorders. 4th edition. Washington, DC: American PsychiaState-Trait Anxiety: Van der Ploeg HM, Defares PB, Spielberger CD. Handleiding bij de Zelfbeoordelingsvragenlijst: een Nederlandstalige bewerking 

Trait Anxiety Inventory. Lisse: Swets and Zeitlinger, 1980. 

Cancer-specific distress (IES scales): Horowitz MJ, Wilner N, Alvarez W. Impact of Event Scale: a measure of subjective stress. Psychosom Med 197

Hospital anxiety and depression scale (HAD): Goldberg D, Williams P. A User’s Guide to the General Health Questionnaire. Windsor: NFER-Nelson
110 

Key Question 6a: What Are the Management Options for CRC 

Patients Who Are HNPCC Positive? b: Does the Identification of 

HNPCC Mutations Lead to Improved Patient Outcomes in Terms of 

Early Detection, Mortality/Morbidity, or Management Decisions (e.g., 

Counseling, Surveillance, Treatment, Other Decision Making) by 

Patients and Providers? 

HNPCC mutation 

negative Patient

Outcomes

Medical

Decision 

making

HNPCC

mutation

positive

6,7

Intervention

 Key Question 6 examines the outcomes of using MMR mutation status to make management 

decisions compared to conventional management without MMR mutation status. There are three 

aspects to Key Question 6: 1) Are management options for patients with CRC with a MMR 

mutation different from those without a MMR mutation? 2) Does the knowledge of MMR 

mutation status change management decisions by patients and providers? 3) Does changing 

management options for MMR positive patients with CRC improve outcomes (e.g., prognosis 

and survival) compared to standard approaches for patients with CRC? 

 We encountered a variety of surgical and medical management options in patients with CRC 

who were MMR positive. These included various forms of colonic resection108-110 and 

chemotherapy for patients with stage III tumors.111 However, these studies did not directly 

address the components of Key Question 6 described above. 

 Because of the limited data, we broadened the scope of this question to include studies 

evaluating all forms of cancer related to HNPCC, since patients with CRC from HNPCC families 

are potentially at increased risk for all of these cancers. We also included studies using clinical 

criteria and laboratory testing (such as MSI and IHC) as a surrogate for identification of HNPCC 

mutations. 

 Despite having broadened the inclusion criteria, we did not identify any prospective, 

comparative study (direct evidence) addressing any aspect of Key Question 6. However, two 

retrospective cohort studies indirectly addressed the prognosis of CRC and the survival outcome 

of patients with endometrial cancer from HNPCC families, in relation to MMR mutation 

testing.112,113 Both were of C quality, due to potential selection bias and unclear effects of 

treatments or interventions on the outcomes. 

 In addition, five retrospective cohort studies indirectly addressed mortality or morbidity in 

relation to risk assessments (clinical criteria and laboratory testing) for HNPCC 

mutations108,109,111,114,115 (Table 22). Of these, three studies of B quality described clinical 

outcomes in patients with CRC who were screened for HNPCC using clinical criteria.108,109,115

Another two described clinical outcomes in patients with CRC who were screened for HNPCC 

with laboratory testing.111,114 Both were of C quality and limited by the potential selection bias, 

and/or unclear effects of treatments or interventions on the outcomes. 
111 

Summary of Findings. Indirect evidence from one study suggested that identification of 

HNPCC mutations was associated with better prognosis of CRC. However, there was no data on 

whether management options for CRC differed based on MMR mutation status.

 Indirect evidence from one study showed no difference in survival of patients with 

endometrial cancer, comparing those who were mutation positive to those who were mutation 

negative. 

 In five studies with indirect evidence, there was no evidence in favor or against differences in 

survival, when comparing CRC patients who fulfilled different clinical criteria for HNPCC or 

screened positive for HNPCC by laboratory testing with those who did not (Overview Table 1; 

Table 22). 

Overview Table 1: Key Question 6b 

Reported 

Outcomes 

Body of 

evidence 

(study 

duration) 

Summary 

Prognosis of CRC 1 Retro 

study 

(~8 yr) 

• ⇔ Comparing mutation carriers to non-carriers with MSI 

• Better, comparing mutation carriers to non-carriers with MSS 

Survival of patients 

with endometrial 

cancer 

1 Retro 

study 

(ND) 

• ⇔ Comparing MMR mutation carriers to non-carriers 

Survival of CRC 

patients 

5 Retro 

studies 

(12-153 

mo)a

• Results were inconsistent among studies that compared survival in 

patients with CRC who fulfilled Amsterdam I criteria with those considered 

to have sporadic CRC 

• ⇔ Comparing Amsterdam I to Japanese A criteria 

• Å Comparing Amsterdam I to Japanese B criteria 

• ⇔ Comparing MSH2 and/or MLH1 status by IHC in patients with sporadic 

CRC 

• ⇔ Comparing MSI-H to MSI-L/MSS CRC 

• ⇔ 5-year cumulative survival comparing patients with endometrial cancer 

who were mutation positive to those who were mutation negative. 

• ⇔ Comparing stage III CRC patients receiving adjuvant chemotherapy 

with MSI tumors to those with MSS/MSI-L. 

Å Statistically increased ⇑ Increased, but not statistically significant 

⇔ No statistically significant differences 

Æ Statistically decreased ⇓ Decreased, but not statistically significant 

a

 Only two studies reported the study duration 

Gaps in the Literature. Some studies described the management of patients with HNPCC but 

surprisingly, there were no data directly exploring how MMR mutation status influenced the 

choice among these management options or their outcomes. As a result, the available data do not 

directly answer whether knowledge of MMR mutation status changes management decisions by 

patients and providers or whether changes in management for MMR positive patients improves 

their outcomes compared to standard CRC management. Issues related to MMR mutation testing 

in influencing decisions to undergo cancer screening procedures is discussed below with Key 

Question 10.
112 

Table 22. Key Question 6b. Does the identification of HNPCC mutations lead to improved patient outcomes in terms of early dmortality/morbidity or management decisions (e.g., counseling, surveillance, treatment, other decision making) by patients and Author, year Country 

Study 

Design 

(f/up 

duration) 

Target population for 

genetic testing 

(Eligible/Enrolled N) 

N 

evaluated 

% 

Positive 

HNPCC 

test 

Description 

of HNPCC 

tests 

% Received 

interventions or 

treatments 

Ou

Mutation testing 

Group A: 

361 100% 

Group B: 

241 0% 

Benatti, 2001 Italy 

Retro 

(8.2, 6.7, and 

9.4 yrs for 

group A, B, 

and C 

respectively) 

29 HNPCC families met inclusion 

criteria: 10 carried MMR gene 

mutations (Group A), 10 were 

characterized by MSI phenotype 

but not by MMR gene mutation 

(Group B), and 9 did not show 

mutations or MSI (Group C) Group C: 355 0% 

MMR gene 

mutation ND Prognosis of CRC 

Boks, 2002 Netherlands 

Retro 

(ND) 

Patients with endometrial cancer 

from 46 HNPCC families with a 

mutation or that met revised 

Amsterdam criteria 

(ND/66) 

50 75% MMR gene mutation ND 

⇔ 5-year cum

Clinical criteria and laboratory testing Bertario, 1999 Italy 

Retro 

(60 mo) 

3 CRC groups: 

144 HNPCC 

161 FAP 

2035 sporadic 

(ND/2,340) 

2,340 5% Amsterdam criteria 

“Adjuvant-treatment 

protocols were the 

same b/w the 3 

groups” 

HNPCC and FAP 

patients underwent 

periodic examinations 

for different “spectrum 

of the diseases” 

⇔ Survival b/w and sporadic Cadjusting for aand tumor locaHNPCC was 11.39) compare
113 

Table 22. Key Question 6b. Does the identification of HNPCC mutations lead to improved patient outcomes in terms of early dmortality/morbidity or management decisions (e.g., counseling, surveillance, treatment, other decision making) by patients and Author, year Country 

Study 

Design 

(f/up 

duration) 

Target population for 

genetic testing 

(Eligible/Enrolled N) 

N 

evaluated 

% 

Positive 

HNPCC 

test 

Description of 

HNPCC tests 

% Received 

interventions or 

treatments 

Outcomes 

8 /1000 Amsterdam I criteria 

17 /1000 Japanese criteria A Fujita, 1996 Japan Retro (ND) CRC patients who underwent surgery (ND/3,356) 1785 

56 /1000 Japanese criteria B 

100% surgery 

5-year survival rate (30 day mor

Amsterdam I=92.3% Japanese A=81.2% Japanese B=66.5% Sporadic=60% 

Better survival for Amsterdam a

Japanese B and Sporadic gr

Tomoda 1996 Japan 

Retro 

(12-153 mo) 

Nonpolyposis CRC 

patients who underwent 

resection 

(ND/1042) 

1042 3.7% Japanese criteria B 100% resection 

Mean age was significantly youn

meeting Japanese criteria (ocases who didn’t (55.9 yr vs. 

Å Metachronous (postoperative

cases with HNPCC to those (10.2% vs. 3.5%, p=0.0001) 

Å Survival, compared cases wi

without HNPCC (p=0.02) 

⇑ Survival, compared cases wit

without HNPCC with stage II

Perrin, 2001 France 

Retro 

(ND) 

Patients diagnosed with 

sporadic CRC 

(ND/225) 

208 13% 

Lack of 

expression of 

MSH2 and/or 

MLH1 by IHC 

ND 

⇔ Disease-free survival compa

MLH1 status by IHC. Subpoptumors, MSH2 and/or MLH1 associated with longer disea

19 100% Sporadic MSI-H tumors 

15 100% 

Hereditary 

MSI-H tumors b Kruhoffer, 2005 

Denmark 

Retro 

(ND) 

Stages II and III CRC 

patients with sporadic 

MSI, hereditary MSI, or 

MSS a tumors 

(151/101) 67 0% MSS or MSI-L tumors 

65 patients with 

Stage III tumors 

receiving adjuvant 

chemotherapy 

Overall survival highly significan

classification in 36 Stage II ppatients who died ≤ 5 years MSS/MSI-L group (p=0.0014

16 classified as MSI and 49 as M

65 Stage III patients receivinchemotherapy. 

⇔ overall su

MSI and 30 MSS/MSI-L patie

f/up (p=0.55) 

FAP=familial adenomatous polyposis Å Statistically increased 

⇑ Increased, but not statistically significant 

⇔ No statistically significant differences Æ Statistically decreased 

⇓ Decreased, but not statistically significant 

a The definition of MSS in the study was Including MSI-L. b MSI tumors with HNPCC origin were classified by a maximum likelihood MSI classifier with a “leave-one-out” cross validation scheme basically as 
114 

Key Question 7: What Are the Harms Associated With Subsequent 

Management Options After Identification of HNPCC Mutations in CRC 

Patients? 

HNPCC mutation 

negative Patient

Outcomes

Medical

Decision 

making

HNPCC

mutation

positive

6,7

Intervention

 Due to limited data on harms associated with subsequent management options or 

interventions, we broadened the scope of this question to include studies evaluating all forms of 

cancer related to HNPCC, since patients with CRC from HNPCC families are potentially at risk 

for all forms of HNPCC-related cancers. We also included studies that reported any outcome 

relating to subsequent management options or interventions in these patients. 

 Two retrospective studies of B and one of C quality reported outcomes related to subsequent 

actions or interventions in patients with CRC and gastric cancers, respectively (Table 23).110,116

The validity of the study of C quality (gastric cancers) is questionable because of unclear 

participation rates and an unclear description of how survival/death was ascertained. 

Summary of Findings. No study described harms associated with subsequent management 

options after identification of HNPCC mutations in patients with CRC or other forms of 

HNPCC-related cancers.

 One study (involving two centers) described the types of colorectal surgery performed on 

CRC patients who were part of an Amsterdam criteria-positive family, and compared rates of 

metachronous cancers that followed each type of index operation.110 The overall rate of second 

surgeries for metachronous cancer were 23% in patients who underwent right colectomy, 17% in 

patients who underwent left/sigmoid colectomy or proctosigmoidectomy, 0% in patients who 

underwent total/subtotal colectomy, and 44% in patients who underwent segmental colectomy. 

The two centers had significantly different second resection rates for metachronous cancer. 

 One study described the survival rate of 45 patients with gastric cancer from HNPCC 

families with MMR mutations.116 Many of these patients had already had treatments for other 

HNPCC-related cancers, including CRC. The 5-year survival was higher in patients in whom 

radical surgery was performed (48%) than in patients in whom radical palliative surgery or 

explorative laparotomy alone was performed (15%). 

Gaps in the Literature. There are no data on harms associated with surgical procedures or 

chemotherapy in CRC patients with HNPCC, although it is likely that such patients encounter 

the same spectrum of harms associated with chemotherapy and surgery as patients with CRC (or 

other forms of HNPCC-related cancer) without HNPCC. One cannot assume “no harm” when a 

study did not report data on harms. 

 There were also no data on whether cancer patients with MMR mutations derive a greater 

benefit from specific management options compared with patients without MMR mutations. 
115 

However, several studies have suggested a relatively favorable prognosis of colorectal cancer in 

patients with HNPCC or those who have clinical features associated with HNPCC such as 

tumors that are MSI-H.117-120 These data have also suggested that patients with colorectal cancer 

that demonstrate MSI may not derive a benefit from adjuvant chemotherapy with 5-fluorouracil 

(5-FU), possibly because of a higher cure rate with surgery alone and/or intrinsic resistance to 5-

FU.120 

 However, a 2004 consensus statement issued by the American Society of Clinical 

Oncology121 found insufficient evidence to recommend modification of overall treatment 

recommendations in patients with stage II colorectal cancer with a known mismatch repair 

mutation or tumors that demonstrate MSI, noting the retrospective and inconclusive nature of the 

existing evidence. The report cites an ongoing European trial (FOLFIRI), which is comparing 

irinotecan and 5-FU and is stratifying patients based on tumor MSI status, and a United States 

Intergroup trial that is also incorporating MSI status as a prognostic factor. The final results of 

these trials have not been published. 

 Our review of the literature did not provide additional strong evidence in favor or against the 

use of adjuvant chemotherapy in patients with HNPCC of any stage. However, our literature 

search strategy excluded studies with patients who had tumors that demonstrated MSI unless the 

study performed additional testing for HNPCC (either by clinical criteria, IHC, or genetic 

testing). Thus, the literature search was not comprehensive for establishing the relationship 

between tumor MSI status and the outcome of adjuvant chemotherapy. The ongoing trials 

described above will help clarify this issue (as well as the role of other biologic markers 

proposed for guiding adjuvant chemotherapy in patients with colorectal cancer). 
 116 

Table 23. Key Question 7. What are the harms associated with subsequent management options after identification of HNPCC in CRC patients? Author, year Country 

Study 

design 

(Follow-up 

duration) 

Target population 

(Eligible/Enrolled N) 

% with 

CRC at 

baseline 

N 

evaluated 

% who 

took 

actions 

Description of 

subsequent actions 

or interventions 

Harms Outco

Van Dalen 2003 US 

Retro 

(1-49 yr) 

CRC patients from 39 

HNPCC families met 

Amsterdam I criteria 

(ND/93) 

100% 93 100% 

33% received 

RC, 14% L/SC, 

18% PSC, 25% 

TC, and 10% 

SGC 

ND 

2nd 

resections 

rate 

7

rec

pa

L/S

pa

TC

r

36% Radical surgery 

Aarnio 1997 Finland 

Retro 

(ND) 

Patients with gastric 

cancers from 51 

HNPCC families by 

Amsterdam I or with a 

known MMR mutation 

(ND/45) 

40%a 45 

63% 

Palliative surgery 

or explorative 

laparotomy alone 

with no surgery 

ND 5-year survival 

RC=Right colectomy; L/SC=Left/sigmoid colectomy; PSC=Proctosigmoidectomy; TC=Total/subtotal colectomy; SGC=Segmental colectomy.a 18 (40%) already had been treated for CRC (13 cases), endometrial cancer (2 cases), ovarian cancer (1 case), urinary-tract cancer (1 case) and testicu
117

 Key Questions 8 to 11 summarize what is known about the processes and benefits of 

bringing family members into the testing process. Knowing that a CRC patient is MMR mutation 

positive has implications for family members. As a general rule, family members receive 

counseling and, subsequently, genetic testing for MMR mutations ideally based upon the results 

of MMR testing in the proband. 

Key Question 8b: What is the Accuracy of HNPCC Testing in Family 

Members in Predicting the Risk of CRC? 

Key Question 8c: Do Other Factors, Such as Race/Ethnicity, Age, 

Gender, or Co-Morbidities Affect the Accuracy of the Testing? 

Kindreds with

HNPCC

Family members of HNPCC 

positive CRC patients

Genetic Counseling 

HNPCC testing

HNPCC

mutation

positive

8,9

HNPCC

mutation

negative

HNPCC

mutation

positive

Risk

assessment

 The analytic framework proposed above greatly restricted the pool of potentially eligible 

studies since it required that CRC probands were proven to have a MMR mutation. As a result, 

we broadened the scope of our search to include studies using clinical criteria, laboratory testing 

or mutation testing in predicting the risk of CRC or extracolonic cancers in family members of 

CRC probands with HNPCC (based on clinical or genetic criteria) and/or kindreds with HNPCC 

(based on clinical or genetic criteria). We considered the findings in such studies to be applicable 

to the Key Question. Any measure of relative risk (e.g., the standardized incidence ratio) was 

included as indirect evidence for the accuracy of the testing. 

 We summarize the findings for family members of CRC probands with HNPCC (in Table 

24) and for kindreds with HNPCC (in Table 25) separately, because the risk of CRC and/or 

extracolonic cancers is different between the two groups. 

Six studies reported the accuracy of HNPCC testing in family members of CRC probands 

(based on clinical or genetic criteria) in predicting the risk of CRC20,122-126 (Table 24). Of these, 

only two (both of quality B) reported the accuracy of MMR mutation testing in family members 

of CRC probands with a positive MMR mutation.20,126The other four reported the accuracy of 

HNPCC testing in family members of CRC probands with HNPCC based on clinical criteria122-

125 Half studies were of B quality and half were of C quality. These studies were limited by 

verification bias, selection bias, unclear follow-up duration, and/or bias from post-hoc analyses. 

Six studies indirectly reported the accuracy of the HNPCC testing in kindreds with HNPCC 

(based on clinical or genetic criteria)127-132 (Table 25). Of these, two were of B quality and four 
118

were of C quality. These studies were limited by selection and verification bias. Most studies did 

not adjust for familial clustering of cancers. 

Summary of Findings. Only two studies of quality B reported the risk of CRC in family 

members of probands with positive MMR mutations (the proposed framework). The lifetime risk 

of CRC was 68.7% for men and 52.2% for women with MMR mutations in one study,20 and it 

was 74% and 30% respectively in the other study.126 Men had a higher lifetime risk of CRC in 

both studies. 

 In a study that reported the risk of CRC in family members of probands with HNPCC based 

on clinical criteria, the cumulative risk of CRC by age 75 years old was 57% and 41% in families 

that fulfilled the Amsterdam I and II criteria, respectively.122 The cumulative risk of CRC by age 

75 years old was 42% and 23% for men and women, respectively, from families that fulfilled 

Bethesda criteria. In another study123 family members of CRC probands who were younger than 

50 years old at cancer diagnosis had a higher risk of CRC, compared to family members of CRC 

probands who were 50 years old and older. The risk of CRC was increased three-fold, comparing 

first-degree relatives of CRC probands who developed a second primary in the HNPCC spectrum 

with the single primary group in a third report124 (Overview Table 2; Table 24). 

 In a study that reported the risk of CRC in kindreds with HNPCC based on genetic criteria, 

the cumulative risk of CRC by age 70 yr was 82% in MLH1/MSH2 mutation carriers.132 The 

cumulative incidence of CRC was 100% in men and 54% in women. There was overall a higher 

risk of CRC in men but the sex difference was not consistent among HNPCC kindreds with 

different MMR mutations (Overview Table 2; Table 25). 

Overview Table 2. Key Questions 8b and 8c 

Reported Outcomes 

Body of 

evidence 

(study duration) 

Summary 

CRC risks in family members of 

probands with positive MMR 

mutations; factors that influenced 

the accuracy of testing

2 Retro studies 

(ND)

• In a Finland population, lifetime risk of CRC was 

68.7% for men and 52.2% for women with MMR 

mutations. Mean age of diagnosis of CRC 55.1 for 

men and 60.3 for women. 

• In a Scottish population, cumulative risk of CRC (95% 

CI) by age 70 yr was 74% for men and 30% for 

women with MMR mutations (P=0.0066). The risk of 

CRC was significantly higher at all ages in men than in 

women. 

CRC risks in family members of 

probands with HNPCC based on 

clinical criteria; factors that 

influenced the accuracy of testing

1 Prosp study (2 

yr) and 3 Retro 

studies 

(8 yr)a

• Cumulative risk of CRC (95% CI) by age 75 yr:

Am I families=57.1% (46-68.8%) 

Am II families=41.2% (33.3-50.1%) 

Bethesda families= 

 Men 41.7% (39.4-44.1%) 

 Women 23.3% (22.2-24.4%) 

• SIR of CRC (95% CI): 

Å Proband<50 yr; MSI = 21(12-35) 

Å Proband<50 yr; MSS = 7 (3-14) 

⇔ Proband>50 yr = 1.07 (0.74-1.40) 

• 8% in the group fulfilled Amsterdam I criteria had 

adenomas, while 2% in the group with family history of 

CRC only had adenomas. 

• Å CRC risk, compared 1st degree relatives of CRC 

probands who developed a second primary in the 

HNPCC spectrum with the single primary group 

(RR=3.2, p<0.00001) 
119

Overview Table 2. Key Questions 8b and 8c (continued) 

Reported Outcomes 

Body of 

evidence 

(study duration) 

Summary 

CRC risks in kindreds with 

HNPCC based on clinical criteria 

and/or genetic criteria; factors that 

influenced the accuracy of testing

6 Retro studies 

(ND) 

• Cumulative risk of CRC by age 70 yr was 82% in 

MLH1/MSH2 mutation carriers, compared with only 

1.6% in the Finnish population as a whole.

• Cumulative risk of CRC by age 60 yr:

MLH1 mutation (+): 84% - 94% in men compared to 

63% in women (p=NS) 

MSH2 mutation (+): 71% - 96% in males compared to 

39% in women (p=0.034)

• SIR 95%CI of CRC in HNPCC families based on 

genetic criteria:

MLH1/MSH2 mutation (+): 68 (56–81) 

- SIR was higher in men (83) than in women (48) 

• SIR and 95%CI of CRC in HNPCC families meeting 

either Amsterdam or Bethesda criteria:

Mutation (-): 158.61 (132.8–189.4) 

MLH1 mutation (+): 196.76 (143.0–270.7) 

MSH2 mutation (+): 134.24 (99.1–181.8)

• Comparing family members with pathogenic MSH6 

mutations to those with MLH1/MSH2 mutations: Æ Prevalence of CRC 

Æ Cumulative risk by age to develop CRC or any 

tumor 

• Compared MSH2 carriers to MLH1 carriers:

Å Risk of CRC (p=0.13) 

• Relative risk of developing adenoma before age 54 yr 

compared to reference groupb

:

Å MMR mutation carriers (MSH2, MLH1, or hMSH6)

(RR=4.4. p<0.001)

• Å Risk of CRC comparing male to female MSH2 

mutation carriers (p<0.01) 

• ⇔ Risk of CRC comparing male to female MLH1 or 

MSH6 mutation carriers 

• Comparing family members with pathogenic MSH6 

mutations to those with MLH1/MSH2 mutations:

Å Median age of CRC onset (54 vs. 44 yr) 

• Å Age at diagnosis compared MMR mutation (+) to 

mutation (-) (+ 5 yr) 

• ⇔ Age at diagnosis compared MSH2 mutation (+) to 

MLH1 mutation (+), adjusted for familial clustering. 

• Proportion of patients younger than 50 found to have 

an adenoma: Å Amsterdam I 

SIR=standardized incidence rate. 

Å Statistically increased ⇑ Increased, but not statistically significant 

⇔ No statistically significant differences 

Æ Statistically decreased ⇓ Decreased, but not statistically significant a

 Only one study reported the duration of follow-up. 

b

 Reference group was based on three published forensic autopsy studies used for estimates of adenoma prevalence in the normal 

population. 

Gaps in the Literature. We found only limited evidence on the accuracy of MMR mutation 

testing in predicting the risk of HNPCC-related cancers in family members of CRC probands. 

Only two studies based such predictions on proven MMR mutations in the proband.20,126 The 

study by Hampel20 found that cancers in family members developed at a later age compared with 
120

previous reports that had used clinical criteria to establish the diagnosis of HNPCC, suggesting 

that additional studies are required to fully understand penetrance in family members of CRC 

probands who carry MMR mutations. 

 No study directly examined factors that influenced the accuracy of the HNPCC testing, 

although some found significant differences in the risks of CRC among HNPCC kindreds with 

different MMR mutations. More studies are needed to fully understand the relationships among 

specific MMR mutations and cancer risk in probands with HNPCC-related cancers and their 

family members. 
121

Table 24. Key Question 8b. What is the accuracy of HNPCC testing in family members in predicting the risk of CRC? Key Que

factors, such as race/ethnicity, age, gender, or co-morbidities affect the accuracy of the testing? Author, year Country 

Study 

design 

(follow-up 

duration) 

Target population for HNPCC 

test 

(Eligible /Enrolled N) 

N 

evaluated 

% 

Positive 

HNPCC 

test 

Description of 

HNPCC tests 

Accuracy of HN

predicting risk of C

related cancers / F

the accuracy 

Family members of CRC probands with HNPCC based on genetic criteria Hampel 2005 Finland 

Retro 

(ND) 

Probands and 373 mutationpositive family members from 70 

HNPCC families 

(ND/461) 

461 100% 

Mutations (MLH1 or 

MSH2) and clinical 

criteria, mostly 

Amsterdam I or II 

Lifetime risk of CRC w

and 52.2% for wom

Lifetime risk of endom

approximately 54% 

Mean age of diagnos

CI 52.6-57.6)) for m58.0-62.6) for wom10-15 years older testimates of age aamong HNPCC pa

Dunlop 1997 Scotland 

Retro 

(ND) 

Relatives of 6 probands with 

MMR gene mutations (from the 

Scottish National Cancer 

Registry) 

(156/156) 

156 43% Mutations (MLH1 or MSH2) 

By 70 years of age, th

was 74% while the 30% (p=0.0066). 

The risk of CRC was 

all ages in men tha

In females, the risk of 

exceeded the risk years, giving an es70 years. 

Family members of CRC probands with HNPCC based on clinical criteria 

0.04 /1000 Amsterdam I 0.07 /1000 Amsterdam II 

Bermejo 2005 Sweden 

Retro 

(ND) 

Families from Swedish FamilyCancer Database with ≥4 

generations of cancer. No age 

restrictions for parents but 

maximum age in 2nd generation 

was 70 year. 

(ND/566,877) 

566,877 

families 

8.99 /1000 Bethesda 1-5 

Cumulative risk of CR

75 yr: 

Am I families=57.1Am II families=41.Bethesda families Men 41.7% (39.4 Women 23.3% (2

Cumulative risk of en

(95% CI) by age 7

Am II families=45.Bethesda families

Cumulative risk for ov

CI) by age 75 yr: 

 Bethesda group=4 Gastric cancer= 
122

Table 24. Key Question 8b. What is the accuracy of HNPCC testing in family members in predicting the risk of CRC? Key Que

factors, such as race/ethnicity, age, gender, or co-morbidities affect the accuracy of the testing? (continued) Author, year Country 

Study 

design 

(follow-up 

duration) 

Target population for HNPCC 

test 

(Eligible /Enrolled N) 

N 

evaluated 

% Positive 

HNPCC 

test 

Description of 

HNPCC tests 

Accuracy of HN

predicting risk of C

related cancers / F

the accuracy 

23% Proband<50 yr; MSI+ 

Å CRC risk (SIR =21Å All cancer risk (SIR 

4.5) 

24% Proband<50 yr; MSS 

Å CRC risk (SIR =7; Å All cancer risk (SIR 

3.2) 

18% Proband>50 yr; MSI+ 

⇔ CRC risk ⇔ All cancer risk 

Cederquist, 2001 Sweden 

Retro 

(8 yr) 

1st degree family members of 36 

colon-endometrial caner 

probands and 43 colon-colon 

cancer probands. 

(ND/649) 

649 

34% Proband>50 yr; MSS 

⇔ CRC risk ⇔ All cancer risk 

Multiple primary group: 1st

degree relatives of CRC 

probands, who developed a 

second primary in the HNPCC 

spectrum1

(166/157) 

128 8% 

Brown 1998 

UK 

Retro 

(ND) 

Single primary group: 1st degree 

relatives of single CRC patients 

(595/444) 

444 0.7% 

Amsterdam I and 

modified criteria 

Å CRC risk, compare

primary group (RR

Single primary group

associated and noextracolonic cancefrequency. 

Multiple primary grou

HNPCC cancers hfrequency howeveextracolonic cancefrequency of gener

28% Amsterdam I (or high risk) 64% 

Family history of 

CRC only 

(moderate risk) 

Bradshaw, 2003 UK 

Prosp 

(2 yr) 

Asymptomatic individuals with a 

family history of CRC at a young 

age who received colonoscopy 

(448/190) 

163 

8% Low risk 

Colonoscopy was com

in 92%. 

No cases of CRC we4 patients (8%) in hig

adenomas. 

In moderate group, ov

had an adenoma 

Proportion of patients 

found to have an asignificantly greategroup than those in group (p=0.05). 

1 CRC, stomach, urinary, ovary, or endometrial cancers 
123

Table 25. Key Questions 8b and 8c in kindreds with HNPCC based on clinical and/or genetic criteria Author, year Country 

Study 

design 

(follow-up 

duration) 

Target population for HNPCC 

test 

(Eligible /Enrolled N) 

N 

evaluated 

% Positive 

HNPCC 

test 

Description of 

HNPCC tests 

Accuracy of HNPCC testin

of CRC or other HNPCC

Factors that affected th

testing 

Mutation testing Aarnio, 1998 Finland 

Retro 

(43 yr) 

50 HNPCC families in which a 

MLH1 gene mutation (47 

families) or MSH2 gene 

mutation (3 families) had been 

detected 

(1,763/1,763) 

1,763 100% MLH1/MSH2 

In mutation carriers, cumulat

70 years of age were 82% endometrial cancer, compand 1.3%, respectively, in population as a whole. 

The cumulative incidence of 

men and 54% in women. 

The cumulative incidence for 

13% and, for ovarian cancuroepithelial, kidney and bfor brain tumors, the cumuranged from 2 to 4% by 70 

18% MLH12

Scott, 2001 Australia 

Retro/ 

Prosp 

(ND) 

HNPCC families meeting either 

Amsterdam (34%) or Bethesda 

(66%) criteria 

(ND/95) 

95 families 

16% MSH2f

Relative standardized incide

of CRC: 

 Mutation (-): 158.61 (132.8 MLH1 mutation (+): 196.7 MSH2 mutation (+): 134.2Compared MSH2 mutation–p

MLH1 mutation–positive g

⇔ Trend for lower CRC rat⇔ Frequency of extracolon

Malignancies were overrepre

compared with expected fpopulation 

⇔ For age at diagnosis com

mutation–positive group to positive group, adjusted fo(MSH2 mutation-positive fMLH1 mutation-positive fa

Å For age at diagnosis comp

positive group to mutationyr) 

2 Genetic analysis was performed on a fresh blood specimen from the youngest living affected proband in each family. 
124

Table 25. Key Questions 8b and 8c in kindreds with HNPCC based on clinical and/or genetic criteria (continued) Author, year Country 

Study 

design 

(follow-up 

duration) 

Target population for HNPCC test 

(Eligible /Enrolled N) 

N 

evaluated 

% Positive 

HNPCC 

test 

Description of 

HNPCC tests 

Accuracy o

predicting r

HNPCC-related 

affected the ac

80% MLH1/MSH2 

Plaschke, 2004 Germany 

Retro 

(ND) 

HNPCC families meeting Amsterdam I or II, plus 

Bethesda criteria. Selected members with MSIlow or -high, and abnormal IHC, plus 

MLH1/MSH2 mutation-positive, or MSH6 

mutation-positive 

(706/183 families) 

1,974 

20% MSH6 

Compared family 

pathogenic MSwith MLH1/MS

Æ Prevalence (29% vs. 37.5%Æ Cumulative CRC or any tum

Compared family 

pathogenic MSwith MLH1/MS

Å Median age 

44) 

Å Median age 

vs. 43) 

Vasen, 2001 Netherlands & Norway 

Retro 

(ND) 

Families with HNPCC meeting Amsterdam I or II 

or high suspicion of HNPCC 

(193/193) 

138 

families 25% MLH1 

Compared MSH2 

carriers: 

Å Lifetime risk any age (p<0.0Å Risk of CRC Å Risk of devecancer (p=0.05Å Risk of deveurinary tract by Å Risk of CRC female MSH2 m(p<0.01), 

⇔ fo

mutation carrie

Mean age of CRC 

carriers of MSor MSH2 (50 vrespectively), snot reported 
125

Table 25. Key Questions 8b and 8c in kindreds with HNPCC based on clinical and/or genetic criteria (continued) Author, year Country 

Study 

design 

(follow-up 

duration) 

Target population for HNPCC test 

(Eligible /Enrolled N) 

N 

evaluated 

% Positive 

HNPCC 

test 

Description of 

HNPCC tests 

Accuracy of HNPCC 

risk of CRC or other 

/ Factors that affect

tes

Lin, 1998 US 

Retro 

(ND) 

22 females and 27 males from 2 kindreds 

were identified with MSH2 mutation; 28 

females and 28 males from 2 kindreds were 

identified with MLH1 mutations 

(ND/105) 

105 100% MLH1/MSH2 

The cumulative inciden

was 84% in MLH1 acarriers (p=NS) 

The cumulative inciden

was 96% in MSH2 min MSH2 females (pMLH1 males compafemales (p=NS) 

The cumulative inciden

cancers by age 60 w48% in MSH2 carrie

Other HNPCC testing 

15% HNPCC, at 70% risk316% HNPCC, at 35% risk5

2% HNPCC, at 17% risk68% HNPCC, at 5% risk7

Lindgren, 2002 Sweden 

Retro 

(ND) 

Patients who were undergoing colonoscopy 

surveillance were classified as having 

HNPCC, defined as meeting Amsterdam 

criteria, germline mutation, or MSI, HCC, 

TCR, or OCR 

(ND/304) 

304 

60% HCC, TCR, or OCR 

Relative risk of develop

age 54 yr compare

Å HNPCC, at 70% 

p<0.001) 

Å TCR group (RR

No influence of sex on 

developing adenom

Å Statistically increased ⇑ Increased, but not statistically significant ⇔ No statistically significant differences Æ Statistically decreased ⇓ Decreased, but not statistically significant 3 Germline mutation carriers (MSH2, MLH1, or hMSH6). 4 Reference group was based on three published forensic autopsy studies used for estimates of adenoma prevalence in the normal popu5 Fist generation of HNPCC families. 6 Second generation of HNPCC families. 7 Subjects in HNPCC families who were under surveillance before testing but tested negative for mutation. 
126 

Key Question 9: What Are the Harms Associated With Informing/ 

Counseling Family Members or With Subsequent Testing for HNPCC 

Mutations? 

Key Question 8a: What is the Efficacy of Pre-Test Genetic Counseling 

for Informing Family Members of Potential Risks and Benefits of 

Testing? 

Family members of HNPCC 

positive CRC patients

Genetic Counseling 

HNPCC testing

HNPCC

mutation

positive

8,9

HNPCC

mutation

negative

HNPCC

mutation

positive

 

 Studies were considered eligible for Key Question 8a if they summarized the efficacy of pretest genetic counseling immediately after counseling and before performing genetic testing. 

Studies addressing Key Question 9 were considered together with those addressing Key Question 

8a because there was substantial overlap in the studies addressing these questions. The studies 

addressed the long-term efficacy of pre-test genetic counseling or harms associated with 

screening high-risk individuals (such as by using clinical criteria, MSI, or IHC), genetic testing, 

and informing/counseling family members or with subsequent testing for HNPCC mutations. 

 Four studies addressed the efficacy of pre-test genetic counseling immediately after 

counseling and before performing MMR genetic testing107,133-138 (Table 26). Of these, three were 

of B quality and one was of C quality. 

 Eight comparative and four qualitative studies addressed the harms associated with genetic 

testing for HNPCC mutation or with informing/counseling family members or with subsequent 

testing for HNPCC mutations4,104-106,133-144 (Table 27). Of these, one study was of A quality, six 

of B quality, and five of C quality. 

Summary of Findings. Overall pre-test genetic counseling had good efficacy in improving 

knowledge about HNPCC, and resulted in high likelihood of proceeding with genetic testing, 

satisfaction in decision to undergo genetic testing, and decreasing in depression and distress 

levels among family members of HNPCC probands or among asymptomatic individuals from 

HNPCC families. Family members of HNPCC who received positive MMR mutation test results 

had higher psychological distress levels and anxiety compared to those who received negative 

test results, but this difference generally disappeared with time. However, all of these 

psychological measures were within the normal ranges for the general populations. Available 
127 

evidence did not show differences in quality of life, comparing mutation carriers to non-carriers 

or the general population (Overview Table 3; Table 26-27). 

Overview Table 3. Key Questions 8a and 9 

Reported 

Outcomes 

Body of 

evidence 

(study 

duration) 

Summary 

Decision to genetic 

testing 

2 Prosp 

studies 

(1 mo to 1 yr) 

• 1%-2% family members of HNPCC enrolled in the studies opted not to 

undergo genetic testing. 

Shortened vs. 

extended pre-test 

counseling 

1 RCT 

(3 mo) 

• High satisfaction in both groups. 

• ⇔ Decision making outcomes or knowledge about HNPCC between 

groups. 

• Mutation carriers had higher psychological distress in extended pretest counseling group but lower psychological distress in shortened 

pre-test counseling group, compared to non-carriers. 

Satisfaction in 

decision to undergo 

testing 

1 Prosp study 

& 1 RCT 

(1 mo to 1 yr) 

• No short-term difference in satisfaction in decision to undergo genetic 

testing between carriers and noncarriers, but MMR mutation carriers 

were less satisfied in decision to undergo testing 1 year after testing. 

Anxiety 6 Prosp 

studies 

(2 wk to 1 yr) 

• Æ Anxiety levels immediately after genetic counseling 

• Anxiety levels of HNPCC family members were within normal ranges 

compared to the general population. 

• Generally there was no change or decrease in anxiety level over-time 

among non-carriers, while there was no change or increase in anxiety 

level among mutation carriers over-time. 

• MMR mutation carriers had higher anxiety levels than non-carriers 

shortly after genetic test results were revealed, but this difference 

generally disappeared with time. 

Depression 5 Prosp 

studies 

(2 wk to 1 yr) 

• Æ Depression levels immediately after genetic counseling 

• Depression levels of HNPCC family members were within normal 

ranges compared to the general population. 

• Conflicting results on changes in depression levels over-time 

• Conflicting results comparing MMR mutation carriers to non-carriers 

Distress 5 Prosp 

studies & 1 

RCT 

(2 wk to 1 yr) 

• Psychological distress, cancer-specific distress (including Intrusive and 

avoidant thoughts about CRC or cancer worries), and distress from 

receiving test results generally decreased with time. 

• In general, MMR mutation carriers had higher cancer-specific distress 

levels than non-carriers. 

Quality of life 2 Prosp 

studies 

(2 wk to 1 yr) 

• QOL of HNPCC family members were within normal ranges compared 

to the general population. 

• ⇔ QOL compared MMR mutation carriers to non-carriers 

Å Statistically increased ⇑ Increased, but not statistically significant 

⇔ No statistically significant differences 

Æ Statistically decreased ⇓ Decreased, but not statistically significant 

Gaps in the Literature. The results described above apply only to individuals who agreed to 

participate in the genetic studies and opted for genetic testing. Many individuals from HNPCC 

families chose not to participate in the study or pre-test genetic counseling. The reasons were 

often not described; however, they included fear of knowing the results, fear of insurance 

discrimination, survivor’s guilt and disinterest. 

 The literature did not provide what key elements should be included in genetic counseling, 

and the effectiveness of different counseling strategies. Only a small RCT compared the efficacy 

of shortened to extended pre-test counseling and found no differences in psychological and 

decision making outcomes or knowledge about HNPCC between groups immediately after 
128 

counseling and before revealing the results of genetic testing.133 At 3-month follow-up, mutation 

carriers in the shortened group experienced the least distress along with a high level of decision 

satisfaction, in contrast to carriers in the extended group who reported the most distress and least 

decision satisfaction. However, there were no details regarding baseline familiarity, education 

level and other potentially relevant characteristics across study groups to assure randomization 

was successful. 

 Some studies examined the factors that influenced decisions regarding genetic testing or the 

reasons for pursuing or for not pursuing genetic testing.134-138,145-151 Although these studies (most 

of which were descriptive) were not specifically relevant to any of the Key Questions, they were 

clinically relevant and thus we attempted to summarize them in ancillary tables (Extra Table 1). 

People who were employed, who had higher education levels, who had CRC screening, and who 

had familial cancers were more likely to accept genetic testing. 

 Common reasons for not pursuing genetic testing include worry about losing health 

insurance, and concerns about coping with the emotional reactions if the test were positive. 

Women appeared to need more professional psychological support for coping with the results of 

genetic testing. These findings are important considerations for genetic counseling programs and 

support systems to minimize the harms associated with genetic testing for HNPCC mutations. 

 Findings from two surveys of genetic centers or insurance providers suggested the need for 

more empirical research on which components of pretest counseling are effective in promoting 

good psychological outcomes and the need to prevent insurance discriminations to the persons 

with genetic risk of CRC152,153 (Extra Table 2). 
129

Table 26. Key Question 8a. What is the efficacy of pre-test genetic counseling for informing family members of potential risks Author, year Country 

Study 

design 

(follow-up 

duration) 

Target population for genetic testing 

(Eligible /Enrolled N) N evaluated Efficacy of pre-test gene

Atkan-Collan 2000; 2001a; 2001b; 2001c Finland 

Prosp 

(1 mo & 1 yr) 

Family members from 36 HNPCC 

families with known mutations at 50% 

risk 

(446/401) 

334 

At 1 year, pretest counseliconsidered: 

Very useful: 49% Fairly useful: 40% Slightly useful: 10% 

7 subjects (2%) not opting testing 

Arver, 2004 Sweden 

Prosp 

(1 yr) 

Healthy women from 73 of 80 families 

with an identified HNPCC or BRCA1, 

BRCA2 mutation 

(114/90) 

87 

1 subject (1%) refused genbecause of worries about employment 

Keller, 2002 Germany 

Prosp 

(4-6 wk) 

Unaffected individuals from families at 

risk for HNPCC 

(ND/34) 

34 

Æ Anxiety (HAD) Æ Depression (HAD) ÆÆ Cancer-specific distre

All: 27 

Brain, 2005 UK 

RCT 

(3 mo) 

Unaffected individuals from families with 

a known HNPCC mutation 

(51/34) 

15 in shortened; 11 in 

extended pre-test 

counseling 

High satisfaction in both g⇔ In psychological & decioutcomes or knowledge abbetween groups. 

Å Statistically increased 

⇑ Increased, but not statistically significant (or statistical analysis was not performed) 

⇔ No statistically significant differences Æ Statistically decreased 

⇓ Decreased, but not statistically significant (or statistical analysis was not performed) 
130

Table 27. Key Question 9. What are the harms associated with informing/counseling family members or with subsequent testHNPCC mutations? 

Observed impact (Key 

Author, year Country 

Study 

design 

(follow-up 

duration) 

Target population for 

genetic testing 

(Eligible /Enrolled N) 

N 

evaluated 

% 

Mutation 

positive 

Measures of 

psychological impact 

after revealing 

genetic testing 

results 

Test 

receivers 

vs. 

normal 

ranges8

Follow-up 

vs. baselin

(changes 

over time) 

Quantitative, Comparative Studies 

Anxiety (State-Trait) 

Mutation (+

Å 1 yr 

Mutation (-)

⇔ at all f/up 

Depression (CES-D) 

Mutation (+

Å 1 yr 

Mutation (-)

⇔ at all f/up 

Quality of life (SF-36) ⇔ at all f/up 

Cancer worries10

Mutation (+

⇔ 1 yr 

Mutation (-)

Æ at all f/up 

Gritz, 2005 US 

Prosp 

(2 wk, 6 

mo & 1 yr) 

Unaffected9 persons at 

least 18 years old with 

HNCC defined by 

Amsterdam or 

suggestive family history 

followed by mutation 

testing 

(178/66) 

66 28% 

Distress from receiving 

test results (RIES) Æ

Need for support 

Satisfaction in decision 

to 

undergo testing 

AtkanCollan 2000; 2001a; 2001b; 2001c Finland 

Prosp 

(1 mo & 1 

yr) 

Family members from 36 

HNPCC families with 

known mutations at 50% 

risk 

(446/401) 

334 30% 

Fear of cancer, or fear 

of dying (n=271)11 Æ 

Å Statistically increased ⇑ Increased, but not statistically significant ⇔ No statistically significant differences Æ Statistically decreased ⇓ Decreased, but not statistically significant 8 Published population levels or the normal ranges. 9 “Unaffected” persons included relatives with no personal history of cancer. 10 Lerman C, Trock B, Rimer BK, et al: Psychological and behavioral implications of abnormal mammograms. Ann Intern Med 114:657-611 Subjects who had received genetic testing and returned all 3 questionnaires (n=271, 61% of the whole group). 
131

Table 27. Key Question 9. What are the harms associated with informing/counseling family members or with subsequent testiHNPCC mutations? (continued) 

Observed imp

Author, year Country 

Study 

design 

(followup 

duration) 

Target population for 

genetic testing 

(Eligible /Enrolled N) 

N 

evaluated 

% 

Mutation 

positive 

Measures of 

psychological 

impact after 

revealing genetic 

testing results 

Test 

receivers 

vs. 

normal 

ranges12

Follow-

vs. 

baselin

(chang

over tim

Intrusive and 

avoidant thoughts 

about CRC (Miller 

behavioral style 

scale) 

Mutatio

(+): Å a

wk; ⇔ a

mo & 1 

Mutation 

): Æ at 

f/up 

Depression (HAD) 

Mutatio

(+): Æ a

wk & 4 m

⇔ at 1 

Mutation 

): Æ at 

f/up 

Meiser 2004 Australia 

Prosp 

(2 wk, 4 

mo & 1 yr) 

Individuals from families with 

known HNPCC mutation who 

chose to receive predictive 

genetic testing and had no 

personal history of an 

HNPCC-related cancer 

(134/114) 

11413 28% 

Anxiety (StateTrait) 

Mutatio

(+): ⇔ a

f/up 

Mutation 

): Æ at 

wk; ⇔ a

mo & 1 

Quality of life (SF36) ⇔

Anxiety (HAD) ⇔ Æ 

Arver, 2004 Sweden 

Prosp 

(1 yr) 

Healthy women from 73 of 80 

families with an identified 

HNPCC or BRCA1, BRCA2 

mutation 

(114/90) 

87 36% 

Depression (HAD) ⇔

Æ Mutat

positive

Å Mutat

negativ

12 Published population levels or the normal ranges. 13 N=97 returned 2-week-followup questionnaire, N=98 returned 4-month-followup questionnaire; N=95 returned 1-yr-followup questionn
132

Table 27. Key Question 9. What are the harms associated with informing/counseling family members or with subsequent testiHNPCC mutations? (continued) 

Observed impact 

Author, year Country 

Study 

design 

(follow-up 

duration) 

Target population 

for genetic testing 

(Eligible /Enrolled 

N) 

N 

evaluated 

% 

Mutation 

positive 

Measures of 

psychological impact 

after revealing 

genetic testing 

results 

Test 

receivers 

vs. normal 

ranges14

Follow-up vs

baseline 

(changes 

over time) 

Anxiety (State-Trait) Æ 

Cancer-specific 

distress (IES scales) Claes, Æ 

2005 Belgium 

Prosp 

(1 yr) 

Self-referred 

unaffected persons 

who opted for 

genetic testing for 

HNPCC 

(79/79)15

72 50% Reported >1 

disadvantages of 

knowing genetic test 

results 

Minor depression 

(DSM-III-R and DSMIV scales) 

Murakami, 2004 Japan 

Prosp 

(1 mo) 

Unaffected relatives 

whose family 

members had been 

identified as carrying 

the MLH1/MSH2 

mutation, age ≥20 

years, and opted for 

genetic testing 

(16/15) 

15 33% 

Guilt 

(mutation carriers only) 

Anxiety (State-Trait) Æ 

Claes, 2004 

Belgium 

Prosp 

(1 mo) 

Self-referred 

unaffected persons 

who opted for 

genetic testing for 

HNPCC 

(48/48) 

40 48% Cancer-specific 

distress (IES scales) ⇓ 

14 Published population levels or the normal ranges. 15 Might have some subjects overlapped with Claes, 2004 (337) study. 
133

Table 27. Key Question 9. What are the harms associated with informing/counseling family members or with subsequent testiHNPCC mutations? (continued) Author, year Country 

Study 

Design 

(Followup 

duration) 

Target population for 

genetic testing 

(Eligible /Enrolled N) 

N 

evaluated 

% 

Mutation 

positive 

Results 

All: 27 30% 

Æ S

15 in shortened; 33% Psychological distress16

Å E

Brain, 2005 UK 

RCT 

(3 mo) 

Unaffected individuals from 

families with a known HNPCC 

mutation 

(51/34) 

11 in extended 

pre-test 

counseling 

27% Satisfaction in decision to undergo testing17

inte

Qualitative, Non-Comparative Studies Wagner, 2005 Netherlands 

Prosp 

(6 mo to 

~8 yr) 

1st and 2nd degree 

relatives who were 

germline mutation 

carriers from 24 

HNPCC families 

(115/94) 

70 100% 

84% approved of how they were informed of HNPCC 97% approved information given during counseling 4/10 had life, disability or mortgage insurance problem

health insurance 

Lynch 1997 US 

Prosp 

(ND) 

High-risk members from 

4 extended HNPCC 

families that were 

selected from 

Creighton’s HNPCC 

resource, which 

comprised about 100 

HNPCC extended 

kindreds 

(219/130) 

130 36% 

Of the 89 members who did not enroll in the study, 67

respond to the invitation, 22% had their counseling and 10% refused, citing fear of knowing, fear of insdiscrimination, survivor’s guilt and disinterest. 

When mutation positive, 20% exhibited sadness, part

of concern of the potential risk of transmission of th

their children. Others were concerned about their pdestiny, particularly the possibility of an unfavorabcould lead to their early death and may put their chjeopardy of not having a biological parent to care f

18/130 were concerned about sharing information wit

company. 

Lynch, 1996 US 

Retro 

(ND) 

Family members of 

Native American CRC 

patients who received 

MLH1 genetic testing 

(ND/51) 

44 ND 

Many family members believed the family’s fate was i

factors such as fear, a taboo, or a curse. 4 family metheir heightened cancer genetic risk. 

23 individual (52%) chose to receive genetic test resu

base counseling. All but 1 of the 23 individual counsto understand what was told them. The family who wnegative results exhibited profound relief and happinwith positive results were for the most part stoic. 

16 Goldberg D, Williams P. A User’s Guide to the General Health Questionnaire. Windsor: NFER-Nelson, 1988. 17 Holmes-Rovner M, Schmitt N, Rothert ML et al. Patient satisfaction with health care decisions: the satisfaction with decision scale. Me

58–64. 
134

Table 27. Key Question 9. What are the harms associated with informing/counseling family members or with subsequent testiHNPCC mutations? (continued) Author, year Country 

Study 

Design 

(Follow-up 

duration) 

Target population for genetic 

testing 

(Eligible /Enrolled N) 

N 

evaluated 

% 

Mutation 

positive 

Results 

Mesters, 2005 Netherlands 

Survey 

(N/A) 

Persons from the database of 

the Netherlands Foundation for 

the Detection of Hereditary 

Tumors 

(44/30) 

30 37% 

Disclosure was stimulated if people felt mo

do so or when they anticipated regret ipreventable would happen. Presence o(e.g., professionals) appeared importaas well. 

Disrupted and tense family relations were 

disclose, as well as young age of the mrecipients and negative experience at tto disclose. 

Disclosure was restricted to the nuclear fa

approach was preferred. 

Participant disclosed only the presence of 

defect and the possibility of testing. It wconsidered the recipients’ responsibility choice to obtain further information. 

Peterson 2003 US 

Props 

(15 mo) 

A proband with HNPCC 

mutation and family members 

with >50% risk of carrying a 

mutation from 5 HNPCC families 

(ND/39) 

39/2518 52% 

The identification of a cancer-predisposing 

was new information that confirmed thefamilies were not shocked or surprised This information was not viewed as a m

Compared with mutation carriers, those w

negative for a mutation and those who perceived those topics as less personawere less involved in discussing the mneed for genetic testing. 

Å Statistically increased ⇑ Increased, but not statistically significant ⇔ No statistically significant differences Æ Statistically decreased ⇓ Decreas

References for standardized instruments: Cancer-specific distress (IES scales): Horowitz MJ, Wilner N, Alvarez W. Impact of Event Scale: a measure of subjective stress. Psychosom Med 197CES-D: Center for Epidemiologic Studies Depression. DSM-III-R: Association Psychiatric Association. Diagnostic and statistical manual of mental disorders. 3rd edition, revised. Washington, DC: Americ

DSM-IV: Association Psychiatric Association. Diagnostic and statistical manual of mental disorders. 4th edition. Washington, DC: American PsychiaHospital anxiety and depression scale (HAD): Goldberg D, Williams P. A User’s Guide to the General Health Questionnaire. Windsor: NFER-Nelson

Miller behavioral style scale: Horowitz MJ, Wilner N, Alvarez W. Impact of Event Scale: a measure of subjective stress. PsychosomMed1979: 41: 20

DS, Horowitz MJ. Impact of Event Scale: a cross-validation study and some empirical evidence supporting a conceptual model of stress response s

Psychol 1982: 50 (3): 407–414. 

SF-36: Ware JE Jr, Gandek B. Overview of the SF-36 Health Survey and the International Quality of Life Assessment (IQOLA) Project. J Clin Epidem

Trait Anxiety: Van der Ploeg HM, Defares PB, Spielberger CD. Handleiding bij de Zelfbeoordelingsvragenlijst: een Nederlandstalige bewerking v

Anxiety Inventory. Lisse: Swets and Zeitlinger, 1980.

18 Of the 39 interviewed members, 25 donated blood for genetic testing. 
135

Extra Table 1. Studies examined the factors that might affect the acceptance of genetic testing 

Author, year Findings 

AtkanCollan 

2000; 

2001a; 

2001b; 

2001c 

Acceptors differ from decliners in more often having a spouse or partner, being employed and having 

higher education. On multivariate analysis, the only significant factor for accepting genetic testing 

was employment status. 

Most common reasons cited against undergoing testing were: being old already and having no 

children (23%), would not trust result (15%), timing found unsuitable because of pregnancy 1 (8%), 

no reason given (54%) 

53% indicated that they might have used professional psychological support along with counseling if it 

had been provided. The support might have been used by women with children more than by 

others. 

Berth, 2002 Survey sent to 2076 persons of the general population and 36 patients at high risk for HNPCC or FAP 

(familial adenomatous polyposis) 

“Overall, both the general population and at-risk persons hold a favorable attitude toward genetic 

testing”. 

Brodersen, 

2004 

Cross sectional study to examine anticipated reactions to genetic testing for HNPCC in 437 

asymptomatic people with a family history of colorectal cancer. 

More women anticipated feeling fairly or extremely worried if they did not have the test (36 versus 23 

percent, p =0.009) or if the test was positive (64 versus 40 percent, p =0.008). 

More women anticipated that they would feel fairly or extremely regretful if the test showed that they 

were at high risk (30 versus 20 percent, p =0.03). 

More women thought that they would feel fairly or extremely angry if the test showed that they were at 

high risk (33 versus 13, p <0.001). 

There was only one significant difference between men and women in anticipated reaction following 

genetic testing: if at low risk more women then men would try to adopt a healthier lifestyle 

(p=0.007). 

When asked if they would have more screening if found to be at high risk, 416/432 (96%) said that 

they probably (23%) or definitely (74%) would. 

Codori, 

1999 

Evaluated uptake of genetic testing for HNPCC among first-degree relatives of CRC by comparing test 

acceptors (N=77) and decliners (N=181). 

Significant predictors of acceptance on multivariate analysis were: perceived ability to cope with a 

positive gene test result, risk perception, frequency of thoughts about CRC (not at all or rarely 

versus sometimes or more) and CRC screening history. 

Coping with positive test results: ≥90% confident OR 4.0 (95% CI 1.53-10.3, p=0.005) 

CRC screening: never had a colonoscopy (OR 0.45, 95% CI 0.21-0.93, p=0.030) 

Cancer thoughts: Rarely/never versus sometimes, often, a lot: OR 0.44 95% CI 0.17-1.13, p 

=0.088) 

Graff, 2005 Investigated experiences of 12 individuals with HNPCC (pathogenic mutation) in a semi structured 

telephone interview. All patients had been asked to inform family members. 

12 subjects (4 men and 3 women). Respondents older than nonrespondents (55 versus 49) had more 

cancer diagnoses per individual and were more likely to be female (no statistical comparisons 

made). 

All had told some (mainly immediate family) at-risk family members about predictive testing. 

Family reactions ranged form interest to disinterest. 

Men expressed a need for guidance and support in communicating to relatives more than women. 

Letters and booklets were thought to enhance the quality of information but further aids were unlikely 

to increase the number of relatives made aware of predictive testing by the probands. 

Hadley, 

2003 

Elucidate factors affecting decisions regarding genetic testing in individuals form families with newly 

identified HNPCC. 

104/165 (63%) eligible patients (including index cases and 1st degree relatives agreed to participate. 

This included 54 probands (of whom 87% agreed to participate) and 111 family members of whom 

51% agreed to participate. 

Awareness of genetic testing: those at higher household income levels were more aware of genetic 

testing for cancer (p=0.001) and colon cancer (p=0.009) than those at lower household income 

levels. There was no significant association between participants’ awareness and age, sex, personal 

cancer history or number of 1s-degree relatives. 

Perceived risk of being a carrier: Participant’s feelings about their chances of getting colon cancer 

were significantly associated with their beliefs about the likelihood that they carry a mutation 

(p<0.001) 
136

Extra Table 1. Studies examined the factors that might affect the acceptance of genetic testing (continued) 

Author, 

year Findings 

 Intentions toward genetic testing: The intention to pursue genetic testing was found to have a positive 

association with participant’s beliefs that cancer may be explained by family heredity (p=0.006). 

Concern about the psychosocial effect of genetic testing on the family demonstrated a negative 

association with their intention to pursue testing (p=0.001). Participant’s concerns about their ability 

to handle the emotional aspects of genetic test results demonstrated a negative association with 

their intentions to pursue testing (p<0.001). No association between age, sex or cancer status in 

regard to their intentions toward genetic testing. 

Reasons for pursuing genetic tests: 50% of respondents believe that the most important reason for 

undergoing genetic testing was to learn about their children’s risk. Second most important reason 

(17%) was to guide cancer screening; third (13%) as to confirm their belief that they carry a 

mutation. With respect to the importance of genetic testing for reproductive decision making, a 

statistically significant difference was detected between those younger than the median age of 43 or 

older (p=0.002). 

Reasons for not pursuing testing: Worry about losing health insurance (39%), concerns about how it 

might affect the family (27%), concerns about handling the results emotionally (10%). A statistically 

significant difference was detected between those younger than the median age of 43 compared 

with those older with respect to concerns about handling the emotional aspects of genetic testing 

(p=0.006). 

Testing decisions: 44/54 (81%) of eligible probands eventually chose to undergo genetic testing for 

HNPCC. 56/111 eligible first-degree relatives chose to pursue genetic testing (intention-to-treat 

analysis). 

Of those agreeing to participate in the study, 44/47 (94%) probands agreed to undergo genetic testing 

and 56/57 (98%) of family members agreed. 

Keller, 2004 Of 140 eligible patients and their families, 36 families participated in the information session, 25 

participants were patients, and 11 were spouses or relatives of patients who had died or were too ill to 

attend. Participation rate 26%. Of the 104 nonparticipating patients addressed by mail, 61 responded 

(59%), 12 died, 48 returned a completed questionnaire. Sample here consisted of 73 patients: 25 

participants and 48 nonparticipants. 

Among participants, 60% reported being very worried because of familial cancer; among nonparticipants 35% reported being very worry because of familial cancer (P<0.05). 

28% of participants reported impairment of daily life because of these worries compared to 19% of 

nonparticipants (difference not significant). Similarly, perceived distress of spouse and family from 

patient’s point of view was higher in participants than in nonparticipants (difference not significant). 

Regardless of whether patients participated in the information session, most were in favor of genetic 

testing. Twice as many nonparticipants (25%) as participants (13%) were undecided about whether 

to accept genetic testing. All participants definitely expressed the wish to know whether a mutation 

is present in the family, compared to 81% of nonparticipants. 

Lerman, 

1996 

This was a pilot study in preparation for a prospective study of genetic testing for HNPCC-associated 

mutations. 24 consecutive index colon cancer patients (who did not fulfill the Amsterdam criteria) 

identified 61 first-degree relatives for the study (index patient consent rate = 83%). Of these first 

degree relatives, 44 (72%) completed the interview, 2 (2%) declined, 16 (26%) could not be reached. 

Anticipated reactions to positive and negative results of a genetic test: 

Over 1⁄2 of respondents indicated that they would become depressed or very anxious if they tested 

positive. 

Most subjects reported that they would react by changing their health habits, including diet and 

screening behaviors. 

In response to negative test result, about 1/3 of respondents anticipated they would feel guilty and 

1⁄2 would still worry because the test might be wrong. About 1⁄2 of respondents expected that they 

would decrease their use of screening tests and make fewer attempts to reduce dietary fat if 

they tested negative. 
137

Extra table 2. Surveys of genetic centers or insurance providers 

Author, year Findings 

Brain, 2003 Survey obtained from 16 regional genetic centers involved in predictive testing for HNPCC. Purpose 

was to gain evidence regarding core elements of the pre-test counseling protocol for HNPCC. 16/20 

centers participated. One declined because of few patients counseled. No other details provided. 

Average number of pre-test counseling sessions was 2 (range 1-3) with an average of 4 weeks 

between sessions. 

Minimum number of sessions was 1 (range 1-2). 

Counselors included clinical geneticists and genetic nurse and gastroenterologists. 

Some included home visits, psychiatric consultation, explicit circumstances in which a client would be 

excluded or postponed from testing. Four centers reported that they were considered shortening the 

counseling protocol. 

Centers described a total of 144 topics as being core topics of pre-test counseling. 

Norum 2000 11 health insurers in Norway were mailed a questionnaire to evaluate two hypothetical individuals’ 

requests for insurance; one has HNPCC mutation, the other has BRCA1/BRCA2. 

9/11 insurers responded. 

2 companies reported results on personal indemnity insurance. 

There were no restrictions for individuals with a genetic risk of breast CA when seeking life insurance 

or disability pension. One company raised premium on the one with genetic risk of CRC. 

Another company offered standard or higher rate of premium on personal indemnity insurance in the 

person with genetic risk of CRC. 

Key Question 10a: What Are the Management Options for Family 

Members of CRC Patients Who Have a Positive HNPCC Test? 

b1: Does the Identification of HNPCC Mutations Lead to Improved 

Outcomes in Terms of Decision Making by Patients, Family Members 

and Providers, or Public Health Policy? b2: Does the Identification of 

HNPCC Mutations Lead to Improved Outcomes in Terms of Early 

Detection and Mortality/Morbidity of Patients, and Family Members? 

 

Family Outcomes

Increased early 

detection, 

decreased 

mortality/morbidity, 

improved decision 

making 

HNPCC

mutation

negative

HNPCC

mutation

positive

10,

11

 Below we summarize management options for family members of CRC patients or 

asymptomatic individuals from HNPCC families (based on clinical or genetic definitions) that 

were reported in the literature. This is not necessarily a complete list of management options. 

 Many studies described management options for family members of CRC patients or 

asymptomatic individuals from HNPCC families. We encountered the following in the studies 

included in this report. 

• Annual colonoscopy beginning at age 20 to 25 or 5 years earlier than the youngest affected 

family members.140,154-159
138

• Colonoscopy or double contrast barium enema and sigmoidoscopy at two- or three-year 

intervals.42,155,160-162

• Endometrial screening from age 30 to 35 generally with annual or biennial transvaginal 

ultrasound (TVU).44,45,154,157,158

• Endometrial sampling in premenopausal women and annual TVU and CA 125 testing for 

postmenopausal women.45,154

• Prophylactic oophorectomy and hysterectomy (TAH) from ages 30 to 35 or when 

childbearing was complete.154

• Screening for stomach, duodenum or urinary tract cancers.157

• Prophylactic hysterectomy with or without bilateral salpingo-oophorectomy.163

• 1.5 g calcium daily in a form of calcium carbonate tablet.164

• Biennial breast mammography starting at age 35.158

 We included studies evaluating all forms of cancer related to HNPCC since family members 

of CRC probands are potentially at risk for all such cancers. 

 Six studies examined the impact of mutation testing on the decision to undergo specific 

management recommendations among family members of CRC patients or asymptomatic 

individuals from HNPCC families139-141,154,155,165 (Table 28). Of these, four were of B quality and 

two were of C quality. No study examined the impact of HNPCC mutation testing on public 

health policy or decision making by insurance providers. 

 Two studies (in three publications) of B and C quality indirectly addressed outcomes of early 

detection and mortality/morbidity in relation to the identification of MMR mutations in family 

members of CRC probands or asymptomatic individuals from HNPCC families42,112,160 (Table 

29). These studies were limited by potential selection bias, and/or unclear effects from treatments 

or subsequent interventions. 

Summary of Findings. Identification of HNPCC mutations was associated with improved 

outcomes in terms of decision making to undergo screening for cancers in family members of 

HNPCC. 

 Survival was increased among asymptomatic HNPCC members who received colonoscopy 

screening regularly, regardless of their mutation status (Overview Table 4; Table 28-29). 
139

Overview table 4. Key Question 10 

Reported 

Outcomes 

Body of 

evidence 

(study 

duration) 

Summary 

Decision making 6 Prosp 

studies 

(1 mo & 1 yr) 

• Compared mutation carriers with non-carriers: 

Å Intention to undergo colonoscopy & colonoscopy within 1 yr after 

genetic testing 

Å TVU within 1 yr after genetic testing 

Å Endometrial sampling within 1 yr after genetic testing 

• Following a positive test result, 67% were considering prophylactic 

colectomy. 87% were considering prophylactic total abdominal 

hysterectomy and bilateral salpingo-oophorectomy 

Age at onset CRC 1 Retro study 

(~8 yr) 

• ⇔ Comparing mutation carriers to non-carriers with MSI 

• Younger, comparing mutation carriers to non-carriers with MSS 

Frequency of CRC 1 Prosp 

study 

(15 yr) 

• Æ HNPCC members who received colonoscopy screening regularly, 

regardless of mutation status 

Survival of family 

members 

1 Prosp 

(15 yr) 

• Å Among asymptomatic HNPCC members who received colonoscopy 

screening regularly, regardless of mutation status 

Å Statistically increased ⇑ Increased, but not statistically significant 

⇔ No statistically significant differences 

Æ Statistically decreased ⇓ Decreased, but not statistically significant 

Gaps in the Literature. Identification of HNPCC mutation alone cannot lead to improved 

outcomes in terms of mortality or morbidity unless persons are willing to take subsequent actions 

or interventions. Thus, the Key Questions above have two main components: 1) whether the 

identification of HNPCC mutations led to improved outcomes in terms of decision making by 

patients, family members and providers, or public health policy, and 2) whether the decision 

making subsequently leads to improved outcomes in terms of mortality or morbidity. Studies that 

reported outcomes relating to subsequent management options or interventions in HNPCC 

family members are summarized later with Key Question 11.

 No study examined the impact of HNPCC mutation testing on public health policy or 

decision making by insurance providers on an actual cohort of patients. However, a survey of 11 

health insurers in Norway evaluated two hypothetical individuals’ requests (one who had an 

HNPCC mutation; the other who had a BRCA1/BRCA2 mutation) for insurance153 (Extra Table 

2). The insurers raised premiums on the one with genetic risk of CRC but not in the patient with 

a genetic risk of breast cancer. 

 HNPCC family members who were mutation carriers were more likely undergo screening 

and treatment for preventing cancers than non-carriers. Thus, the data presented above suggest 

that screening and testing for HNPCC mutations improves acceptance of screening procedures, 

at least in individuals who agree to undergo screening and testing. 

 Whether this benefit translates into improved morbidity or mortality is less clear from the 

available literature. A study that attempts to examine whether the identification of HNPCC 

mutations leads to improved mortality or morbidity should ideally control for the differences in 

the rates and intensity of screening or treatments for cancers. However, most studies did not 

provide such details. Furthermore, there may be complex interactions with age, gender, and 

time,since identification of HNPCC mutation, compliance with planed screening programs or 

treatments, and mutation status. No study considered all of these factors together. Large HNPCC 

registries with active data collection for the elements described above are needed. 
140

 Although not specifically asked in the Key Questions, a few studies reported factors that 

influenced compliance with screening (such as socioeconomic status and perceived barriers to 

screening).166,167 We considered such observations clinically important and thus summarize them 

in ancillary tables. However, there were few such studies (Extra Table 3). 
141 

Table 28. Key Question 10b1. Does the identification of HNPCC mutations lead to improved outcomes in terms of decision ma

members and providers, or public health policy? 

Author, year Country 

Study 

design 

(Followup 

duration) 

Target population 

for genetic testing 

(Eligible /Enrolled 

N) 

N 

evaluated 

% 

Mutation 

positive 

% 

Colonoscopy 

before 

genetic 

testing 

Management options 

recommended when 

mutation positive 

Outcome 

measures 

Colonoscopy 

within 1 yr after 

genetic testing 

Å 

a

Transvaginal 

ultrasound 

(TVU) within 1 

yr after genetic 

testing 

Å 

Collins, 2005 Australia 

Prosp 

(1 yr) 

Individuals who had 

never had CRC or 

any of the cancers 

associated with 

HNPCC 

(134/114) 

114/9819 28% 

3/12 (25%) 

<25 yr old 

and 74/101 

(73%) ≥25 yr 

old reported 

a 

colonoscopy 

Annual colonoscopy 

beginning at age 25 or 5 years earlier than youngest affected family members 

Endometrial screening 

considered from age 30-35 or even 25 generally with annual TVU and endometrial sampling, in addition to CA 125 testing for postmenopausal women 

Prophylactic 

oophorectomy and TAH also considered for ages 30 to 35 or when childbearing is complete 

Endometrial 

sampling within 

1 yr after 

genetic testing 

Halbert, 2004, US 

Prosp 

(1 yr) 

Family members 

with a 25% risk of 

having HNPCC 

mutation 

(ND/222) 

98 22% 

Overall: 62% 

 36% (8/22) 

of carriers, 

27% (13/49) 

of 

noncarriers, 

and 26% 

(7/27) testing 

decliners) 

ND 

Colonoscopy 

within 1 yr after 

genetic testing 

Å 

19 Baseline/follow-up N. 
142 

Table 28. Key Question 10b1. Does the identification of HNPCC mutations lead to improved outcomes in terms of decision ma

members and providers, or public health policy? (continued) 

Author, year Country 

Study 

design 

(Followup 

duration) 

Target population for 

genetic testing 

(Eligible/Enrolled N) 

N 

evaluated 

% 

Mutation 

positive 

% 

Colonoscopy 

before genetic 

testing 

Management 

options 

recommended 

when mutation 

positive 

Outcome measure

Colonoscopy within 

yr after genetic testin

Claes, 2005 Belgium 

Prosp 

(1 yr) 

Self-referred unaffected 

persons who opted for 

genetic testing for 

HNPCC 

(79/79)20

72 50% 

65% (69% for 

mutation 

positive; 61% 

for mutation 

negative) 

Colonoscopy 

within 1 year if 

not done in 

previous year 

Compliance to 

recommendation fo

management option

>1 colonoscopy with

1 yr after genetic 

testing 

Colonoscopy use 

Hadley, 2004 US21

Prosp 

(1 yr) 

Members of families 

with newly identified 

HNPCC with any of the 

following features: 

HNPCC-associated 

cancer demonstrating 

MSI or a family history 

suggestive of HNPCC 

and 1st-degree relatives 

at 50% risk of inheriting 

the mutation 

(111/57) 

56 30% 

54% (41% for 

mutation 

positive; 59% 

for mutation 

negative) 

Colonoscopy 

every 1 to 3 

years 

Nonadherent with 

colonoscopy 

recommendations22

Lynch 1997 US 

Prosp 

(ND) 

High-risk members from 

4 extended HNPCC 

families that were 

selected from 

Creighton’s HNPCC 

resource, which 

comprised about 100 

HNPCC extended 

kindreds 

(219/130) 

130 36% ND ND 

Following a positive 

were considering 

colectomy. 87% we

prophylactic tota

hysterectomy and bi

oophorectomy. Th

mutation negative w

Intension to undergo 

colonoscopy 

Claes, 2004 Belgium 

Prosp 

(1 mo) 

Self-referred unaffected 

persons who opted for 

genetic testing for 

HNPCC 

(48/48) 

40 48% 65% ND Intension to undergo 

endometrium 

examination 

Å Statistically increased 

⇑ Increased, but not statistically significant 

⇔ No statistically significant differences 

20 Some subjects may have overlapped with Claes, 2004 (337). 21 Same population as Hadley, 2003 (946). 22 The recommendations were colonoscopy every 1 to 3 years for mutation-positive persons and general population screening for mutati

flexible sigmoidoscopy every 3 to 5 years after the age of 50 years). 
143 

Table 29. Key Question 10b2. Does the identification of HNPCC mutations lead to improved outcomes in terms of early detect

mortality/morbidity, of patients, family members? 

Author, year Country 

Study 

Design 

(Follow-up 

duration) 

Target population 

for genetic testing 

(Eligible/Enrolled 

N) 

N 

evaluated 

% 

Positive 

HNPCC 

test 

Descript

ion of 

HNPCC 

tests 

% Received 

interventions or 

treatments 

Outcome 

measures 

Effe

positi

Mutation testing 

“Study 

group”: 

133 

33% Frequency of CRC

Æ 8 

group 

contro

Æ Am

positiv

(8 o

group 

46) i

Jarvinen, 1995; 2000 Finland 

Prosp 

(15 yr) 

Asymptomatic 

member of 22 

HNPCC families at 

50% risk, meeting 

Amsterdam I criteria 

or other suggestive 

family history 

(ND/252) “Control 

group”:

118a

40% 

MMR 

gene 

mutation 

Study group: Initial 

colonoscopy or double 

contrast barium enema 

and sigmoidoscopy and 

then repeated 

examinations at 3-year 

intervals. 

4 subjects underwent 

total abdominal 

colectomy (3 in study 

group; 1 in control 

group), and 1 subject 

(study group) received a 

segmental resection of 

the sigmoid colon. 

Mortality

0/133 

vs. 

co

Æ (R

Group A: 

361 100% 

Group B: 

241 Benatti, 0% 2001 

Italy 

Retro 

(8.2, 6.7, 

and 9.4 yrs 

for group 

A, B, and C 

respectivel

y)

29 HNPCC families 

met inclusion 

criteria: 10 carried 

MMR gene 

mutations (Group 

A), 

10 were 

characterized by 

MSI phenotype but 

not by MMR gene 

mutation (Group B), 

and 9 did not show 

mutations or MSI 

(Group C) 

Group C: 

355 0% 

MMR 

gene 

mutation 

ND 

Age at 

onset 

CRC 

⇔ Co

Youn

Gro

Å Statistically increased 

⇑ Increased, but not statistically significant 

⇔ No statistically significant differences 

Æ Statistically decreased 

⇓ Decrea

a Of those invited to participate, 78 eligible declined colonoscopy screening and 40 could not be traced; these patients constituted the control group. A

mutation-positive subjects were recommended the screening program, whereas endoscopies were discontinued in mutation-negative subjects in whom 

20% received screening when requested.
144

Extra Table 3. Factors that might affect the compliance with CRC screening 

Author, year Findings 

Adams, 2003 Secondary analysis of data recorded for a study on polyp prevention (a database) on 

chemopreventive effects of aspirin. Study had included 600 patients with HNPCC; of these 154 

were available for analysis and full data were available on 134. Of these 134, 49 had a 

personal history of CRC. 

Socioeconomic status assessed using the Town-Deprivation Score (TDS) of the current 

enumeration district of residence. This score is based on relative material deprivation based on 

census data. 

There was a statistically non-significant correlation between the TDS and the age of first resection 

indicating that individuals in less deprived areas tended to have their colorectal cancer resected 

at an earlier age than those living in more deprived areas. 

Bleiker, 2005 178 patients at high risk for CRC (27 Amsterdam I criteria; 43 familial CRC occurrence). All had 

received counseling from a geneticist and were advised to have periodic colonoscopy. 

Objective and self-reported compliance. 

149/178 returned a completed questionnaire. Respondents significantly older than 

nonrespondents. 

Average f/u 5.8 years 

61% of study sample fulfilled Amsterdam I 

Objective compliance data available for 132/149. Of these 95 (72%) were found to be compliant 

with screening advice. 

Of 37 (28%) noncompliant, 20 had postponed screening for at least 1 year, 13 were screened 

only once, and 4 never underwent screening. 

Compliers were significantly younger than noncompliers and there was a nonsignificant trend to 

increased level of education, mutation status, and type of colon screening. 

No difference between perceived colon cancer risk. 

Noncompliers significantly more likely to perceive barriers to screening. 

Noncompliers rated screening as being more embarrassing. 

On multivariate analysis only the perceived number of barriers to screening was associated 

significantly with noncompliance (OR 1.2, 95% CI 1.1-1.3). 

Of self-reported data, noncompliance was associated with embarrassment with screening 

procedure and sigmoidoscopy rather than colonoscopy. 

Compliers were significantly more likely to have remembered receiving a reminder letter. 

Key Question 11: What Are the Harms Associated With Subsequent 

Actions or Interventions for Family Members? 

Family Outcomes

Increased early 

detection, 

decreased 

mortality/morbidity, 

improved decision 

making 

HNPCC

mutation

negative

HNPCC

mutation

positive

10,

11

 

 Due to limited data on harms associated with subsequent management options or 

interventions, we broadened the scope of this question to include studies that reported any 

outcome relating to subsequent management options or interventions in HNPCC family 

members. 

 Nine studies reported outcomes related to subsequent actions or interventions among family 

members. Of these, six were of B quality and three were of C quality. Four of these nine studies 

reported harms or adverse events associated with subsequent actions or interventions for family 
145

members. In addition, one study of C quality examined the psychological impacts associated 

with colonoscopies44,45,156-159,161-164 (Table 30). Some of these studies did not have a control 

group of subjects who declined to undergo surveillance, and most results did not adjust for 

potential confounders such as age, personal history of cancer, and educational levels. 

Summary of Findings. Fewer than 0.5 percent of family members experienced harms 

associated with screening or surveillance examination or surgical procedures. There was some 

negative psychological impact associated with colonoscopies. 

 HNPCC family members who took subsequent actions or interventions had a lower risk of 

developing HNPCC-related cancers and lower mortality rates, compared to those who did not 

take actions (Overview Table 5; Table 30). 

Overview Table 5. Key Question 11 

Reported 

Outcomes 

Body of evidence 

(study duration) Summary 

Harms 3 Retro studies 

(0.1-35 yr), 1 

Prosp study 

(6 mo to ~8 yr), & 

1 RCT (12 wk) 

• 1 patient (0.25%) had a cardiac arrest due to screening exams. 

• 1 patient (0.3%) had surgical complications from hysterectomy.

• ⇔ Anxiety but Å depression and Æ metal health & vitality scores 

associated with colonoscopies. These scores were within the 

normal ranges for the general population. 

• Colonoscopy unpleasant 57%, fearful 32%, painful 51%, 

shameful 16%. Worry about complications of colonoscopy 14% 

• No side effect was reported for 1.5 g calcium supplementation 

daily 

Outcomes relating to 

subsequent actions 

or interventions 

6 Retro (0.1-82 

yr), 1 Prosp study 

(6 mo to ~8 yr), & 

& 1 RCT (12 wk) 

• Colonoscopy and/or sigmoidoscopy: 

Æ Mortality 

⇓ CRC mortality 

⇔ Risk of developing CRC 

• TVU, TAH, and/or breast mammography: 

Æ Risk of extracolonic cancer 

Æ Risk of endometrial cancer 

Æ Risk of ovarian cancer 

• 1.5 g calcium supplementation daily: Cytolytic activity decreased 

with calcium but difference was not statistically significant. 

• ⇔ Survival b/w HNPCC, FAP, and sporadic CRC groups 

adjusting for age, gender, stage and tumor location 

• 5-year survival rate in patients with gastric cancers (40% also had 

CRC) was 48% vs. 15%, in those who received radical surgery 

and those who received palliative surgery or explorative 

laparotomy alone, respectively. 

• No cases of endometrial carcinoma detected by ultrasound but 

one case of endometrial carcinoma was found (in a woman with 

an MLH1 mutation) 

TVU=transvaginal ultrasound; TAH= Prophylactic hysterectomy 

Å Statistically increased ⇑ Increased, but not statistically significant 

⇔ No statistically significant differences 

Æ Statistically decreased ⇓ Decreased, but not statistically significant 

Gaps in the Literature. There was little information on harms associated with screening and 

surveillance examinations or surgical procedures in CRC patients and their family members. 

However, the risks of these procedures are likely to be similar to the non-HNPCC setting. Only 

five studies reported data on harms or adverse events. One cannot assume “no harm” when a 

study did not report data on harms; therefore harms associated with the screening procedures 

may be underreported. 
146

Table 30. Key Question 11. What are the harms associated with subsequent actions or interventions for family members? Author, year Country 

Study design 

(Follow-up 

duration) 

Target population 

(Eligible/Enrolled N) 

N 

evaluated 

% who 

took 

actions 

Description of 

subsequent 

actions or 

interventions 

Harms 

Outcom

receive

vs. thos

CRC mort

Vasen, 1995 Netherlands 

Retro 

(1-20 yr) 

“Cases”: high-risk 

relatives of 50 HNPCC 

families who met 

Amsterdam I criteria and 

accepted surveillance 

(579/493) 

388a 100% 

Surveillance 

program included 

colonoscopy or 

sigmoidoscopy with 

barium enema every 

2-3 years 

1 patient 

(0.25%) had a 

cardiac arrest 

due to screening 

exams 5-yr surv

Risk o

endomet

cance

Schmeler, 2006 US 

Retro 

(0.1-35 yr) 

Women with documented 

MLH1, MSH2 or MSH6 

gemline mutations 

(380/315) 

315 

19% TAH 

alone; 15% 

TAH&BSO 

Prophylactic 

hysterectomy with or 

without bilateral 

salpingooophorectomy 

1 patient (0.3%) 

had surgical 

complications 

from 

hysterectomy c

Risk o

ovaria

cance

Cumulat

CRC-spe

survival 1

after surg

RenkonenSinisalo, 2000 Finland 

Retro 

(Median 3 yr 8 

mo in 

surveillance 

group; 5 yr 3 

mo in nonsurveillance 

group) 

All new CRC cases 

occurring in known 

HNPCC families with 

mutation or met 

Amsterdam criteria 

(137/137) 

137 24% 

Surveillance 

program involved 

colonoscopy or 

double-contrast 

barium emema and 

sigmoidoscopy. 

Non-surveillance 

group underwent 

colonic examinations 

due to symptoms of 

cancer. 

ND 

Cumulat

overal

surviva
147

Table 30. Key Question 11. What are the harms associated with subsequent actions or interventions for family members? (conAuthor, year Country 

Study 

design 

(Followup 

duration) 

Target population 

(Eligible/Enrolled N) 

N 

evaluated 

% who 

took 

actions 

Description of 

subsequent actions or 

interventions 

Harms 

Outcom

received 

those 

Wagner, 2005 Netherlands 

Prosp 

(6 mo to 

~8 yr) 

1st and 2nd degree relatives 

who were germline mutation 

carriers from 24 HNPCC 

families 

(115/94) 

70 100% 

Colonoscopy every 1-2 

years from age 20-25 years 

Female carriers offered 

gynecological screening by US and CA125-measurements of blood from age 30-35 years and on 

Screening for stomach, 

duodenum or urinary tract as advised 

Colonoscopy 

unpleasant 57%, fearful 32%, painful 51%, shameful 16% 

Worry about 

complications of colonoscopy 14% 

88% (n=37

carriers h

screening 

Rijcken, 2003 Netherlands 

Retro 

(5 mo to 

11 yr) 

Women from HNPCC 

families (MMR gene mutation 

or Amsterdam II criteria) 

identified by physicians and 

national registry 

(ND/103) 

41 100% 

Annual screening started 

between 30 and 35 

years old consisting of 

gynecological exam, 

TVU, serum levels of 

CA125 (normal 

≤35 

kU/L). Endometrial 

sampling as indicated. 

ND 

179 TVU p

patient yesampling TVU resusymptomendometshowed npatholog

115 serum 

ranged frmedian o

No ovarian 

by screeannual sc

Cats, 1995 Netherlands 

RCT 

(12 wk) 

First-degree relatives of 

patients with HNPCC 

participating in regular 

screening program. At 50% 

lifetime risk of developing 

CRC. 

(53/30) 

30 50% 

1.5 g calcium daily in a 

form of calcium 

carbonate tablet 

No side effect was 

reported 

Cytolytic a

with calci

was n

significan

sigmoid 
148

Table 30. Key Question 11. What are the harms associated with subsequent actions or interventions for family members? (conAuthor, year Country 

Study 

design 

(Followup 

duration) 

Target population 

(Eligible/Enrolled N) 

N 

evaluated 

% who 

took 

actions 

Description of subsequent 

actions or interventions Harms 

Outco

received 

thos

Risk 

developin

Arrigoni, 2005 Italy 

Retro 

(21-82 yr) 

Individuals from 22 

HNPCC families that met 

Amsterdam I criteria 

(ND/331) 

331 60% 

Surveillance program included 

colonoscopy starting at age 25; 

GYN exam, and transvaginal US 

starting at age 30; Urinary 

ultrasonography and urine 

analysis starting at age 30; 

Endoscopy starting at age 30; 

Thyroid ultrasonography starting 

at age 35; Breast 

mammography starting at age 

35. All tests repeated every 2 

years. 

ND 

Risk 

develo

extraco

canc

Liljegren 2004 Sweden 

Retro 

(10 yr) 

Individuals at high-risk for 

CRC (>10% lifetime risk) 

who were either mutation 

positive, suggestive family 

history but not fulfill 

Amsterdam I, or having 2 

close relatives affected by 

CRC 

(304/265) 

240 100% Colonoscopies were performed every 2-3 years 

⇔ Anxiety 

(HAD) but 

Å

depression 

(HAD) and 

Æ

metal health & 

vitality scores 

(SF-36), 

compared to 

general 

population 

DoveEdwin, 2002 Netherlands 

Retro 

(ND) 

Women from 38 HNPCC 

families (Amsterdam I or II 

criteria, or mutation 

testing) 

(ND/118) 

118 100% Annual or biennial TVU from age 30-35 years. ND 

No cas

carcino

ultrasou

endomet

found (in 

ML
149

Table 30. Key Question 11. What are the harms associated with subsequent actions or interventions for family members? (conAuthor, year Country 

Study 

design 

(Follow-up 

duration) 

Target population 

(Eligible/Enrolled N) 

N 

evaluated 

% who 

took 

actions 

Description of 

subsequent 

actions or 

interventions 

Harms Outcomes, those who rece

vs. those who d

Green, 1995 UK 

Retro 

(ND) 

HNPCC family members 

fulfilling Amsterdam I criteria 

and undergoing a 

colonoscopy 

(ND/61) 

61 100% Colonoscopy screening ND 

2 patients (3 %) had a malignDukes B and the other Dukes 

41, respectively. Another pati

an obstructing sigmoid car

patients had adenomas (mea

26-63). 

Å Statistically increased ⇑ Increased, but not statistically significant ⇔ No statistically significant differences Æ Statistically decreased ⇓ Decreased, but not statistically significant a Surveillance detected 11 CRC cases (out of 388 high-risk individuals) during follow-up. Mortality and 5-yr survival of the surveillance-detected CRC

controls (or symptomatic CRC patients who were not under surveillance). 

b The 11 surveillance detected CRC cases were compared to 238 “controls” subjects, who were symptomatic CRC patients who were not under surveic Ureteral injury, ureterovaginal and ureteroeneteric, and then rectovaginal fistula. d Compared with age-matched, mutation positive women who had not undergone either procedure (n=210 for endometrial cancer and n=223 for ovaria

151

Chapter 4. Discussion 

Our report was based upon an analytic framework that begins with a patient with CRC and 

explores the spectrum of implications of screening, testing, and management of the patient and 

their family members considering the perspectives of the patient, provider, family member and 

the public health. The Key Questions we addressed reflected the main considerations along this 

pathway. We have explored a large and varied literature and have found a wide range of quality 

of the evidence across these areas. 

 

Analytic Validity 

A previous review sponsored by the CDC and EGAPP found that 13 commercial laboratories 

in the United States offered some form of laboratory testing for HNPCC. (The report is available 

at http://www.cdc.gov/genomics/gtesting/file/print/FBR/CC_AnalyticValidity.pdf, accessed on 

August 5th, 2006). The report noted that there were no data from which to assess the analytic 

sensitivity, specificity, or reliability of genetic tests used to diagnose HNPCC. We also 

summarized commercial laboratories performing HNPCC-related tests that had been included on 

a federally sponsored website (www.genetests.org). This likely represents only a partial list, but 

highlights the variability in the testing available at the participating centers. 

 The published literature regarding the analytic validity of laboratory testing in HNPCC is 

extremely limited. Most of the studies we reviewed were only indirectly related to analytic 

validity and had major methodologic limitations, particularly in use and definitions of reference 

standards. However, there was some evidence that there may be variability across testing 

facilities at least for immunohistochemistry (IHC).56 Furthemore, a study published after our 

literature review found a “learning curve” in laboratories performing MSI testing.168 Thus, 

analytic validity is not merely a theoretical concern but may have important clinical impact in the 

care of probands with HNPCC-related cancer or their families. 

 A better understanding of analytic validity could be achieved by incorporating non-literature 

based methods. This might include contact of commercial laboratories performing such testing. 

In addition, an external proficiency-testing program for MSI was introduced in 2006 (by the 

College of American Pathologists, information accessible at http://www.cap.org/apps/cap.portal) 

but results have not been published. Finally, some of the laboratory tests used in evaluation of 

HNPCC are also used for other disorders. A literature review based upon a search for these 

methods would not be practical. However, a committee of experts involved in these laboratory 

procedures could help evaluate their strengths and limitations in detecting the spectrum of 

genotypes/phenotypes associated with HNPCC. 

Clinical Validity 

 We summarized the literature describing the relationships among clinical predictors of 

HNPCC using a clinical or genetic definition of the disorder, depending upon the parameter that 

we were estimating. Our analysis underscored the difficulty in comparing studies with one 

another because of important differences in study characteristics such as inclusion criteria, the 

quality and extent of laboratory testing performed, and the degree to which the diagnosis was 
152

verified among participants. Despite these difficulties, we have clarified the accuracy of the 

clinical predictors and critically evaluated study characteristics that may have a bearing upon 

them. 

 We used the estimates of test accuracy to develop a decision-tree model to understand how 

these predictors can be used to identify MMR mutations among unselected patients with CRC. 

We performed sensitivity testing across the range of estimates for key parameters. The point 

estimates (and ranges) tested were derived from our systematic review of the data and thus 

confidently demonstrate the range of possibilities based upon the best available evidence. 

 We found that a set of three simple criteria i.e., 1) age less than 50 years old at diagnosis 2) 

or a history of colorectal or endometrial cancer in a first degree family member, 3) or the 

presence of multiple, synchronous or metachronous colorectal or endometrial cancers) performed 

as well as the more complex criteria (such as the Bethesda guidelines) when combined with MSI 

or IHC testing of tumor tissue. However, our analysis did not consider costs or utilities and thus 

a more formal cost-effectiveness analysis would be required to more fully understand the 

implications of the various strategies. 

 Our model was based upon parameters obtained from the published literature. As a result, it 

may not fully account for practical considerations that are encountered in the care of patients 

with CRC and family members with suspected HNPCC. For example, it may not be possible to 

obtain tumor tissue to perform laboratory testing. In addition, there may not be uniform access to 

expert laboratory and pathology facilities or qualified genetic counselors. Economic and other 

barriers may prevent access to care and surveillance in some settings. Obtaining a family history, 

even if abbreviated, may not always be feasible. As noted in Chapter 1 and 3, the patients’ report 

of the family history may not be accurate, particularly for cancers other than colorectal that are 

potentially related to HNPCC.30 Issues of uncertain paternity may also be relevant in some 

families while some families may be too small (or have insufficient contact among family 

members) to obtain a clinically meaningful family history. 

 Furthermore, testing for HNPCC in patients with CRC represents only one of the diagnostic 

considerations when evaluating patients for a genetically susceptibility to cancer. To fully 

understand the implications, diagnostic strategies for other forms of hereditary cancer (such as 

MUTYH-associated polyposis and attenuated familial adenomatous polyposis) also need to be 

considered. For example, it is possible that certain combination of clinical history and laboratory 

testing of tumor tissue may be superior to the strategy proposed above when attempting to 

optimize the yield in uncovering all recognizable forms of genetically-based CRC predisposition. 

We identified many gaps in the literature. For example, there were relatively more studies 

demonstrating test characteristics of MSI testing compared with IHC and thus greater precision 

in the estimates of sensitivity and specificity of MSI testing. There were insufficient data to 

understand confidently whether MSI testing had better sensitivity and specificity compared with 

IHC and even fewer studies that addressed benefits of using both approaches concurrently. An 

advantage of IHC compared with MSI testing is that it is easier to perform and less expensive, 

both features offering important benefits from a public health perspective. Nevertheless, more 

studies are needed to clarify whether IHC should be preferentially adopted based upon these 

considerations. 

The majority of studies provided data on patients with CRC who were implicitly or explicitly 

selected based upon heightened suspicion for HNPCC while there were few studies providing 

estimates on characteristics of these tests in an unselected population-based sample of patients 

with CRC. More studies on such patients are needed. 
153

Benefits and Harms 

We identified and critically evaluated studies that considered the range of outcomes (harms 

and benefits) in probands with HNPCC-related cancers and their family members from the 

perspectives of the patient, their family members, providers and the public health. Where there 

was limited evidence, we expanded the scope of the Key Questions to include indirect evidence, 

and variable definitions of HNPCC (e.g., clinical or genetic) to provide as comprehensive an 

appraisal of the knowledge base as possible. The major conclusions and gaps in the literature are 

summarized in the Executive Summary, Chapter 3, and (more briefly) below. 

We found only limited data on harms associated with testing for MMR mutations in probands 

with CRC but the available data do not suggest severe psychological impact in most patients. In 

addition, pre-test genetic counseling had good efficacy in improving knowledge about HNPCC 

and resulted in a high likelihood of proceeding with genetic testing, satisfaction in the decision to 

undergo genetic testing and decreasing depression and distress levels among family members of 

HNPCC probands and among asymptomatic individuals form HNPCC families. Although there 

was little information regarding how pre-test counseling should optimally be performed, the 

consistent benefits observed across studies underscore the effectiveness of current programs. 

Testing of family members also had the potential to exclude the presence of HNPCC and thereby 

excuse family members from the need for intensive cancer surveillance and the psychosocial 

concerns related to a cancer predisposition. 

There was limited direct evidence regarding whether the identification of MMR mutations 

led to improved outcomes for the proband in terms of early detection, mortality/morbidity or 

management decisions by patients or providers. However, identification of HNPCC mutations 

was associated with an increase in the likelihood that family members of probands with CRC 

would undergo cancer-screening procedures. Survival was increased among asymptomatic 

HNPCC family members who received colonoscopy screening, regardless of their mutation 

status. Family members who underwent cancer-screening procedures had a lower risk of 

developing HNPCC-related cancers and lower mortality rates than those who did not take 

actions. The literature most strongly supported surveillance for CRC while there were only a few 

studies that focused on prevention of other HNPCC-related tumors. However, all of the relevant 

studies suggesting these benefits had important limitations. 

There are a multitude of ethical, social,and medical issues that require clarification. There 

was very little information regarding surveillance approaches in patients who were considered at 

risk for HNPCC, but had a negative or equivocal genetic test result. We found very few moderate 

or high quality studies from which to confidently understand the clinical expression (penetrance) 

of HNPCC among family members of CRC probands found to have MMR mutations through 

predictive genetic testing programs. There was also limited information regarding the 

relationship of specific genotypes to phenotypes. 

Summary 

• There is very little published information related to the analytic validity of laboratory testing 

in HNPCC. However, the available data suggest there may be important variability among 

testing facilities. Additional unpublished information about analytic validity is available 

through an external proficiency-testing program for MSI conducted by the College of 
154

American Pathologists, and through internal testing performed by individual laboratories. In 

addition, the analytic validity of the specific testing methods used for other genetic disorders 

may be applicable to HNPCC. 

• Among CRC fulfilling Amsterdam I criteria, approximately 44% (95% CI: 35, 52%) had a 

MMR (MLH1 or MSH2 in most studies) gene mutation when considering all studies 

together. The prevalence was 51% (95% CI: 35, 66%) among studies performing relatively 

comprehensive genetic testing. However, the true prevalence of MMR mutations among 

Amsterdam I patients may be different (possibly higher) than these estimates for several 

reasons: 

1. Testing for MMR genes other than MLH1 and MSH2 (particularly MSH6) may 

increase the proportion of patients with MMR by around 10% (this is an uncertain 

estimate). 

2. Genomic rearrangements and large deletions are missed when only sequencing and 

gene screening is performed. Limited evidence suggests that approximately onefourth to one-third of the identified MMR mutations are large genomic 

deletions/rearrangements. 

3. The prevalence estimates are influenced by the certainty that identified genotypes are 

classified as being pathogenic. 

4. The strategy used for selecting patients with CRC can have a substantial impact on 

the prevalence estimates. Very few studies focused on unselected patients with CRC. 

Most identified cases from cancer registries or used other selection strategies that may 

have increased the proportion of patients with MMR mutation included in the study. 

This report examined various features of studies evaluating these estimates (such as 

the selection criteria, specific type of laboratory and genetic testing, sample size and 

methodologic quality), but did not identify consistent associations among these 

features and the prevalence estimates. 

Accounting for MMR genes other than MLH1 and MSH2 and for the routine use of methods 

to detect large genomic deletions/rearrangements, one may calculate that approximately 70% 

to 75% for Amsterdam I patients are MMR mutation carriers. 

• The same considerations apply to other clinical criteria such as the Amsterdam II criteria or 

the Bethesda Guidelines (original and revised). Among CRC patients who fulfilled the 

Amsterdam II criteria, 39% (95% CI: 30, 49%) had a MLH1 or MSH2 gene mutation when 

considering all studies together. The prevalence was 40% (95% CI: 30, 52%) when 

considering two studies that performed gene sequencing in all patients. Accounting for MMR 

genes other than MLH1 and MSH2 and for the routine use of methods to detect large 

genomic deletions/rearrangements, one may calculate that approximately 55% to 59% for 

Amsterdam II patients are MMR mutation carriers. 

• 71% (95% CI 63, 78%) of tumors from patients who fulfilled the Amsterdam I criteria were 

MSI-H. The corresponding prevalence for Amsterdam II was 68% (95% CI: 58, 76%). 

However, these estimates may be influenced by selection bias (both in selecting patients for 

study and in the availability of tumor tissue for testing). We examined several other studyrelated features but did not observe consistent associations with the prevalence estimates. 
155

• Approximately 40% of colorectal cancers (95% CI: 28, 53%) from patients who fulfill the 

Amsterdam I criteria demonstrate loss of protein expression for MLH1 or MSH2 by IHC. 

Testing for loss of expression of MSH6 may increase the proportion by about 6% but the data 

were limited. The potential biases described above for the prevalence of MMR mutations 

may also apply to these estimates. 

• The sensitivity and specificity of clinical criteria for predicting the presence of MMR 

mutations is described in Chapter 3. A decision model based upon these estimates 

characterizes the yield of various strategies in identifying MMR mutations among unselected 

patients with CRC. An abbreviated family history plus tumor tissue testing for MSI or IHC 

appeared to identify a similar number of patients with MMR mutations as strategies 

involving a more complex clinical history. 

• No studies described harms of the risk assessment process (e.g., using the Amsterdam 

criteria, Bethesda guidelines, MSI or IHC testing) in identifying CRC patients at increased 

risk for CRC. 

• There was limited data on harms associated with testing for MMR mutations in probands 

with CRC. However, the available data do not suggest severe psychological impact in most 

patients. 

• There was limited direct evidence regarding whether the identification of MMR mutations 

led to improved outcomes for the proband in terms of early detection, mortality/morbidity or 

management decisions by patients or providers. 

• No study described harms associated with the subsequent management options after 

identification of MMR mutations in patients with CRC or other forms of HNPCC-related 

cancers.

• Several studies described the accuracy of HNPCC testing in predicting the risk of CRC in 

family members of probands with HNPCC. However, in most, the diagnosis of HNPCC was 

not based upon genetic testing. There was limited evidence regarding variables associated 

with penetrance in family members. 

• Pre-test genetic counseling had good efficacy in improving knowledge about HNPCC and 

resulted in a high likelihood of proceeding with genetic testing, satisfaction in the decision to 

undergo genetic testing and decreasing depression and distress levels among family members 

of HNPCC probands or among asymptomatic individuals form HNPCC families. 

• Identification of HNPCC mutations was associated with an increase in the likelihood that 

family members of probands with HNPCC would undergo cancer-screening procedures. 

However, such patients were self-selected based upon their willingness to undergo predictive 

genetic testing and thus, this observation may not be applicable to all family members. 

Survival was increased among asymptomatic HNPCC family members who received 

colonoscopy screening, regardless of their mutation status. 
156

• There was limited direct evidence related to harms of the cancer-screening procedures in 

family members of probands with HNPCC. Complication rates associated with these 

procedures in other settings are probably similar. 

• HNPCC family members who underwent cancer-screening procedures had a lower risk of 

developing HNPCC-related cancers and lower mortality rates than those who did not take 

actions. However, all of the relevant studies suggesting these benefits had important 

limitations. 

Implications for Future Research 

 Our report identified several areas for future research; we considered the following to be 

particularly important priorities: 

• There is very little information regarding the analytic validity of tests used in the diagnosis of 

HNPCC. Studies specifically addressing sensitivity, specificity, and reliability of all of the 

laboratory and genetic testing methods in HNPCC are needed. Such studies should focus on 

contemporary testing methods and compare them against well-defined reference standards in 

tissue samples representative of the spectrum of genotypes associated with HNPCC. 

Unpublished information regarding analytic validity is also available; it may be feasible to 

obtain information from commercial or private laboratories performing such testing. It may 

also be possible to obtain data from the College of American Pathologists regarding their 

MSI proficiency program once experience has accumulated. Experience with genetic testing 

methods in other conditions is likely to be relevant to HNPCC; a review of such information 

could be conducted by groups of experts on genetic testing techniques. 

• Additional studies are needed to clarify the validity of specific clinical and laboratory 

predictors of HNPCC in patients with CRC who are representative of the general population 

of patients with CRC. 

• Future studies should consider all forms of genetically based CRC cancer predisposition to 

fully understand the effectiveness of various diagnostic strategies. Such studies should 

consider all known genetic causes of cancer predisposition and the accuracy of clinical and 

laboratory testing in identifying these disorders in individuals who are representative of the 

general population. 

• Additional studies are needed to establish the availability of genetic testing centers that can 

provide adequate counseling and whether there are barriers to access them. Such studies may 

involve electronic, mail, or telephone surveys. 

• More studies are needed to understand what forms of surveillance should be offered to MMR 

mutation carriers for HNPCC-related cancers other than CRC. Well-designed controlled 

trials comparing various surveillance (or other management) strategies could be helpful. 

 
157

• More studies are needed to clarify the risk of cancer in family members of probands with an 

HNPCC-related cancer who are found to carry MMR mutations. Such studies would ideally 

be prospective, fully account for interventions (such as cancer screening procedures) in those 

at-risk, and have a well-defined control population of individuals at average risk for cancer. 

• Standards for reporting studies of genetically based diseases (including those addressing all 

aspects of the ACCE model) should be developed. A consensus development process with 

publication of a guideline(s) could be helpful. 

159

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List of Acronyms and Abbreviations 

Acronym/Abbreviation Meaning 

ACCE Four components of test evaluation—analytic validity, clinical 

validity, clinical utility and associated ethical, legal and social 

implications. 

AM1, AM2 Amsterdam I criteria, Amsterdam II criteria 

AHRQ Agency for Healthcare Research and Quality 

BethR-All Testing strategy - Perform MMR testing only among those fulfilling 

the revised Bethesda guidelines 

BethR-IHC Testing strategy - Perform IHC testing on patients fulfilling the 

revised Bethesda guidelines; perform MMR only among those with 

suggestive IHC test 

BethR-MSI Testing strategy - Perform MSI testing on patients fulfilling the 

revised Bethesda guidelines; perform MMR only among those with 

suggestive MSI test 

CDC Centers for Disease Control and Prevention 

CI Confidence interval 

3Clinical-All Testing strategy - Perform MMR testing only among those fulfilling 

at least one of the three simple clinical criteria (age <50y at diagnosis, 

1st degree family history of CRC or endometrial cancer or multiple, 

synchronous or metachronous, CRC or endometrial cancer in the 

same patient) 

3Clinical-IHC Testing strategy - Perform IHC testing on patients fulfilling at least 

one of the three simple clinical criteria (age <50y at diagnosis, 1st

degree family history of CRC or endometrial cancer or multiple, 

synchronous or metachronous, CRC or endometrial cancer in the 

same patient); perform MMR only among those with suggestive IHC 

test 

3Clinical-MSI Testing strategy - perform MSI testing on patients fulfilling at least 

one of the three simple clinical criteria (age <50y at diagnosis, 1st

degree family history of CRC or endometrial cancer or multiple, 

synchronous or metachronous, CRC or endometrial cancer in the 

same patient); perform MMR only among those with suggestive MSI 

test 

CRC Colorectal cancer 

CSGE Conformation sensitive gel electrophoresis 

DGGE Denaturing gradient gel electrophoresis 

DHPLC Denaturing high-pressure liquid chromatography 

DNA Deoxyribonucleic acid 

EGAPP Evaluation of Genomic Applications in Practice and Prevention 
180

Acronym/Abbreviation Meaning 

(EGAPP) Project, Office of Genomics and Disease Prevention, 

Centers for Disease Control and Prevention 

HNPCC Hereditary Nonpolyposis Colorectal Cancer 

hMLH1 MMR gene mutation in the human mutL homolog 1 gene 

hMSH2 MMR gene mutation in the human mutS homolog 2 gene 

hMSH6 MMR gene mutation in the human mutS homolog 6 gene 

hPMS2 MMR gene mutation in the human postmeiotic segregation 2 

IHC Immunohistochemistry 

IHC-All Testing strategy - Perform IHC testing on all patients; followed by 

MMR testing only among those with suggestive IHC test 

MLPA Multiplex ligation-dependent probe amplification 

MMR Mismatch repair 

MMR-All Testing strategy – perform MMR testing on all colorectal cancer 

patients 

MSI Microsatellite instability 

MSI-All Testing strategy - Perform MSI testing on all patients; followed by 

MMR testing only among those with suggestive MSI test 

MSI-high High microsatellite instability 

MSI-low Low microsatellite instability 

MSS Microsatellite stable 

NCI National Cancer Institute 

ND No data 

ROC Receiver operating curve 

SSCP Single-stranded conformation polymorphism 

SROC Summary receiver operating curve 

TEP Technical Expert Panel 
APPENDIXES: 

to 

“Hereditary Nonpolyposis Colorectal Cancer: 

Diagnostic Strategies and Their Implications” 

Prepared by the Tufts New England Medical Center 

Evidence-based Practice Center 

(Contract #290-02-0022) 
A-1

Appendix A. Search Strategy 

"colorectal neoplasms, hereditary nonpolyposis"[MeSH Terms] 

hereditary non-polyposis[tw] 

(HNPCC[tw] 

(MLH1[tw] OR MSH2[tw] OR MSH6[tw] 

(hMSH2[tw] OR hMLH1[tw] OR hPMS1[tw] 

hPMS2[tw] OR hMSH6[tw] OR hMLH3[tw] 

English[Lang] AND "humans"[MeSH Terms] 
B-1

Appendix B. Sample Data Abstraction Forms 

Analytic Validity 

Data Extraction for Analytic Validity 

Study: (Author, year, UI) 

Inclusion criteria (all must be yes) 

Yes No 

Did study evaluate biological material from patients with CRC considered to be at risk for 

HNPCC? 

 

Did the study report ANY of the following? (check which one below) 

1) Proportion MSI-H with NIH markers versus other markers 

2) Sensitivity or specificity of MSI-H using NIH markers compared with a reference 

standard that the study claims is better 

3) Sensitivity or specificity of IHC compared with an immunohistochemical standard that 

study claims is better 

4) Sensitivity or specificity of a genetic technique compared with a reference standard (or 

combination of standards) 

 5) Reliability of MSI/IHC/genetic method across laboratories or within a laboratory 

Are data (proportions or 2 X 2 tables) extractable or reliability data extractable? 

*NIH markers are BAT-25, BAT-26 D2S123, DS346 and D17S250 

Exclusion criteria (exclude if yes) 

 Yes No 

Did the study include the index test in the reference standard? 

Describe source of 

biological materials (and 

whether patients were 

known to have an 

HNPCC phenotype) 

Summarize how 

materials were processed 

and analyzed 

MSI Proportion (add additional 2 X 2 tables where relevant) 

Proportion MSI-H using NIH markers (≥2 markers) Proportion MSI-H using other markers 

 
B-2

MSI with a reference standard 

 MSI-H using another 

reference standard 

Describe 

 Positive Negative 

MSI-H using NIH 

markers 

MSI-H (≥2 markers) 

 MSI-S or MSI-L 

IHC with a reference standard 

 IHC using another 

immunohistochemical 

reference standard 

Describe 

 Positive Negative 

IHC Positive 

Describe Negative 

Genetic technique with a reference standard 

 Reference standard 

genetic technique 

Describe 

 Positive Negative 

Index genetic technique Positive 

Descirbe Negative 

Intra or inter-hospital reliability data Describe 

Of 18 participating centers 2 were excluded: one because 

slides were damaged in transit and the other because of 

insufficient staining. 

Sensitivity for detecting loss of hMSH2 2 expression 

ranged from 84 to 100%; 10 centers identified all six. 5/6 

false positive results were in the same case suggesting 

that staining or interpretation were not random. 

14/16 laboratories showed 100% specificity (one 

laboratory had 93% specificity due to staining failure on 

one slide and one lab demonstrated 45% specificity due to 

weak or absent staining in most cases. 

Re-review of returned hMSH2 slides shoed lack of 
B-3

Intra or inter-hospital reliability data Describe 

internal positive control staining in at least 2 of the 6 

hMSH2-negative cases from 8 of 16 centers. The other 8 

centers had 100% sensitivity and 93-100% specificity on 

re-review. The slides that lacked internal positive control 

staining wre largely accounted for by two cases 

suggesting the possibility of fixation or processing 

variation. 

Variation of staining quality and interpretation was much 

greater for hMLH1 than for hMSH2. individual centers 

reported 0 to 100% sensitivity and 40 to 100% specificity. 

Re-review of the returned slides resulted in sensitivities of 

0-90%. 12 centers experienced difficulty with lack of 

internal positive control or high background. 

Overall, four laboratories performed relatively well with 

both hMLH1 and hMSH2 staining protocols. The key 

element common to these and distinguishing them from 

the rest was a heated antigen retrieval step. Steam 

treatment in the presence of EDTA provided the best 

results although steam and citrate buffer also provided 

acceptable results. 

 Study Quality Yes No Unc 

1 Was the description of how MSI or IHC and other genetic techniques described in sufficient 

detail that others could replicate it (e.g. either a full description or relevant references)? 

 

2 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

 

3 Was MSI, IHC, other genetic testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

 

4 Was there a clear description of which mismatch repair mutations were being tested for? 

5 Were quality control methods described for the molecular and genetic tests? 

6 Did the authors attempt to address the reproducibility of results (reliability of tests)? 

7 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled 

and did not fulfill clinical criteria or those who did or did not have MSI) included in each 

batch analyzed? (i.e. this helps minimize the effect of random errors). 

 

8 Was microdissection performed? 

9 Did the study specify whether the biological tissues were from patients known to have 

HNPCC clinically? 

 

10 Did the study include a control group in which biological material was obtained from 

patients known not to have HNPCC clinically 

 

11 Overall rating (A B C) 
B-4

Clinical Validity Study: (Author, year, UI) Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? Was a minimum of hMLH1 and hMSH2 sequencing performed? 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

 Y N ? 

≥5 MSI markers used? MSI-H defined by ≥ 2 markers? Microdissection? Gene screening? Deletion analysis? Conversion analysis? 

Am 1 

Am R Beth 1 Beth R MSI-H MSI-L IHC Age <50 Suggestive family history Specify Other Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsattelite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
B-5

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 

Amsterdam R Other (specify) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 Amsterdam R Other (specify) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 
B-6

If yes, which clinical criteria (check all that apply)? Am 1 + Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) Other (specify) COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST 

What was the population 

(i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE

Index test Number with

MMR+ 

Number with

MMRAm 1 + + (A) (B) 

Am R + - (C) (D) Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 
B-7

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based u

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification using 

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eithe

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study group

the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who underwent 

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or t

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
B-8

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
B-9

Benefits and Harms 

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl 

Outcomes Dropouts, explanations, other comments Study Conclusions 
B-10

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 5 9 8a 8b,8c 

Public health or policy 

2,3 5 9 8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A)Most overall quality rating scores are an “A” and the results of the study are considered to provide (B) Most overall quality rating scores are a “B” and the results of the study are considered to provide 

(C)Most overall quality rating scores are a “C” and the results of the study are considered to provide w

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 ND NA Allocation Bias A 

(stro
B-11

(RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes No Were data collection tools shown or are they known to be reliable? Yes No Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 ND NA 

Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially No Is there a statistically significant difference between groups? Yes No ND Are the statistical methods appropriate? Yes No ND Indicate the unit of allocation Community Organization/group Provider Client Institution Indicate the unit of analysis Community Organization/group Provider Client Institution If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND NA 
B-12

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No Can’t 

tell 
Appendix C. Evidence Tables 
C - 1 

Study: Aaltonen 1998 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? X Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? X Was a minimum of hMLH1 and hMSH2 sequencing performed? X Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

509 consecutive patients with CRC from 9 hospitals in Finland between May 1994 and April 1996 ie does not overlap with Salovaara mean age 67 

MSI wad done in all patients (Battery of MSI was not common to all), founder mutation 1 in MLH1 was looked for in all patients; MLH1 and MLH2 sequencing was done in MSI positive and founder mutations negative patients 

Founder mutation 1 in MLH1 and other mutations in MLH1 and MLH1 (NB Salovaara tested founder mutation 2 also) 

 Y N ? 

≥5 MSI markers used? 

 x 

MSI-H defined by ≥ 2 markers? 

 x 

Microdissection? x Gene screening? x Deletion analysis? 

 x 

Conversion analysis? 

 x 

Genetic testing varied between first 198 and subsequent 311 pts. MSI testing differed between pts, all however received the BAT26 test and correlation was OK. 

Am 1 

Am R Beth 1 Beth R MSI-H MSI-L X IHC Age <50 Suggestive family history Specify Other x MSI 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 2 

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

509 mean age ? males ? 

10 MMR/509 patients with CRC 9 MLH1 and 1 MSH2 NOT COUNTING THE NONPATHOGENIC MUTATIONS 

4/509 (0.8%) Am I 

Other miniSATs  + - BAT + 58 6 26 - 5 440 

Large scale molecular screening for HNPCC can be done by 2-stage procedure: screening for founders mutations, then MSI, then sequencing for those with + MSI To select who to screen for MSI, use Age <50, family Hx of EC or CRC or multiple tumor in same pt. 

no 

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam 

Amsterdam 1 X Combined 4/4 
C - 3 

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

criteria) what proportion had an MMR gene mutation? 

Amsterdam R 

Other (specify) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 X Amsterdam R X Other (specify) 

Combined 0/4 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

NA 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

 X 

If yes, which clinical criteria (check all that apply)? 

X 

Am 1 + Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) Other (specify) 
C - 4 

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with 

MMRAm 1 + + 4 0 

Am R + - 6 499 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Consecutive CRCs 

Combined Am1&R X 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylat
C - 5 

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + 10 53 

Am R + - 0 446 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Consecutive CRCs 

MSI, Other Sats X 

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + 10 54 

Am R + - 0 443 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Consecutive CRCs 

MSI, BAT26 X 
C - 6 

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 7 

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 8 

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Aarnio, 1997 RefID 2366 Multicenter, Finland 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Focused on same patients as Aarnio 1995 Ref ID 2666 but reported different outcomes. Focused on 51 families with HNPCC affected by gastric cancer. Of 92 families registered in HNPC registry, a mutated MLH1 gene segregated in 45 and a mutated MSH2 in 2 while four met Amsterdam I criteria. In these 51 families, there were 570 family members affected by cancer of whom 62 (10.9%) had gastric cancer and formed the basis for the study. 

Suspected HNPCC by Amsterdam I, some families with known mutation. 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl X 

History of gastric cancer and member of an HNPCC family. 

Examined 24 histology and Performed IHMSI on 20 cain a total of 4
C - 9 

Outcomes Dropouts, 

explanations, 

other comments 

Study Conclusions Quality scor

45 patients with gastric cancer (24 men, 21 women) mean age 56 (range 31-85). Metachronous other cancer occurred in 23 (51%). Total number of tumors 90. There was variation in the occurrence of gastric cancer in families with different MLH1 or MSH2 genes but difference was not statistically significant. 19/24 were intestinal type; 6 (32%) were poorly differentiated, four others (17%) classified as diffuse or mucinous. 7/15 samples (47%)with suitable tissue were MSI-H H. pylori positive in 3/15. Overall 5-year survival 15%; 48% in patients in whom radical surgery was performed. 

ND Gastric cancer was an HNPCC-associated tumor on 

average in 11% of affected family members. Predominant tumor type was intestinal. Existence of this tumor in patients younger than 60 suggests HNPCC in the background. Relative rarity in HNPCC families makes cost-effectiveness of endoscopic screening questionable. 

Significance of survival d

no information on stag

selected for treatment. 

tissues samples, and tissu

in a subset of patients. U

eligible population agreed 

survival/death 
C - 10 

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 x 5 9 8a 8b,8c 

Public health or policy 

2,3 5 9 8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 11 

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely X 

Somewhat likely 

Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 X 

ND 

NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes X 

No 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes X 

No 

NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No X 

ND 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 X 

ND 

NA 

Analysis A (stro
C - 12 

Domain/question Place an “X” in one Is there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? Yes No X 

ND 

Are the statistical methods appropriate? X 

Yes 

No ND 

Indicate the unit of allocation Community X 

Organization/group 

Provider Client Institution 

Indicate the unit of analysis Community X 

Organization/group 

Provider Client Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No X 

Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 X 

ND 

NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes X 

No 

ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

X 

Yes 

No Can’t 

tell 
C - 13 

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Adams, J 2003, RefID 653 Multicenter UK 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Secondary analysis of data recorded for a study on polyp prevention (a database) on chemopreventive effects of aspirin. Study had included 600 patients with HNPCC; of these 154 were available for analysis and full data were available on 134. Of these 134, 49 had a personal history of CRC. Socioeconomic status assessed using the Town-Deprivation Score (TDS) of the current enumeration district of residence. This score is based upon relative material deprivation based upon census data. 

Amsterdam II criteria 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

Inclusion criteria included age >25 and, in those with a previous diagnosis of CRC, at least five years since resection. 

Primary outcbetween TDS colorectal can

Outcomes Dropouts, explanations, other comments Study Conclusions 

There was a statistically non-significant correlation between the TDS and the age of first resection indicating that individuals in less deprived areas tended to have their colorectal cancer resected at an earlier age than those living in more deprived areas. 

Most dropouts from missing data due to wrong postal codes 

More affluent individuals from HNPCC families tended to undergo surgical resection for CRC earlier than less affluent individuals. This relationship bordered on statistical significance aprobably represents socio-economic variations in access to treatment. 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence 
C - 14 

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely Somewhat likely X 

Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 ND X 

NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes X 

No 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes X 

No 

NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes X 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No X 

ND 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 X 

60-79 

<60 ND NA 

Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? Yes X ND 
C - 15 

Domain/question Place an “X” in one 

No 

Are the statistical methods appropriate? X 

Yes 

No ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No X 

Can’t tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 X 

ND 

NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No X 

ND 

NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

X 

Yes 

No Can’t 

tell 
C - 16 

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Aktan-Collan 2000 Ref ID 1798, same patients as Atkan-Collan 2000-2001, Ref ID 866, 1419 and 1835 but studied different outcomes 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Counseling offered to members of 36 families in which a genetic mutation had been identified. Questionnaires filled out before, counseling and then 1 month and 1 year afterward. Of 446, 90% consented to participate, 381 (85%) returned a baseline questionnaire. Education/counseling attended by 347 of whom 333 (96%; 75% of total population) opted for predictive testing. Of these, 271 filled out all three questionnaires. These form the basis of the study group. 

Mutation identified in families 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

As per study description Surveys 
C - 17 

Outcomes Dropouts, explanations, other comments Study Conclusions 

334/381 accepted genetic testing of whom 30% were mutation-positive. Acceptors differ from decliners in more often having a spouse or partner, being employed and having higher education. On multivariate analysis, the only significant factor for accepting genetic testing was employment status. At 1-month, there was no difference in satisfaction in decision to undergo testing in mutation positive and negative subjects. At one-year, subjects who had not inherited a mutation were slightly more satisfied. Most common reasons cited against undergoing testing were: being old already and having no children (23%), would not trust result (15%), timing found unsuitable because of pregnancy 1 (8%), no reason given (54%) 

ND The possibility of early detection a

of colorectal tumors makes predictHNPCC beneficial. The test was wthe majority of individuals at risk fand few reported having regretted tto make it. Thus, we suggest that a

families with HNPCC should be acinformed about the predictive test bphysician. 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 18 

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

X 

Very likely 

Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 X 

60-79 

<60 ND NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

X 

Yes 

No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

X 

Yes 

No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding X A 

(stro

Was (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND X 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 X 

60-79 

<60 ND NA 

Analysis A 
C - 19 

Domain/question Place an “X” in one 

(stro

Is there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? X 

Yes 

No ND 

Are the statistical methods appropriate? X 

Yes 

No ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND X 

NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

X 

Yes 

No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No 

X 

Can’t 

tell 
C - 20 

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Atkan-Collan, 2000 RefID 1835 Multicenter Finland, Same patients as Atkan-Collan 2001, Ref ID 866 and 1419 but different outcomes 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

446 family members from 36 HNPCC families with known mutations at 50% risk offered counseling and genetic testing. Families contacted by letter. Along with counseling and testing, all sent questionnaires before counseling, 1 month, and 1 year after testing. Of 446 eligible, 401 (90%) agreed to participate and 381 (85%) returned a baseline questionnaire. Educational counseling attended by 347, of whom 333 (96%, 75% of whole group) opted for predictive genetic testing. Study focused on 271 subjects (61% of whole group) who had been tested and returned all questionnaires. 

Families known to have a genetic mutation. 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

See study description. Surveys 
C - 21 

Outcomes Dropouts, explanations, other comments Study Conclusions 

Mean age 43 (range 19-77, SD 13) At one month 88% indicated counseling was easily comprehended. 10% wanted minor changes, most commonly asking for more written material. Those with a university education suggested changes more than those without At one year, pretest counseling considered very useful by 49%, fairly useful by 40% and slightly useful by 10%. Women and those with a lower level of education were significantly more likely to consider counseling useful. At post-test follow-ups, 89 and 95%, respectively, considered a single post-test session sufficient. 53% indicated that they might have used professional psychological support along with counseling if it had been provided. The support might have been used by women with children more than by others. Mutation positive subjects more often reported the greatest need for support soon after hearing the results. 

As per study description` A protocol that includes one-comp

test counseling session and a test dsession, supplemented with option professional psychological supportsufficient for both the educational supportive needs of counselees. Onexpressed a need for post-test follosessions. 
C - 22 

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 5 x 9 x 8a x 8b,8c 

Public health or policy 

2,3 5 9 8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 23 

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

X 

Very likely 

Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 X 

60-79 

<60 ND NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

X 

Yes 

No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

X 

Yes 

No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding X A 

(stro

Was (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND X 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 X 

60-79 

<60 ND NA 

Analysis A 
C - 24 

Domain/question Place an “X” in one 

(stro

Is there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? X 

Yes 

No ND 

Are the statistical methods appropriate? X 

Yes 

No ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND X 

NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

X 

Yes 

No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No 

X 

Can’t 

tell 
C - 25 

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Aktan-Collan 2001, Ref ID 1419, Multicenter Finland. Same patients as Aktan-Collan 2001 Ref ID 866 but studied different outcomes 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Counseling offered to members of 36 families in which a genetic mutation had been identified. Questionnaires filled out before, counseling and then 1 month and 1 year afterward. Of 446, 90% consented to participate, 381 (85%) returned a baseline questionnaire. Education/counseling attended by 347 of whom 333 (96%; 75% of total population) opted for predictive testing. Of these, 271 filled out all three questionnaires. These form the basis of the study group. 

Mutation in participating families 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Unclr  X 

See study description and Aktan-Collan 2001 

Surveys 
C - 26 

Outcomes Dropouts, explanations, other comments Study Conclusions 

Data analyzed according to mutation positive or negative. Fear of cancer, fear of dying soon and satisfaction were life were all modestly (but significantly) different between those who turned out to be mutation positive and negative even after adjusting for age. Mutation positive subjects who had just received their results had significantly higher scores than mutation-negative subjects. After 1 year, the fear of cancer had decreased from baseline in both groups. Mutationpositive subjects were slightly, although significantly, more afraid of cancer at every measurement. 

 As per study description Counseling and testing relieve fear 

harmful emotional impact was deteyear follow-up. These findings shoafter a longer interval. 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 27 

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

X 

Very likely 

Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 X 

60-79 

<60 ND NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

X 

Yes 

No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

X 

Yes 

No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding X A 

(stro

Was (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND X 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 X 

60-79 

<60 ND NA 

Analysis A 
C - 28 

Domain/question Place an “X” in one 

(stro

Is there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? X 

Yes 

No ND 

Are the statistical methods appropriate? X 

Yes 

No ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND X 

NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

X 

Yes 

No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No 

X 

Can’t 

tell 
C - 29 

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Aktan-Collan 2001, Ref ID 866 Multicenter Finland 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Described insurance behavior of 271 patients who had undergone predictive genetic testing for HNPCC. 446 family members from 36 HNPCC families with known mutations at 50% risk offered counseling and genetic testing. Families contacted by letter. Along with counseling and testing, all sent questionnaires before counseling, 1 month, and 1 year after testing. Of 446 eligible, 401 (90%) agreed to participate and 381 (85%) returned a baseline questionnaire. Educational counseling attended by 347, of whom 333 (96%, 75% of whole group) opted for predictive genetic testing. Study focused on 271 subjects (61% of whole group) who had been tested and returned all questionnaires. 

Mutation identified in family. 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

See study description. Surveys 
C - 30 

Outcomes Dropouts, explanations, other comments Study Conclusions 

Mutation-positive subjects reported that they already had a life insurance before entering study more often than mutation-negative subjects (36 versus 28%) but results were not statistically significant. The difference in possessing a health insurance plan before genetic counseling between mutation-positive and negative individuals was significant (21 versus 11%). 3% indicated that they had purchased a life insurance policy and 2% correspondingly a health insurance policy before they were tested. 

See study description Mutation-positive subjects did not 

others in purchase of life or health policies. However, the mutation-poindividuals reported that they possinsurance policies before entering tmore often than their counterparts. 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 31 

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

X 

Very likely 

Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 X 

60-79 

<60 ND NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

X 

Yes 

No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

X 

Yes 

No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding X A 

(stro

Was (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND X 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 X 

60-79 

<60 ND NA 

Analysis A 
C - 32 

Domain/question Place an “X” in one 

(stro

Is there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? X 

Yes 

No ND 

Are the statistical methods appropriate? X 

Yes 

No ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND X 

NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

X 

Yes 

No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No 

X 

Can’t 

tell 
C - 33 

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Arrigoni, A 2005, Ref ID 276 Multicenter, Italy 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Individuals form families fulfilling Amsterdam I criteria advised to take place in a surveillance program as follows (all tests repeated every two years) Colonoscopy starting at age 25 GYN exam, and transvaginal US starting at age 30 Urinary ultrasonography and urine analysis starting at age 30 Endoscopy starting at age 30 Thyroid ultrasonography starting at age 35 Breast mammography starting at age 35 

Amsterdam criteria I 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

As per study description Surveillance 

study descrip
C - 34 

Outcomes Dropouts, explanations, other comments Study Conclusions 

22 families identified with a total of 331 atrisk living relatives of whom 40 had already developed CRC. 199 accepted surveillance while 132 refused. Patients who accepted or refused did not differ significantly for by sex or age. Six individuals accepting surveillance were found to have CRC (3%). Overall incidence of CRC was 7/199 in patients undergoing surveillance and 5/132 in those not (p=NS). However, CRC detected in those undergoing surveillance was significantly less advanced. Seven at-risk individuals were found to have extracolonic cancer during surveillance. Extracolonic cancers were detected in six of 132 who refused. 

As per outcomes. Our study confirms the importance 

a careful family history for each paCRC. Data on a relatively short folto support the efficacy of repeat coin reducing CRC rate in at-risk meHNPCC. Studies with longer followneeded. 
C - 35 

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 x 5 9 8a 8b,8c 

Public health or policy 

2,3 5 9 8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 36 

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely X 

Somewhat likely 

Not 

likely 

What % of selected individuals agreed to participate? 80-100 X 

60-79 

<60 ND NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No X 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes X 

No 

NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes X 

No 

ND NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 X 

ND 

NA 

Analysis A 
C - 37 

Domain/question Place an “X” in one 

(stro

Is there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? X 

Yes 

No ND 

Are the statistical methods appropriate? Yes X 

No 

ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND X 

NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No X 

ND 

NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No X 

Can’t 

tell 
C - 38 

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Arver B 2004 Ref ID 433 Multicenter Sweden 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

1000 families underwent “oncogenetic counseling” between 1995 and 1995. In those where an autosomal dominant breast/ovarian or colon cancer gene mutation was suspected, genetic screening of BrcA1 and BRCA2 or MLH1 and MSH2 offered. A total of 295 BRCA1 and BRCA2 and 57 MLH1 and MSH2 offered. 114 healthy men and women from 73 of the 80 families with an identified mutation opted for genetic testing. Study sample consisted of 90 healthy women. 

Women at risk for a genetic syndrome as defined in study description. HNPCC defined genetically. 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

Healthy females belonging to a family with a known mutation in BRCA1, BRCA2, MLH1 and MSH2 desiring genetic testing, age>18 and Swedish speaking. Men were excluded except if they had a personal history of cancer. 

Surveys cond1 week, 2, 6 were given. Hospital anxSwedish SF-
C - 39 

Outcomes Dropouts, explanations, other comments Study Conclusions 

Al but one woman agreed to genetic testing. The one exception worried about consequences of insurance and employment in the future. Mean age in 87 women was 38.1 in mutation carriers and 41 in non-mutation carriers. A statistically significant decrease in anxiety mean scores over time was observed in total sample irrespective of tested gene or carrier status. Anxiety scores were similar to a normative Swedish population. Women tested for breast cancer predisposing genes showed an average lower levels of depression measured over time than women tested for colon cancer genes but difference was not statistically significant. QOL subscores did not change significantly during observation. Women tested for colon cancer genes showed increased vitality scores over time. However, carriers of colon cancer genes had a decrease in vitality scores two to six months after the result of disclosure, followed by increased levels while levels in non-carriers were more stable. The difference between carriers and non-carriers over time was statistically significant. No differences were found between study group and Swedish normative data on any of the five QOL parameters. 83% thought decision to undergo genetic counseling was “easy”. All but one woman would have redone the testing again. 

1 because of worries about insurance and employment, 1 non Swedish-speaking, 1 did not receive surveys 

Predictive testing for hereditary bror colon cancer gene mutations wilimpaired mental health in motivatewho receive thorough pre- and posinformation and support and for whpreventive surveillance is offered. 
C - 40 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely X 

Somewhat likely 

Not 

likely 

What % of selected individuals agreed to participate? 80-100 X 

60-79 

<60 ND NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes X 

No 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No X 

NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No X 

ND 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? X 

Yes 

No 

Were data collection tools shown or are they known to be reliable? X 

Yes 

No 

Withdrawals and Dropouts X A 
C - 41 

Domain/question Place an “X” in one 

(stro

Indicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

X 

80-100 

60-79 <60 ND NA 

Analysis X 

A 

(stro

Is there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? X 

Yes 

No ND 

Are the statistical methods appropriate? X 

Yes 

No ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No X 

Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND X 

NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

X 

Yes 

No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No X 

Can’t 

tell 
C - 42 

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Benatti, 2001 RefID 1411 Italy, multicenter 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

HNPCC families from the data of a population-based CRC registry were identified in 1984. All incident CRC cases diagnosed in the period 1984-1997 were collected and then stratified according to the presence of 6 clinical features. When a patient had 2 or more of these features, an extended genealogic pedigree was traced and then analyzed for the presence of diagnostic criteria for HNPCC. Once identified, families were considered eligible for the study if they met the inclusion criteria. 29 families met the inclusion criteria: 10 carried MMR gene mutations (group A), 10 were characterized by MSI phenotype but not by MMR gene mutation (group B), and 9 did not show mutations or MSI (group C). Time of followup was calculated from the date of HNPCC diagnosis in the index case to 12/31/1999. Mean duration was 8.2, 6.7, and 9.4 years for group A, B, and C, respectively. 

1) fulfillment of AM-II for HNPCC diagnosis, (2) MSI testing in at least 1 case of CRC or endometrial cancer in the family, or (3) availability of hMLH1 and hMSH2 mutation analysis results. 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl x 

(1) fulfillment of AM-II for HNPCC diagnosis, (2) MSI testing in at least 1 case of CRC or endometrial cancer in the family and (3) availability of hMLH1 and hMSH2 mutation analysis results. 

Observation 
C - 43 

Outcomes Dropouts, explanations, other 

comments 

Study Conclusions 

Baseline clinical features of the 3 groups examined: 

 Group A: 

MMR+ 

Group B: MSI&MMRGroup C: MSS&MMR- P value 

No. of family 10 10 9 Subjects (M/F) 

361 (188/173) 

241 (129/112) 

355 (169/186) ns 

Tumor spectrum  - Colon (% of total)  - Extracolonic HNPCCrelated sites 

66 (58.9) 29 (25.9) 

38 (78.0) 6 (12.0) 

47 (66.2) 5 (7.0) 

<0.005 

Mean age at diagnosis (years)  - Colon  - HNPCCrelated sites  - ExtraHNPCC sites 

47.6 48.4 52.5 

49.0 61.0 53.2 

63.4 65.6 57.6 

<0.05 <0.05 ns 

MSI-H tumors 

25/25 (100%) 

23/25 (92%) 2/26 (7.7%) 

I. Cancer occurrence in the follow-up of the 3 groups: In the follow-up, among HNPCC-related neoplasms, endometrial cancer was more frequent for gastric carcinoma in groups A and B, whereas in group C the stomach was the only extracolonic organ involved. Patients developing CRC in the follow-up were significantly younger (p=0.001) in groups A and B than in group C. II. CRC-specific Survival: The log-rank test revealed a significantly better prognosis for patients from families with a germline MMR gene mutation or MSI (p=0.0001). Furthermore, the presence of MMR gene mutations or MSI, together with stage, appeared as an independent prognostic factor by Cox regression multivariate analysis. 

Retrospective cohort analyses. No dropouts described. Due to significant differences in many factors between the 3 groups at baseline, followup results from univariate analyses (such as analyses for cancer occurrence and log-rank analyses) are not useful. Only results from Cox regression multivariate analyses (variables in the model: groups, sex, age ≤50 vs. >50, Stage, and site) might have some implications for the benefit-and-harm question. Our interpretation of the Cox survival analyses results: After controlling for sex, age, cancer stage, and site of CRC tumor, hazard ratio of dying is significantly higher for Group C (MSS and MMR negative) patients (HR=2.51 95%CI 1.32-4.79), when compared to the Group A (presence of MMR gene mutations) patients. There was no statistical difference in the hazard ratio of dying between group A and group B patients after controlling for sex, age, cancer stage, and site of CRC tumors. 

Authors’ conclusions: “Our results support the hypothesis biomolecular heterogeneity of HNPreflected in heterogeneous clinical confirm the value of MSI analysis marker for MMR gene mutations. Tsimilarities between group A and gpatients indicate the MSI testing coto better define clinical HNPCC faidentifying groups with different ricarrying genetic defects.” 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 44 

Domain/question Place an “X” in one Selection Bias A 

(stron

Are individuals selected to participate likely to be representative of target population? 

Very likely Somewhat likely 

x 

Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 ND X NA Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stron

Is the method of random allocation stated Yes No If the method of random allocation is stated, is it appropriate Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stronPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes 

x 

No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No 

x 

NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes 

x 

No 

Blinding A (stronWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND 

x 

NA 

Data Collection methods A x 

(stron

Were data collection tools shown or are they known to be valid? Yes x No Were data collection tools shown or are they known to be reliable? Yes x No Withdrawals and Dropouts A (stronIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 ND 

x 

NA 

Analysis A (stronIs there a sample size calculation or power calculation Yes Partially No Is there a statistically significant difference between groups? Yes No ND Are the statistical methods appropriate? Yes No ND Indicate the unit of allocation Community Organization/group Provider Client Institution Indicate the unit of analysis Community Organization/group Provider Client Institution If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No NA 

Is the analysis performed by intervention allocation status (i.e. Yes No Can’t 
C - 45 

Domain/question Place an “X” in one intention to treat) rather than the actual intervention received? tell Intervention Integrity A (stronWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No Can’t 

tell 
C - 46 

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Bermejo 2005 UI 16344057 Sweden Multicenter 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

The families of the Swedish FamilyCancer Database with at least 4 generations (N=566,877) were classified according to Amsterdam or Bethesda criteria. Survival methods were used to assess the risk of cancer in the classified families, the prognosis of cancer patients, and the risk of subsequent malignancies after CRCs and after CRCs/endometrial carcinomas. 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  x 

Families with at least 4 generations were included. If a founder parent of the family was missing or if they had married several times, the family was excluded. Overlap between families was possible, eg, individuals in the 4th generation could 

belong to 4 different families. The ages of the parents were unlimited, but the maximum age in the second generation was 70 years. 

Outcomes Dropouts, explanations, other comments Study Conclusions 

N=566,877 families 21 fulfilled Am I; 42 fulfilled Am II; 5095 fulfilled Beth (0.9%) 

1. By age 75 yr, the cumulative risk of CRC in Am I families was 57.1% (95% CI, 46-68.8%); the 

cumulative risk of CRC in Am II families was 41.2% (95%CI, 33.3-50.1%) 

2. The cumulative risk of CRC in Beth families was 41.7% (95%CI, 39.4-44.1%) in men, and 23.3% 

(95%CI, 22.2-24.4%) in women. 

3. The cumulative risk of endometrial cancer in Am II by age 75 yr was 45.4% (95%CI, 34.1-58.5%), 

and it was 8.7% (95%CI, 8-9.5%) in Beth. 

4. For ovarian cancer, the cumulative risk by age 75 yr in Beth was 4.7% (95%CI, 4.2-5.3%); for gastric 

cancer, it was 2.3% (95%CI, 2-2.7%). 

5. Families that fulfilled the Beth criteria showed increase risk of cancer in the colorectum, 

endometrium, small bowel, ovary, stomach, bile ducts, renal pelvis, and ureter; members of Beth 

criteria families were at decreased risks of lung and cervical cancers. 

6. The prognosis of cancer in the ureter, renal pelvis, stomach, ovary, and colorectum, but not in the 

endometrium, was better in Beth criteria than in nonclassified families. 

“Most malignancies in the classifiereflect typical features of HNPCC. presented should help to define surstrategies for members of families criteria for HNPCC testing.” 
C - 47 

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 5 9 8a 8b,8c 

Public health or policy 

2,3 5 9 8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 48 

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 ND NA Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes No Were data collection tools shown or are they known to be reliable? Yes No Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 ND NA 

Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially No Is there a statistically significant difference between groups? Yes No ND Are the statistical methods appropriate? Yes No ND Indicate the unit of allocation Community Organization/group Provider Client Institution Indicate the unit of analysis Community Organization/group Provider Client Institution If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No NA 
C - 49 

Domain/question Place an “X” in one Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No Can’t 

tell 
C - 50 

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Bertario 1999, Ref ID 2106 Multicenter, Italy 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

2340 CRC patients consisting of 3 groups (144 HNPCC, 161 FAP, 2035 sporadic). Comparative survival estimated. FAP=familial adenomatous polyposis

Amsterdam criteria 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

As per study description Cumulative p

survival. For HNPCC up procedureprotocols wewith sporadic Taking into crisk of extrachereditary-coalso subjecteexaminations spectrum of t

Outcomes Dropouts, explanations, other comments Study Conclusions Q

No substantial survival advantage for patients with HNPCC and FAP versus sporadic group after adjustment for age, gender, stage and tumor location. 

ND No substantial survival advantage for HNPCC 

and FAP compared with sporadic group after adjustment for age, gender, stage, and tumor location. HR for HNPCC was 1.01 (95% CI 0.72-1.39) compared with sporadic. 

Shoreliamay 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence 
C - 51 

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely X 

Somewhat likely 

Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 X 

ND 

NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

X 

Yes 

No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

X 

Yes 

No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No X 

ND 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 X 

ND 

NA 

Analysis A 
C - 52 

Domain/question Place an “X” in one 

(stro

Is there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? Yes X 

No 

ND 

Are the statistical methods appropriate? X 

Yes 

No ND 

Indicate the unit of allocation Community X 

Organization/group 

Provider Client Institution 

Indicate the unit of analysis Community X 

Organization/group 

Provider Client Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 X 

ND 

NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No X 

ND 

NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No X 

Can’t 

tell 
C - 53 

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Berth 2002 Ref ID 985 Germany single center 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Survey sent to 2076 persons of the general population and 36 patients at high risk for HNPCC or FAP. FAP=familial adenomatous polyposis

ND 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

As per study description Survey regar

testing. 

Outcomes Dropouts, explanations, other 

comments 

Study Conclusions Qualit

“overall, both the general population and at-risk persons hold a favorable attitude toward genetic testing. 

ND Attitudes of persons at risk for HNPCC and 

FAP similar to general population. 

Validity of su

details prov

differences 

responders, d

or any o

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 54 

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

X 

Very likely 

Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 X 

ND 

NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No X 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND X 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 X 

ND 

NA 
C - 55 

Domain/question Place an “X” in one Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? X 

Yes 

No ND 

Are the statistical methods appropriate? X 

Yes 

No ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 X 

ND 

NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No X 

ND 

NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

X 

Yes 

No Can’t 

tell 
C - 56 

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Bleiker, EM 2005, Ref ID 268 Multicenter, Netherlands 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

178 patients at high risk for CRC (27 Amsterdam I criteria 43 familial CRC occurrence). All had received counseling from a geneticist and were advised to have periodic colonoscopy. 

As per study description. Selected patients who fulfilled Amsterdam were tested for mutations. 21% had been treated for CRC and/or endometrial cancer in the past. 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

As per study description Objective an
C - 57 

Outcomes Dropouts, 

explanations, other 

comments 

Study Conclusions 

149/178 returned a completed questionnaire. Respondents significantly older than nonrespondents. Average f/u 5.8 years 61% of study sample fulfilled Amsterdam I Objective compliance data available for 132/149. Of these 95 (72%) were found to be compliant with screening advise. Of 37 (28%) noncompliant, 20 had postponed screening for at least 1 year, 13 were screened only once and 4 never underwent screening. Compliers were significantly younger than noncompliers and there was a nonsignificant trend to increased level of education, mutation status and type of colon screening. No difference between perceived colon cancer risk. Noncompliers significantly more likely to perceive barriers to screening. Noncompliers rated screening as being more embarrassing. On multivariate analysis only the perceived number of barriers to screening was associated significantly with noncompliance (OR 1.2, 95% CI 1.1-1.3). Of self-reported data, noncompliance was associated with embarrassment with screening procedure and sigmoidoscopy rather than colonoscopy. Compliers were significantly more likely to have remembered receiving a reminder letter. 

ND Although few high-risk individuals abstain from 

screening entirely, approximately 1 in 4 deviates significantly from the recommended frequency of screening. Increased compliance may be achieved by reducing the discomfort and embarrassment associated with the procedure and the use of reminder letters. 

S

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 58 

Domain/question Place an “X” in one Selection Bias X 

A 

(stro

Are individuals selected to participate likely to be representative of target population? 

X 

Very likely 

Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? X 

80-100 

60-79 <60 ND NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes X 

No 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

X 

Yes 

No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No X 

ND 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts X 

A 

(stro

Indicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

X 

80-100 

60-79 <60 ND NA 

Analysis X 
C - 59 

Domain/question Place an “X” in one 

A 

(stro

Is there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? X 

Yes 

No ND 

Are the statistical methods appropriate? X 

Yes 

No ND 

Indicate the unit of allocation Community Organization/group X 

Provider 

Client Institution 

Indicate the unit of analysis Community Organization/group X 

Provider 

Client Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity X 

A 

(stro

What % of participants received the allocated intervention or exposure of interest? 

X 

80-100 

60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

X 

Yes 

No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes X 

No 

Can’t 

tell 
C - 60 

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Boks, DE 2002, Ref ID 1059 Multicenter, Netherlands 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

66 patients with endometrial cancer from 46 families in an HNPCC registry. 

Had an HNPCC mutation OR met revised Amsterdam criteria 38/50 patients included in final analysis had a mutation (19 MLH1, 16 MSH2 and 3 MSH6) 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl 

X 

Cancer diagnosis verified by medical record or pathology report. 

Survival commatched contcase) of patiefrom a populother malign

Outcomes Dropouts, explanations, other 

comments 

Study Conclusions 

No significant difference in distribution of tumor histologic types between cases and control. Survival rates for cases and controls were not significantly different. Cumulative 5-years survival 88 versus 82% for HNPCC versus sporadic. No difference in survival was observed between patients in the study group with a mutation identified and those without an mutation.No significant difference in survival for age or stage matching. 

16 patients excluded from survival analysis because stage of cancer could not be verified or because a matched control 

The outcomes in survival for endomin the general population and womfamilies with HNPCC do not differ 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 61 

Domain/question Place an “X” in one Selection Bias X 

A 

(stro

Are individuals selected to participate likely to be representative of target population? 

X 

Very likely 

Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 X 

ND 

NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

X 

Yes 

No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

X 

Yes 

No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No X 

ND 

NA 

Data Collection methods X 

A 

(stro

Were data collection tools shown or are they known to be valid? X 

Yes 

No 

Were data collection tools shown or are they known to be reliable? X 

Yes 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 X 

60-79 

<60 ND NA 

Analysis X 

A 

(stro
C - 62 

Domain/question Place an “X” in one Is there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? Yes X 

No 

ND 

Are the statistical methods appropriate? X 

Yes 

No ND 

Indicate the unit of allocation Community X 

Organization/group 

Provider Client Institution 

Indicate the unit of analysis Community X 

Organization/group 

Provider Client Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes X 

No 

Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND X 

NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND X 

NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No 

X 

Can’t 

tell 
C - 63 

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Bradshaw 2003, Ref ID 692 Single center UK 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

A two year cohort study described results of colonoscopy surveillance in a total of asymptomatic patients with a family history of CRC at a young age but who do not fulfill the Amsterdam criteria and those who fulfill the Amsterdam criteria. 

Amsterdam I Criteria 

Did all patients have a 

personal history of an 

HNPCC-related 

cancer? 

Check one 

Yes No Uncl  X 

Asymptomatic individuals attending an appointment for 

CRC risk assessment with a genetic counselor between 2000 and 2002. Risk definitions: Low risk - Anyone not fulfilling medium or high risk criteria. Moderate risk – with family history of CRC but not fulfill Amsterdam criteria High risk - with a family history fulfilling the Amsterdam criteria 1 Fifty three individuals with a high risk family history and 123 individuals assessed at moderate risk were eligible for and had colonoscopic evaluation.

Excluded patients with FAP. 

Indifulf1 (hrecocolofrom MocoloyeayouCRC in candnormyea

Outcomes Dropouts, explanations, other 

comments 

Study Conclusions Qualit

No cases of CRC were detected. 4 patients (8%) in high-risk group had adenomas. In moderate group, overall five individuals had an adenoma (2%). Proportion of patients younger than 50 found to have an adenoma was significantly greater in the high risk group than those in the moderate risk group (p=0.05). 

448 attended genetic service for risk assessment. 53 with a high risk family history and 123 at moderate risk had a colonoscopy. Colonoscopy was complete to the cecum in 92%. See Figure 1 in original paper for detailed accounting of dropouts. 

These findings indicate that the prevalence of significant neoplasia in groups defined by family history is low, particularly in younger age groups. These prospective data call into question the value of colonoscopy before the age of 50 in moderate risk individuals. 

Low comp

suggests exam

optimal

endoscopists

post-hoc. Stud

Short followu

endoscopis
C - 64 

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC-related 

cancers/factors such as race, 

age sex, comorbidities that 

may be related to 

accuracy/penetrance 

Be

o

mu

dete

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 5 9 8a 8b,8c 

Public health or policy 

2,3 5 9 8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 65 

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely X 

Somewhat likely 

Not 

likely 

What % of selected individuals agreed to participate? X 

80-100 

60-79 <60 ND NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No X 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No X 

NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No X 

ND 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts X A 

(stro

Indicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

X 

0-100 

60-79 <60 ND NA 
C - 66 

Domain/question Place an “X” in one Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? X 

Yes 

No ND 

Are the statistical methods appropriate? Yes X 

No 

ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

X 

80-100 

60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No X 

ND 

NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes X 

No 

Can’t 

tell 
C - 67 

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Brain, K, 2003, Ref ID 895 Single center UK 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Survey obtained from 16 regional genetic centers involved in predictive testing for HNPCC. Purpose was to gain evidence regarding core elements of the pre-test counseling protocol for HNPCC. 

ND 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

Center performs genetic testing for HNPCC. 

Questionnairprotocol for p

Outcomes Dropouts, explanations, 

other comments 

Study Conclusions Qu

Average number of pre-test counseling sessions was 2 (range 1-3) with an average of 4 weeks between sessions. Minimum number of sessions was 1 (range 1-2). Counselors included clinical geneticists and genetic nurse and gastroenterologists. Some included home visits, psychiatric consultation, explicit circumstances in which a client would be excluded or postponed from testing. Four centers reported that they were considered shortening the counseling protocol. Centers described a total of 144 topics as being core topics of prêt-test counseling. 

16/20 centers participated. One declined because of few patients counseled. No other details provided. 

“The present findings suggest that although some UK centers are favorable towards shortening pretest counseling for HNPCC, there is some uncertainty regarding the key elements of genetic counseling. There is currently little empirical evidence regarding which components of pretest counseling are effective in promoting good psychological outcomes. Controlled trials are needed.” 

Ver

char

popu

that m

for sp
C - 68 

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC-related 

cancers/factors such as race, 

age sex, comorbidities that 

may be related to 

accuracy/penetrance 

Be

o

mu

dete

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 5 9 X 

8a 

8b,8c 

Public health or policy 

2,3 5 9 X 

8a 

8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 69 

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely X 

Somewhat likely 

Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 ND X 

NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No X 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No X 

NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes X 

No 

ND NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts X A 

(stro

Indicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

X 

80-100 

60-79 <60 ND NA 
C - 70 

Domain/question Place an “X” in one Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? Yes No X 

ND 

Are the statistical methods appropriate? Yes No ND Indicate the unit of allocation Community X 

Organization/group 

Provider Client Institution 

Indicate the unit of analysis Community X 

Organization/group 

Provider Client Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity X 

A 

(stro

What % of participants received the allocated intervention or exposure of interest? 

X 

80-100 

60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

X 

Yes 

No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes X 

No 

Can’t 

tell 
C - 71 

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Brain, K 2005, Ref ID 74 Single center UK 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Randomized controlled trial involving 26 high-risk individuals for HNPCC who were randomly assigned to extended or shortened genetic counseling. 

Mutation 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

Unaffected individuals from families with a known HNPCC mutation. 

Extended (twreflection helshortened (a genetic counQuestionnairimmediately house” measumeasures” of satisfaction aAlso qualitat
C - 72 

Outcomes Dropouts, explanations, other 

comments 

Study Conclusions 

At baseline, counseling preferences were significantly more likely to be for information than for support (Z=-298, p=0.003). Participants were very positive in their evaluations of genetic counseling, reporting high levels of satisfaction with the information and support received in both groups (p=0.13). There were high levels of decision satisfaction regardless of study group (p=0.40). Levels of psychological distress were not significantly different (p=0.98). The effect of counseling protocol on knowledge was not significant. Analysis of counseling sessions indicated that counselor’s reflective questions occurred equally in both protocols. 

51 family members of proband invited, 34 agreed to participate, consent form returned by 31 of whom four subsequently declined and one withdrew for “practical” reasons. 

This exploratory study indicates that shortening gcounseling may be an appropriate means of suppdecisions already made by individuals about HNtesting. However, participants would benefit from preparatory information to help them reflect on ipreviously considered, which can then be explorefully as part of a tailored counseling approach. 
C - 73 

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC-related 

cancers/factors such as race, 

age sex, comorbidities that 

may be related to 

accuracy/penetrance 

Be

o

mu

dete

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 5 9 X 

8a 

8b,8c 

Public health or policy 

2,3 5 9 X 

8a 

8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 74 

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

X 

Very likely 

Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 X 

<60 

ND NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes X 

No 

If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes X 

No 

Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No X 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No X 

NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No X 

ND 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts A (stro
C - 75 

Domain/question Place an “X” in one Indicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 X 

<60 

ND NA 

Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? Yes X 

No 

ND 

Are the statistical methods appropriate? X 

Yes 

No ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 X 

<60 

ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No X 

ND 

NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No X 

Can’t 

tell 
C - 76 

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Brodersen, N 2004, Ref 385 Multicenter UK 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Cross sectional study to examine anticipated reactions to genetic testing for HNPCC in 437 asymptomatic people with a family history of colorectal cancer 

Amsterdam 1 criteria/mutations 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

Registered with St. Mark’s family cancer clinic as being at increased risk for CRC because of a family history. All receiving regular colonoscopy screening. No FAP in family. No personal history of cancer. 

Questionnairanticipated emintended actiresults. 
C - 77 

Outcomes Dropouts, explanations, other comments Study Conclusions 

A number of significant differences between men and women in anticipated emotional response. More women anticipated feeling fairly or extremely worried if they did not have the test (36 versus 23 percent, p =0.009) or if the test was positive (64 versus 40 percent, p =0.008). More women anticipated that they would feel fairly or extremely regretful if the test showed that they were at high risk (30 versus 20 percent, p =0.03). More women thought that they would feel fairly or extremely angry if the test showed that they were at high risk (33 versus 13$, p <0.001). There was only one significant difference between men and women in anticipated reaction following genetic testing: if at low risk more women then men would try to adopt a healthier lifestyle (p=0.007. When asked if they would have more screening if found to be at high risk, 416/432 (96%) said that they probably (23%) or definitely (74%) would. 

743 questionnaires sent out, 516 returned (71%), 79 who had cancer excluded. 

The emotional effects of receiving are unlikely to be much more severeffects of choosing not to have a geand in some cases not having a genlikely to result in a poorer emotion

Women anticipate more extreme emreactions than men. 
C - 78 

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately convey 

risks and options, 

minimize anxiety, 

inform decisions 

to take tests) 

Accuracy/penetrance testing 

for HNPCC-related 

cancers/factors such as race, 

age sex, comorbidities that 

may be related to 

accuracy/penetrance 

Be

o

mu

dete

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 5 X 

9 

8a 8b,8c 

Public health or policy 

2,3 5 X 

9 

8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 79 

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely X 

Somewhat likely 

Not 

likely 

What % of selected individuals agreed to participate? 80-100 X 

60-79 

<60 ND NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes X 

No 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No X 

NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No X 

ND 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 X 

60-79 

<60 ND NA 

Analysis A 
C - 80 

Domain/question Place an “X” in one 

(stro

Is there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? X 

Yes 

No ND 

Are the statistical methods appropriate? X 

Yes 

No ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 X 

60-79 

<60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

X 

Yes 

No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No X 

Can’t 

tell 
C - 81 

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Brown, S 1998, Ref ID 2147 Single center, UK 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

157 with a history of CRC who developed a second primary in the HNPCC spectrum (CRC, stomach, urinary, ovary, endometrial) compared with 444 with a single colorectal cancer. 

Amsterdam I criteria and modified criteria. 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl X 

History of CRC Second primary in HNPCC spectrum Second CRC between 1992-1995 Comparison group: history of CRC but no evidence of second primary cancer in HNPCC spectrum, colitis, or FAP. 

Interview/famCompared obdegree relativof CRC for thage and sex. 

Outcomes Dropouts, explanations, other 

comments 

Study 

Conclusio

For multiple primary group, 24 probands (16%) had one 1st-degree relative with CRC, five 

(4%) had two, and five (4%) had three or more first degree relatives. The frequency of familial cancer was similar to that of the comparison group. In the study group, 10 (8%) fulfilled the Amsterdam criteria and six additional were considered to be likely to have HNPCC. This compared with only three families (0.7%) from the comparison group who were considered likely to have HNPCC. RR of CRC among 1st degree relatives was 3.2 (p<0.00001). 

RR of extracolonic cancers: In single primary group, both HNPCC associated and nonHNPCC associated extracolonic cancers were seen at the expected frequency. In the multiple primary group, extracolonic non-HNPCC cancers were also seen at the expected frequency. However, HNPCC associated extracolonic cancers were seen at over twice the frequency of the general population. 

Control patients: 595 approached, 4 excluded because of IBD, 1 because of FAP, 4 because they were adopted. 56 had died and had not traceable next of kin while 34 patients and 15 next of kin refused to give a history, leaving 478 patients. Of these 34 had documented multiple primaries and were excluded leaving 444. 166 met multiple primary cancer criteria of whom 157 were approached and 128 provided a complete family history. 

This study highlights timportance taking a family histoin patients with multipprimary cancers and indicates thrisk of malignancy their relativ
C - 82 

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC-related 

cancers/factors such as race, 

age sex, comorbidities that 

may be related to 

accuracy/penetrance 

Be

o

mu

dete

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 5 9 8a X 

8b,8c 

Public health or policy 

2,3 5 9 8a X 

8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 83 

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely X 

Somewhat likely 

Not 

likely 

What % of selected individuals agreed to participate? 80-100 X 

60-79 

<60 ND NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No X 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes X 

No 

NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No X 

ND 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 X 

60-79 

<60 ND NA 
C - 84 

Domain/question Place an “X” in one Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? X 

Yes 

No ND 

Are the statistical methods appropriate? Yes No X 

ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 X 

60-79 

<60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

X 

Yes 

No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No X 

Can’t 

tell 
C - 85 

Study: Calistri, D 2000 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? X Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? X Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

Multicenter, Italy 45 unrelated patients with CRC either: fulfilling Amsterdam criteria, from families meeting 2/3 Amsterdam criteria, CRC < 50 but no family history, at least one-first degree relative with CRC, or multiple neoplasms 

Tissue samples from cancer analyzed for MSI. DNA from peripheral blood samples analyzed for hMSH2 and hMLH2 

hMSH2 and hMLH2 Y N ? 

≥5 MSI markers used? 

X 

MSI-H defined by ≥ 2 markers? 

X 

Microdissection? X Gene screening? X Deletion analysis? 

 X 

Conversion analysis? 

 X 

Am 1 

Am R Beth 1 Beth R MSI-H X MSI-L X IHC Age <50 Suggestive family history 

X 

Specify See inclusion criteria Other Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 86 

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

45 enrolled, no dropouts 25/45 male this is a convenience sample; dropouts do not apply, mean age 51.5 

8 mutations noted 4 hMLH1 and 4 hMSH2 

16/45 tumors MSI-H 4/45 tumors MSI-L 25/45 MSS 

ND 

As the most likely mutation carriers, HNPCC subjects [i.e. Amsterdam criteria] might be directly analyzed for gene mutations while in non-HNPCC subjects, MSI should be used to select for MMR. 

ND 
C - 87 

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 X 

Amsterdam R Other (specify) 

7/13 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 X Amsterdam R Other (specify) 

11/13 MSI-H 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 X 

Amsterdam R Other (specify) 

 ND 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

X 

If yes, which clinical criteria (check all that apply)? Am 1 + X Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) X Other (specify) COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAmsterdam 1 criteria versus Am 1 + X + (A) 7 (B) 6 
C - 88 

Am R + - (C) 1 (D) 31 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) 

other family history as above 

Other (specify)** 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 7 (B) 11 

Am R + - (C) 1 (D) 26 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** X 

>2/3 Amsterdam criteria OR “partially documented” Amsterdam criteria versusother family history as above What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 6 (B) 5 

Am R + - (C) 0 (D) 2 Beth 1 + Beth R + MSI-H* X MSI-L* 

Amsterdam criteria 

Age <50 
C - 89 

IHC (no staining) Other (specify)** 

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 1 (B) 1 

Am R + - (C) 0 (D) 15 Beth 1 + Beth R + MSI-H* X MSI-L* Age <50 IHC (no staining) Other (specify)** 

“Family history” What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 0 (B) 0 

Am R + - (C) 0 (D) 7 Beth 1 + Beth R + MSI-H* X MSI-L* Age <50 IHC (no staining) Other (specify)** 

“Juvenile cancer”  Note, did not extract for “multiple tumors” since only one patient. 
C - 90 
C - 91 

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 92 

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 93 

Data Extraction for Analytic Validity 

Study: (Casey, 2005, RefID 258) Inclusion criteria (all must be yes) Did study evaluate biological material from patients with CRC considered to be at risk for HNPCC? Did the study report ANY of the following? (check which one below) 

1) Proportion MSI-H with NIH markers versus other markers 2) Sensitivity or specificity of MSI-H using NIH markers compared with a reference standard that the study claims is bett3) Sensitivity or specificity of IHC compared with an immunohistochemical standard that study claims is better 4) Sensitivity or specificity of a genetic technique compared with a reference standard (or combination of standards) 

 5) Reliability of MSI/IHC/genetic method across laboratories or within a laboratory Are data (proportions or 2 X 2 tables) extractable or reliability data extractable? *NIH markers are BAT-25, BAT-26 D2S123, DS346 and D17S250 Exclusion criteria (exclude if yes) 

 Yes No 

Did the study include the index test in the reference standard? x 
C - 94 

Describe source of biological 

materials (and whether patients 

were known to have an HNPCC 

phenotype) 

Summarize how materials were processed and analyzed 

Genomic DNA was isolated from lymphoblastoid cell lines from each participant using a blood kit (Qiagen Inc, Valencia, Calif ). The majority of cases (85 of 89) were selected because they also had prior evidence of a defect in mismatch repair due to having either a tumor with high microsatellite instability or loss of expression of a mismatch repair protein by immunohistochemistry.

Goal: Conversion analysis vs. conventional analysis and DNA sequencing (MLH1, MSH2, and Mwere selected for study based on a number of clinical characteristics and the presence of defective 

repair either by microsatellite instability testing or immunohistochemistry, or both. Cases included patients from HNPCC Amsterdam 1 Criteria Families, 8 HNPCC-like cases (at least 2 first- or secrelatives with colorectal cancer or 1 relative with endometrial cancer and at least 1 other relative wcancer who did not meet Amsterdam 1 criteria), and 17 colorectal cancer cases diagnosed prior to 

All patients received both conversion analysis and conventional analysis and DNA sequencing for identity of the participant samples, family history of cases, and data on MSI or IHC were blinded study.DNA sequencing: PCR technique. DNA sequencing was performed on all cases for mutations in M

MSH2. MSH6 sequencing was performed only on those cases (n=23) that were negative for deletein MLH1 or MSH2. Mutations were confirmed by repeating the PCR amplification reaction and seConversion analysis: Hybrid cell lines were generated following conversion technology protocols lymphoblastoid cells from participants with E2 mouse cells. The E2 cell line is an immortal mousline. The markers used for genotyping were derived from the linkage mapping set (version 2.5, MBiosystems). Polymerase chain reaction amplification of the DNA markers was performed with Tbuffer (Invitrogen, Carlsbad, Calif). The PCR products were fractionated by capillary electrophoreABI 3100 automated DNA sequencer. Total RNA was isolated using the RNeasy Kit (Qiagen) and generated using Super Script II reverse transcriptase (Invitrogen). Both positive and negative revecDNA reactions were performed from each RNA source. Conversion analysis combines the separinto hybrids along with analysis of cDNA sequence changes and effects on mRNA expression. ChmRNA transcript size or levels of mRNA expression of MSH2 and 

MLH1 genes were determined by reverse transcriptase PCR from hybrids containing chromosome
C - 95 

Genetic technique with a reference standard Overall results; results for each subgroup of patients can be segregated. 

Reference standard genetic technique: 

conventional analysis and DNA 

sequencing

Positive: deleterious Negative 

Index genetic technique: Positive: deleterious 35 18 Conversion analysis Negative 28 8 (5 no mutation and 3 insufficient data reported 

as unclassified variant)

*Mutations were considered pathogenic (or deleterious) if the change met any of the following criteria. a frameshift mutation that woula truncated protein; nonsense mutations; missense mutations if additional mRNA expression data revealed aberrant splicing or exon ski

mutations if additional data revealed aberrant splicing; large genomic deletions that removed at least 1 exon; or duplication of exons. Fo

missense mutations of unknown clinical significance were not classified as deleterious because additional data are needed.

Intra or inter-hospital reliability data Describe 

ND 

 Study Quality 

1 Was the description of how MSI or IHC and other genetic techniques described in sufficient detail that others could replicate it (e.

either a full description or relevant references)? 

2 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

3 Was MSI, IHC, other genetic testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

4 Was there a clear description of which mismatch repair mutations were being tested for? 5 Were quality control methods described for the molecular and genetic tests? 6 Did the authors attempt to address the reproducibility of results (reliability of tests)? 7 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or tho

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 

8 Was microdissection performed? 9 Did the study specify whether the biological tissues were from patients known to have HNPCC clinically? 

10 Did the study include a control group in which biological material was obtained from patients known not to have HNPCC clinical11 Overall rating (A B C) 
C - 96 

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Cats 1995; Ref ID 2753, Multicenter, Netherlands 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

30 patients at risk for HNPCC randomly assigned to calcium carbonate (1.5g ) or placebo three times daily for 12 weeks. Primary endpoint was epithelial cell proliferation in colon and rectum. 

Unclear 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

1st-degre relatives of patients with 

HNPCC participating in regular screening program. At 50% lifetime risk of developing CRC 

Calcium supp

Outcomes Dropouts, explanations, other comments Study Conclusions 

Cytolytic activity decreased with calcium but difference was not statistically significant. No difference in sigmoid colon or rectum. 

53 invited 30 participated, reasons for not participating not described. 

Oral calcium was shown to cause ononstatistically significant reduction epithelial cell proliferation in the recompared with placebo and have no sigmoid and descending colon. Thecast doubt on the value of calcium supplementation in the prevention oespecially in individuals already coadequate amount of dietary calcium

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 97 

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely X 

Somewhat likely 

Not 

likely 

What % of selected individuals agreed to participate? 80-100 X 

60-79 

<60 ND NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes X 

No 

If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes X 

No 

Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes X 

No 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes X 

No 

NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes X 

No 

Blinding X A 

(stro

Was (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

X 

Yes 

No ND NA 

Data Collection methods X 

A 

(stro

Were data collection tools shown or are they known to be valid? X 

Yes 

No 

Were data collection tools shown or are they known to be reliable? X 

Yes 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs 80-100 X <60 ND NA 
C - 98 

Domain/question Place an “X” in one by groups, record the lowest). 60-79 Analysis X A 

(stro

Is there a sample size calculation or power calculation? X 

Yes 

Partially No 

Is there a statistically significant difference between groups? Yes X 

No 

ND 

Are the statistical methods appropriate? X 

Yes 

No ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity X A 

(stro

What % of participants received the allocated intervention or exposure of interest? 

X 

80-100 

60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

X 

Yes 

No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes X 

No 

Can’t 

tell 
C - 99 

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Cederquist, 2001 Ref ID 1536 Multicenter, Northern Sweden 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Family histories were obtained on persons with colon and endometrial cancer. The cancer risks in their first degree relatives were analyzed; they were estimated in relation to MSI status and age at diagnosis in proband. Regional Cancer Register identified 89 cases; in 10 cases, unable to localize the patient, to verify the diagnosis or to obtain approval to contact the family. Thus, 36 colon-endometrial cancer cases and 43 colon-colon cases were included in this study. 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl X 

First degree families of CRC and endometrial cases (N=649) Probands- Colon-endometrial cancer cases (n=36) and colon-colon cases (n=43) 
C - 100

Outcomes Dropouts, explanations, other comments Study Conclusions 

In ≤ 50-year-old cohorts with MSI positive 

status, standardized incidence ratio (SIR) for colon cancer 21.13 (95% CI 11.82-34.85). In ≤ 50-year-old with MSI negative status, 

significant increase in SIR for stomach and uterus cancer. In > 50-year-old with MSI positive status, significant increase in SIR for rectal and uterus cancer. In > 50-year-old with MSI negative status, no increased risk at all was observed. In total, 649 FDR were analyzed (328 males and 321 females) with a total of 17,088 person years.

 Among first degree relatives, 111 cancers were observed with an expected number of cancers of 65.86, yielding a SIR of 1.69 (95% CI, 1.39-2.03); colon, rectal and uterus cancer exhibited a significantly increased risk. In the ≤ 50-year-old cohort, the overall risk was 2.67 (95% CI 2.08-3.38) compared with the >50-year-old cohort where no increased risk was observed (SIR 1.04, 95% CI 0.74-1.40). In the ≤ 50-year-old cohort, significant increased risk were found for most other HNPCC-associated tumors such as stomach, colon, rectal, uterus and ovary. Colon cancer had a pronounced increased risk with a SIR of 12.57 (95% CI 7.96-18.86). 

 “A first degree relative to a person 

primary cancer of the colorectum ocolon/endometrium have a significincreased risk of having a colorectaHNPCC-associated cancers if the pdiagnosed with one of the cancers 50.” 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 101

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely 

X 

Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 

X 

60-79 <60 ND NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated Yes No If the method of random allocation is stated, is it appropriate Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No Can’t 

tell 

X 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No 

X 

ND NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes No 

X 

Were data collection tools shown or are they known to be reliable? Yes No 

X 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 

X 

60-79 <60 ND NA 

Analysis A (stroIs there a sample size calculation or power calculation Yes Partially No 

X 

Is there a statistically significant difference between groups? Yes No ND 
C - 102

Domain/question Place an “X” in one 

X 

Are the statistical methods appropriate? Yes 

X 

No ND 

Indicate the unit of allocation Community Organization/group Provider Client Institution Indicate the unit of analysis Community Organization/group Provider Client Institution If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No Can’t 

tell 
C - 103

Study: Christensen, M 2002 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of strategy 

used for testing 

patients with CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply

Multicenter, Denmark 42 patients with colorectal cancer selected based upon clinical and family history meeting either Amsterdam I criteria (n=11) or a suggestive family history. 

MSH2 and MLH1 genes sequenced in 31 patients. MSI obtained in 35 patients IHC performed in 40 patients. Compared sensitivity/specificity of these tests against sequencing as the reference standard. 

hMLH1 and hMSH2 Y N ? 

≥5 MSI markers used? 

X 

MSI-H defined by ≥ 2 markers? 

X 

Microdissection? X Gene screening? X Deletion analysis? 

 X 

Conversion analysis? 

 X 

Am 1 

Am R Beth 1 Beth R MSI-H X MSI-L IHC X Age <50 Suggestive family history Specify Other Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 104

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

42 enrolled, all analyzed as above, sex not described, age not described 

MMR detected in 13/42 patients [Note that only 31 pts got gene sequencing!] 8 hMSH2 5 hMLH1 (one patient had both) 

11/42 Amsterdam I 11/25 tested had MSI-H 9/29 tested had lack of staining on IHC 

There was concordance between sequencing and the combined MS/IHC approach for 94% (16/17) tumors. Concordance among the three methods was found for 74% (17/23) tumors 

IHC should be used in combination with MSI analysis to prescreen suspected HNPCC patients for selection for sequencing. 

ND 
C - 105

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 X 

Amsterdam R Other (specify) 

4/11 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 X Amsterdam R Other (specify) 

8/11 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

4/11 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

X 

If yes, which clinical criteria (check all that apply)? Am 1 + X Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) X Other (specify) 
C - 106

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 11 (B) 6 

Am R + - (C) 1 (D) 16 Beth 1 + Beth R + MSI-H* X MSI-L* Age <50 IHC (no staining) Other (specify)** 

Patients who fulfilled Amsterdam I or were otherwise considered at increased risk for HNPCC *Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with 

MMR+ 

Number with

MMRAm 1 + + (A) Only one patient has MSI-L 

so not extracted 

(B) 

Am R + - (C) (D) Beth 1 + Beth R + MSI-H* MSI-L* X Age <50 IHC (no staining) 

Patients who fulfilled Amsterdam I or were otherwise considered at increased risk for HNPCC 

Other (specify)** 
C - 107

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 9 (B) 3 

Am R + - (C) 4 (D) 15 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) X Other (specify)** 

Patients who fulfilled Amsterdam I or were otherwise considered at increased risk for HNPCC Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 
C - 108

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
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Genetic and molecular testing methods

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 110

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Claes, E 2004, Ref ID337 Single center, Belgium 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Describe motivation, recall of cancer risks and illness representations of 40 individuals who had a predictive test for HNPCC. 

Mutation 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

Self-referred unaffected persons who had a predictive test for HNPCC. 

Surveys prete
C - 111

Outcomes Dropouts, explanations, other comments Study Conclusions 

Motivation for predictive testing: Most common motivations cited were early detection of CRC (95%), early detection of endometrial cancer (93%), knowledge of children’s risk (70%), reduction of uncertainty (38%). Recall and perception of risk: 36/40 correctly recalled the 50% risk of being a mutation carrier Awareness of cancer risk: The majority provided an “accurate” estimation of the lifetime risk of developing CRC. Distress (STAI-Trait scale): Differences between carriers and non-carriers regarding mean pre- and post-test IES scores for colorectal cancer and Stateanxiety were not statistically significant. Although there was a decrease of distress over time in the total group, the decrease was only significant for Stateanxiety and avoidance for colorectal cancer. The difference between pre- and post-test scores in the subgroup of carriers was not statistically significant while the decrease in the group of non-carriers was statistically significant for State-anxiety (Wilcoxon signed ranks test, P < 0.01) Early detection behavior: 26/40 had at least one colonoscopy before predictive testing. All carriers had the intention of having colonoscopies on a regular basis. All female carriers had intention of having yearly examinations of the endometrium. The majority of noncarriers either did not have the intention to have a colonoscopy or only when indicated. 

40/48 included 1 patient who had difficulty concentrating 1 patient did not fill in survey 2 patients who were >70 4 did not fill out survey at 1 month 

“Our results in the first applicants ogenetic testing for HNPCC who camon their own initiative within our clgenetic testing program illustrate thgenetic testing did not induce major 

psychological problems and/or reluadopt health-related behavior in the it is offered in the context of a multapproach.” 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence 
C - 112

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely X 

Somewhat likely 

Not 

likely 

What % of selected individuals agreed to participate? X 

80-100 

60-79 <60 ND NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes X 

No 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No X 

NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND X 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

X 

80-100 

60-79 <60 ND NA 

Analysis A (stro
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Domain/question Place an “X” in one Is there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? X 

Yes 

No ND 

Are the statistical methods appropriate? X 

Yes 

No ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

X 

80-100 

60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes X 

No 

ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No X 

Can’t 

tell 
C - 114

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Claes, E 2005 Ref ID 185. Note same patients as Claes, E 2004, Ref ID337 Single center, Belgium 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Evaluated illness representations, distress, and health-related behavior one-year after disclosure of predictive genetic tests results for HNPCC in 36 carriers and 36 noncarriers. 

Mutations 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

Predictive testing offered to unaffected persons from families in which a DNA MMR mutation had been identified. Excluded age >70 

Surveys befopredictive tes

Outcomes Dropouts, explanations, other comments Study Conclusions 

General distress: Mean scores within normal ranges or lower than population means. Carriers and noncarriers did not differ significantly before testing but carriers had significantly higher scores than noncarriers on cancer-specific distress scores for CRC and endometrial cancer. Impact of genetic testing result: about 1⁄2 of carriers (19/36) and less than 20% of noncarriers (6/36) reported at least one disadvantage of knowing the predictive test result. The main reported disadvantages for carriers were “burden of regular medical examinations” (22%) and the psychological burden (20%). The main disadvantage for noncarriers was that they experienced difficulties due to their favorable test results toward carrier relatives (e.g. survivor guilt). 

One excluded because of difficulty concentrating others because age >70. 72/79 participated. 

“Our study did not reveal major psyproblems, measured by psychometrmeasurements, as a consequence of testing when offered in the context multidisciplinary counseling contexinterview data indicated some indivdifferent problems specifically relatpredictive test (e.g. worry, difficultirelation to other relatives burden of follow-up). The presented data are pregarding the impact on health-relatbehavior.” 
C - 115

Outcomes Dropouts, explanations, other comments Study Conclusions 

All but one carrier and all but 2 noncarriers reported at least one advantage. The two advantages most cited by carriers were “instrumental advantage” (89%) and certainty/reduction of uncertainty (33%). For noncarriers, the two most cited advantages were “reassurance” (50%) and the fact that the children were not at risk anymore (39%). Health-related behavior within the year after the predictive test: 11 carriers (31%) never had a colonoscopy prior to genetic testing. Within the year after disclosure, 27 carriers had a colonoscopy. The three who did not were <25. Thus, compliance with recommendation was 100%. 22 noncarriers (61%) had at last one colonoscopy prior to genetic testing. None had a colonoscopy within the year after testing. *Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 116

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely X 

Somewhat likely 

Not 

likely 

What % of selected individuals agreed to participate? X 

80-100 

60-79 <60 ND NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes X 

No 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No X 

NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND X 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

X 

80-100 

60-79 <60 ND NA 

Analysis A 
C - 117

Domain/question Place an “X” in one 

(stro

Is there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? X 

Yes 

No ND 

Are the statistical methods appropriate? X 

Yes 

No ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

X 

80-100 

60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes X 

No 

ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No X 

Can’t 

tell 
C - 118

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Codori, A 1999, Ref ID 2053 Single center, USA 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Evaluated uptake of genetic testing for HNPCC among 1st-degree relatives of CRC by comparing 77 test acceptors and 181 decliners. 505 1st-degree relatives of CRC from 118 kindreds invited to participate. Final sample included 77 acceptors and 181 decliners (see intervention for definitions). 

Amsterdam criteria I or mutation in family. 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

>18, no personal history of CRC, at least one 1st-degree relative with CRC. 

Flow: 505 invited; 233 interested 264 not interested and 8 undeliverable. Of the 233 Interested, 104 completed phase I (8/104 withdrew, 19/104 entered phase 2 awaiting blood draw and 77/104 entered phase II and had blood draw). Of the 264 not interested 247 were called for interview (the remaining have not been interviewed yet); 181/147 completed the interview. 

Invitation to Acceptors = testing Decliners = dtesting Decliners had acceptors fill
C - 119

Outcomes Dropouts, explanations, other comments Study Conclusions 

Significant predictors of acceptance on multivariate analysis were: perceived ability to cope with a positive gene test result, risk perception, frequency of thoughts about CRC (not at all or rarely versus sometimes or more) and CRC screening history. Coping: ≥90% confident OR 4.0 (95% CI 1.53-10.3, p=0.005) CRC screening: never had a colonoscopy (OR 0.45, 95% CI 0.21-0.93, p=0.030) Cancer thoughts: Rarely/never versus sometimes, often, a lot: OR 0.44 95% CI 0.17-1.13, p =0.088) 

ND “Our study showed that people wh

will get CRC, think about getting ithey can cope with the “bad news,” engage in cancer prevention behavlikely to be tested.” 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 120

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

X 

Very likely 

Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 X 

<60 

ND NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders X A 

(stro

Prior to the intervention, were there between group differences for important confounders reported in the paper? 

X 

Yes 

No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

X 

Yes 

No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes X 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND X 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 X 

<60 

ND NA 

Analysis A (stro
C - 121

Domain/question Place an “X” in one Is there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? X 

Yes 

No ND 

Are the statistical methods appropriate? X 

Yes 

No ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 X 

<60 

ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No X 

ND 

NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No X 

Can’t 

tell 
C - 122

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Collins, V 2005, Ref ID 140 Multicenter, Australia 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

134 invited to participate, 114 returned the baseline questionnaire, 98/114 returned a follow-up questionnaire Evaluate screening and preventive behaviors following predictive testing for HNPCC. Annual colonoscopy beginning at age 25 or 5 years earlier than youngest affected family members recommended in those at risk for HNPCC. Those not carrying the mutation are advised to follow population screening guidelines. Endometrial screening “can be considered” from age 30-35 or even 25 generally with annual transvaginal ultrasound and endometrial sampling in premenopausal women and annual transvaginal ultrasound and CA 125 testing for postmenopausal women. Prophylactic oophorectomy and TAH also considered for known mutation carriers ages 30 to 35 or when childbearing is complete. 

Mutation. 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

Individuals undergoing predictive genetic testing at 1 of 5 familial cancer clinics in Australia between1998-2002. Individuals were eligible who had never had CRC or any of the cancers associated with HNPCC. They were excluded if they had limited literacy in English. 

Surveys 
C - 123

Outcomes Dropouts, 

explanations, 

other comments 

Study Conclusions Qu

Of 114 participants, 32 identified as MMR carriers and 82 as noncarriers. At baseline, 3/12 <25 and 74/101 ≥25 reported a colonoscopy. On multivariate analysis, perceived risk of developing bowel carcinoma was the only variable significantly associated with having a colonoscopy (OR 103, 95% CI 1.00-1.05, p=.03) At 12-month follow-up, 71% of carriers reported having a colonoscopy in the 12 months since receiving their genetic results. Among noncarriers, 8 (12%) also reported a colonoscopy. A significantly higher proportion of those who reported a colonoscopy were mutation carriers or women. This group also had higher levels of perceived risk. However, only carriers status was statistically significant in a multivariate model (OR 20, 95% CI 5.8-68, p<.0001). At baseline 19/63 women (30%) reported ever having a transvaginal ultrasound and 4/58 (7%) an endometrial sampling. At 12 months, 47% (8 of 17)of carriers reported having a transvaginal ultrasound and 53% (9 of 17) endometrial sampling. 39 noncarriers reported either a transvaginal ultrasound (10%) or endometrial sampling (5%). At baseline, no one reported having a prophylactic colectomy; at 12 months, one indicated intention to do so. 

ND The majority of individuals reported 

appropriate screening behaviors after predictive testing for HNPCC. The small group of noncarriers who had screening after genetic testing might benefit from additional counseling. 

12 mon

Effect

at veri

of acc

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 124

Domain/question Place an “X” in one Selection Bias X 

A 

(stro

Are individuals selected to participate likely to be representative of target population? 

X 

Very likely 

Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? X 

80-100 

60-79 <60 ND NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes X 

No 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No X 

NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND X 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts X A 

(stro

Indicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

X 

80-100 

60-79 <60 ND NA 

Analysis X A 
C - 125

Domain/question Place an “X” in one 

(stro

Is there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? X 

Yes 

No ND 

Are the statistical methods appropriate? X 

Yes 

No ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

X 

80-100 

60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

X 

Yes 

No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No X 

Can’t 

tell 
C - 126

Study: Colombino 2005 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of strategy 

used for testing patients 

with CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

Multicenter, Italy 362 patients proven to be of Sardinian origin with histologically proven CRC. All are consecutive, unselected CRC patients 

Family history assessed. Patients classified as sporadic if no CRC in 1st

and 2nd-degree relatives, 

low familial recurrence if one additional family member, familial if at least three family members. HNPCC diagnosed based on Amsterdam criteria. Patients with familiar CRC underwent gene sequencing Sporadic were screened for mutations identified in “familial” 

hMLH1 and hMSH2 

 Y N ? 

≥5 MSI markers used? MSI-H defined by ≥ 2 markers? Microdissection? Gene screening? X Deletion analysis? 

 X 

Conversion analysis? 

 X 

Am 1 X 

Am R Beth 1 Beth R MSI-H MSI-L IHC Age <50 X Suggestive family history 

X 

Specify As in inclusion criteria Other Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 127

Results 

N enrolled, 

Mean age, 

%male 

dropouts, 

reasons for 

dropouts 

Proportion with a 

mismatch repair 

mutation (describe 

which MMR gene). 

(e.g. 23 patients with 

MMR/1000 patients 

with CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

179/362 men=49% Median age at dx 60 (27-87) 

Of 48 patients tested, 14 had a germline MMR: 12 on MSH2, 2 in MLH1 

103/362 “familial” 55/162 two cases 48/362 had at least three family members 13/362 fulfilled Amsterdam 

ND 

Strongly reaffirms several previous predictors (earlier diagnosis age, presence in family of three or more CRC) for occurrence of MLH1 and MSH2 mutations 

ND 

 How was Lynch Syndrome defined 

(check all that apply)? 

Specify numerator and de

comments (ND if n

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 X 

Amsterdam R Other (specify) 

 10/13 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 Amsterdam R Other (specify) 

ND 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

ND 
C - 128

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

X 

If yes, which clinical criteria (check all that apply)? Am 1 + Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) X Other (specify) COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test 

Number with 

MMR+ 

Am 1 + + (A) 16 (B) 87 Am R + - (C) No data (5) (D) No Beth 1 + In parentheses, because the “sporadi

for the mutations found in the “fami

Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** X 

Patients with one additional family member with CRC vs sporadic 

Among 103 with >=2 CRC in family 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 129

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 2 (B) 53 

Am R + - (C) No data (D) No data Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Two cases in family What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 14 (B) 35 

Am R + - (C) No data (D) No data Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

3 cases in family 
C - 130

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + X + (A) 10 (B) 3 

Am R + - (C) (D) Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 6 (B) 55 

Am R + - (C) No data (D) No data Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Age <= 45 
C - 131

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 132

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 133

Study: Curia 1999 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? x Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? x Was a minimum of hMLH1 and hMSH2 sequencing performed? x 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

30 unrelated HNPCC patients from the files of the University of Florence; 17 has Amsterdam criteria 26 have CRC and 15 are Am1 among them 

SSCP and sequencing were done in all patients; IHC were done in 24 patients; MSI were done in 22 patients We caution that the MSI and IHC data are completely convenience-based 

HMLh1 and hMSH2 Y N ? 

≥5 MSI markers used? 

 X* 

MSI-H defined by ≥ 2 markers? 

X 

Microdissection? X Gene screening? X Deletion analysis? 

 X 

Conversion analysis? 

 X 

They screened with 3 and if none was unstable up to a total of 7. 

Am 1 

Am R Beth 1 Beth R MSI-H MSI-L IHC Age <50 Suggestive family history Specify Other Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsattelite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 134

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

30 unrelated patients, but only 26 have CRC 

7 hMLH1 and 5 MSH2 (3 pathogenic, 4 uncertain, 5 polymorphisms) in 30 unrelated patients Pathogenic: 3 (2mlh1 , 1 msh2) 

15/26 eligible were AM1 Unclear if 2 or 1 they refer to “Am criteria” 23/26 were <50 y 12/26 were <40y 

Notably, the lack of hMLH1 or hMSH2 immunostaining and microsatellite instability were observed in tumors from patients in whom a pathogenic mutation could not be identified by SSCP screening of the coding sequence. 

Constitutional alterations in hMLH1 and hMSH2 transcript expression may represent genetic markers for HNPCC carrier status also in cases in which mutational analysis did not detect a definite pathogenic variant. 

 
C - 135

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 x 

Amsterdam R Other (specify) 

1/15 Am1 Eligible has pathogenic 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 Amsterdam R Other (specify) 

12/15 were MSI-H (Am1) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

7/15 had IHC (Am1) 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

 x 

If yes, which clinical criteria (check all that apply)? Am 1 + Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) Other (specify) 
C - 136

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + X + 1 16 

Am R + - 2 11 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Unrelated patients with HNPCC 

MSI only 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test 

Number with

MMR+ 

Number wit

MMRAm 1 + + 1 13 

Am R + - 0 10 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) X Other (specify)** 

Unrelated patients with HNPCC with available specimens 

MSI only 
C - 137

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test 

Number with

MMR+ 

Number wit

MMRAm 1 + + 1 18 

Am R + - 0 3 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Unrelated patients with HNPCC with available specimens 

MSI unstable X 
C - 138

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 139

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 140

Study: De Abajo 2005 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? X Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? X Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

Referrals to the clinic for familial cancer at the San Carlos University Hospital in Madrid. All index cases were CRC 

All tested for MLH1 and MSH2 and the ones who were negative were tested for MSH6 too. 

MLH1 MSH2 MSH6 (among negative for the above) 

 Y N ? 

≥5 MSI markers used? 

X 

MSI-H defined by ≥ 2 markers? 

X 

Microdissection? X Gene screening? X Deletion analysis? 

 X 

Conversion analysis? 

 X 

The NCI/Bethesda marker ser was used 

Am 1 X 

Am R X Beth 1 X Beth R MSI-H MSI-L IHC Age <50 Suggestive family history Specify Other Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 141

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

132 nd on age and sex dropouts NA (only 19 not MSI-typed ) 

24 mlh1 17 msh2 3 msh6 

56/132 AM1 67/132 AM2 NA 

MSH6 disease causing mutations are very infrequent in Spanish CRC referral cases 

ND 

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 

Amsterdam R Other (specify) 

AM1 Æ 32/56 (MLH1 and MSH2AM2 Æ 36/67 (MLH1 and MSH2

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 Amsterdam R Other (specify) 

Am1 Æ 42/55 analyzed were MSIAm2 Æ 44/62 analyzed were MSI

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 
C - 142

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

 X 

If yes, which clinical criteria (check all that apply)? Am 1 + Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) Other (specify) COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + X + 33 23 

Am R + - 11 65 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Referrals to the clinic for familial cancer at the San Carlos University Hospital in Madrid. All index cases were CRC MSH6 is included *Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 143

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + 39 28 

Am R + X - 5 60 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Referrals to the clinic for familial cancer at the San Carlos University Hospital in Madrid. All index cases were CRC MSH6 is included *Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + 44 60 

Am R + - 0 28 Beth 1 + X Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) 

Referrals to the clinic for familial cancer at the San Carlos University Hospital in Madrid. All index cases were CRC MSH6 is included 

Other (specify)** 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 144

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + X + 33 23 

Am R + - 6 5 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Among AM2 patients MSH6 is included *Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check 

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + 39 28 

Am R + X - 5 37 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) 

Among Beth patients MSH6 is included 

Other (specify)** 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 145

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + X + 33 23 

Am R + - 11 37 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Among Beth patients MSH6 is included *Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 146

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 147

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 148

Study: Debniak 2000 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

168 consecutive patients with CRC in whom FAP was excluded. GROUP B: 25were SHNPCC based on age <=40, familial HNPCC-related Ca or synchronous or Metachronous Ca 143 were the rest, apparently sporadic: GROUP A: 43/143 Patients apparently sporadic , ie a) lateonset (>40) no family history of HNPCCrelated tumors and no syn-or metachornous cancer presumably sporadic Poland Center 

IHC performed in all patients MSI examined in all Sequencing performed in all from group B and those from group A that showed abnormal IHC or MSI 

MLH1, MSH2 Y N ? 

≥5 MSI markers used? 

X 

MSI-H defined by ≥ 2 markers? 

X 

Microdissection? X Gene screening? X Deletion analysis? 

 X 

Conversion analysis? 

 X 

Am 1 

Am R Beth 1 Beth R MSI-H X MSI-L IHC X Age <50 Suggestive family history 

X 

Specify Absence of sporadic criteria Other Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsattelite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 149

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

168 but only 68 analyzed, age ND, dropouts ND 

6 mutations detected of the 68 included 4 MLH1 2 MSH2 

Group A: normal protein expression in 42/43 1 patient probably negative staining MSI present in 4/43 tumors Group B: Normal protein expression 18/25 tumors Staining definitively negative MLH1 3 cases, MSH2 in two while in 2 patients, MLH1 was probably absent MSI was present in 9/25 cancers 

Among 9 patients with MSI 2 were “probably abnormal” for protein staining. 

Performing pedigree analysis/clinical data (for exlusion of lateonset sporadic CRC) in conjunction with IHC was lowest cost but missed 1/6 mutations. Additional preselection by IHC and MSI was required to detect all mutations. 

ND 
C - 150

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 X 

Amsterdam R Other (specify) 

1/3 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 Amsterdam R Other (specify) 

2/3 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

2/3 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

X X 

If yes, which clinical criteria (check all that apply)? Am 1 + Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) Other (specify) Absence of criteria that suggested sporadic disease above: Also performed testing on a sample ? random of low-risk population 

x 
C - 151

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with 

MMR+ 

Number with

MMRAm 1 + + (A) 5 (B) 20 

Am R + - (C) 0 (D) 43 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Selected patients considered to Have sporadic colorectal cancer Because of absence of clinical Features suggestive of HNPCC PLUS a group of patients considered To be at high risk for HNPCC 

Suggestive family History as above 

X 

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 5 (B) 4 

Am R + - (C) 1 (D) 15 Beth 1 + Beth R + MSI-H* X MSI-L* Age <50 IHC (no staining) Other (specify)** 

Patients with clinical history Suggestive of Lynch syndrome *Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 152

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 1 (B) 1 

Am R + - (C) 5 (D) 18 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) X Other (specify)** 

Patients with clinical history Suggestive of Lynch syndrome What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 5 (B) 2 

Am R + - (C) 1 (D) 17 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Patients with clinical history Suggestive of Lynch syndrome 

IHC OR MSI X 
C - 153

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with 

MMR+ 

Number with

MMRAm 1 + + (A) 0 (B) 1 

Am R + - (C) 0 (D) 42 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) X Other (specify)** 

Patients without a clinical history Suggestive of the Lynch syndrome What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with 

MMR+ 

Number with

MMRAm 1 + + (A) 0 (B) 5 

Am R + - (C) 0 (D) 38 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Patients without a clinical history Suggestive of the Lynch syndrome 

IHC OR MSI X 
C - 154

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based u

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification using 

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eithe

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study group

the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who underwent 

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or t

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 155

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 156

Study: Dieumegard 2000 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

 Multicenter, France 34 patients with CRC who represented one of three groups: 1) Amsterdam I criteria 2) Incomplete Amsterdam (missing at least one criteria but strong family history) 3) Age <50 and absence of HNPCC-related cancer sin family 

All patients tested for MSI. Patients with MSI-H and nine MSS cases underwent germline testing for MMR. IHC was performed in all but 4 patients. 

hMLH1 and hMSH2 Y N ? 

≥5 MSI markers used? (20 markers) 

X 

MSI-H defined by ≥ 2 markers? 

X 

Microdissection? X Gene screening? X Deletion analysis? 

 X 

Conversion analysis? 

 X 

Am 1 X 

Am R Beth 1 Beth R MSI-H X MSI-L IHC X Age <50 Suggestive family history 

X 

Specify See inclusion criteriaOther Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 157

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

34: 10 group 1, 7 group 2 and 17 group 3, sex not described Age at cancer diagnosis group 1 26-55 (range), group 2 37-56, group 3 17-50 

6 patients with MMR in group 1, 3 in group 2 and none in group 3. In total, 9 MMRs detected 6 inMLH1 and 3 in MSH2 

15/34 MSI-H Lack of protein expression observed in 10 tumors (of 30 who had complete testing) 

Lack of protein 

expression in 10 

tumors all being 

MSI-H. All sporadic 

MSS tested had 

normal staining. 

Simple tests such as MS study combined with hMSH2 and hMLH1 protein immunostaining performed on tumor tissues may provide valuable information to distinguish between familial and probably hereditary, and sporadic CRC cases 

ND 
C - 158

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 X 

Amsterdam R Other (specify) 

6/10 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 X Amsterdam R Other (specify) 

9/10 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 X 

Amsterdam R Other (specify) 

4/8 (not all tested) 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? If yes, which clinical criteria (check all that apply)? Am 1 + X Am R + Beth 1 + Beth R + Age <50 X Suggestive family history (specify) Amsterdam I except missing 1 criteria 

X 

Other (specify) 
C - 159

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 6 (B) 3 

Am R + - (C) 0 (D) 1 Beth 1 + Beth R + MSI-H*+L X MSI-L* Age <50 IHC (no staining) Other (specify)** 

Patients fulfilling Am 1 *Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 2 (B) 2 

Am R + - (C) 2 (D) 2 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) X Other (specify)** 

Patients fulfilling Am 1 
C - 160

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 3 (B) 3 

Am R + - (C) 0 (D) 1 Beth 1 + Beth R + MSI-H*+L X MSI-L* Age <50 IHC (no staining) Other (specify)** 

Patients fulfilling Am 1 minus 1 criteria What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 2 (B) 3 

Am R + - (C) 1 (D) 0 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) X Other (specify)** 

Patients fulfilling Am 1 minus 1 criteria 
C - 161

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 0 (B) 0 

Am R + - (C) 0 (D) 7 Beth 1 + Beth R + MSI-H* X MSI-L* Age <50 IHC (no staining) Other (specify)** 

Patients age <50 and no family History of HNPCC cancers What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 0 (B) 0 

Am R + - (C) 0 (D) 7 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) X Other (specify)** 

Patients age <50 and no family history of HNPCC cancers 
C - 162

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 163

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 164

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Dove-Edwin, 2002 UK, The Netherlands Multicenter 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

UK family clinic: 184 women from HNPCC (67) or HNPCC-like families (117) had annual or biennial endometrial carcinoma surveillance by ultrasound. Amsterdam Criteria had not yet been established so modification included no cancer cases under 50 years, or cases of endometrial carcinoma The Netherlands registry: 108 women from 38 HNPCC families had transvaginal ultrasound scan annual or biennial. 25 families were AC positive, 3 ACII positive, 10 families suggestive of HNPCC and HNPCC mutations present 

Amsterdam Criteria 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

See study description Annual or bie

transvaginal unavailable trecommendeNetherlands advised to hultrasound sfrom age 30

Outcomes Dropouts, explanations, other comments Study Conclusions 

522 scans carried out with ration of 0.63 for years scanned/years of risk. No cases of endometrial carcinoma detected by ultrasound but one case each of endometrial carcinoma from each group. Both women had hMLH1 mutation 

292 women offered screening – data on 269 (171 from HNPCC families, 98 from HNPCC-like families). Limited data on dropouts – 8 died from unrelated causes 

“Endometrial carcinoma surveillanhereditary colorectal carcinoma maobvious clinical benefits.” 
C - 165

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 5 9 8a 8b,8c 

Public health or policy 

2,3 5 9 8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 166

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 ND NA Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes No Were data collection tools shown or are they known to be reliable? Yes No Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 ND NA 

Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially No Is there a statistically significant difference between groups? Yes No ND Are the statistical methods appropriate? Yes No ND Indicate the unit of allocation Community Organization/group Provider Client Institution Indicate the unit of analysis Community Organization/group Provider Client Institution If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No NA 
C - 167

Domain/question Place an “X” in one Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No Can’t 

tell 
C - 168

Study: Durno C 2005 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

Multicenter, Canada Patients with CRC at ≤

age 24 (selected from a total of 1382 patients in a cancer registry. 

Tumors analyzed for MSI, IHC and blood for MMR 

hMLH1 and hMSH2 and PMS2 (select patient not described who) 

 Y N ? 

≥5 MSI markers used? 

X 

MSI-H defined by ≥ 2 markers? 

X 

Microdissection? X Gene screening? X Deletion analysis? 

 X 

Conversion analysis? 

 X 

Am 1 X 

Am R X Beth 1 Beth R MSI-H X MSI-L IHC X Age <50 Suggestive family history 

X 

Specify Age ≤ 24 Other Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 169

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

N=16, all analyzed, 6/16 = 38% male 

MMR 6 of 12 tested; 2 with MLH1, 3 with MSH2 and 1 with PMS2 

8/16 fulfilled revised Amsterdam criteria MSI-H 11/16 IHC abnormal in 2 of 8 tested 

ND 

Patients with early onset CRC often have an inherited predisposition to the disease. Tumors with MSI-H and germline MMR mutations are sufficiently common in this population that they should be considered even though family histories may not satisfy the stringent Amsterdam criteria for HNPCC. 

7/16 patients 

developed a secon

cancer during 

followup (mean 1

years). Majority (7

%), were in the G

tract. 
C - 170

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 X 

Amsterdam R X Other (specify) 

3/5 Amsterdam 1 (not all patients 3/6 Amsterdam 2 (not all patients w

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 X Amsterdam R X Other (specify) 

4/6 (not all tested) 4/6 Am2 (ie the Am1) no other Am R were tested 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 X 

Amsterdam R X Other (specify) 

1/5 Amsterdam I 1/5 Am2 (ie the Am1) no other Am R were tested 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? If yes, which clinical criteria (check all that apply)? Am 1 + Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) X Other (specify) 
C - 171

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 5 (B) 3 

Am R + - (C) 0 (D) 1 Beth 1 + Beth R + MSI-H* X MSI-L* Age <50 IHC (no staining) Other (specify)** 

CRC ≤ age 24 *Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 3 (B) 1 

Am R + - (C) 1 (D) 3 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) X Other (specify)** 

CRC ≤ age 24 Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar
C - 172

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 173

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 174

Study: Farrington, SM 1998 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

50 unrelated patients with CRC at age <30 identified retrospectively from cancer registrations since 1970 compared with 26 age matched volunteers without cancer. Edinburgh, Scotland, Single center 

Detailed family history obtained from cases, paraffinembedded archival tumor material obtained along with matched normal tissue from 42 patients. Tumor and normal tissue analyzed for MSI. Genomic sequencing done on all patients and controls using peripheral blood. 

hMSH2 and hMLH1 Y N ? 

≥5 MSI markers used? 

X 

MSI-H defined by ≥ 2 markers? 

X 

Microdissection? “Only areas with >90% tumor tissue used” 

X 

Gene screening? “genomic sequencing and IVSP analysis” 

X 

Deletion analysis? 

X 

Conversion analysis? 

 X 

Am 1 X 

Am R Beth 1 Beth R MSI-H MSI-L IHC Age <50 Suggestive family history 

X 

Specify 4 categories: 0= no family hx; 1= relative with nonCRC; 2 = relative with CRC but not Amsterdam,; 3 =Amsterdam 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 175

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g., of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

50 patients enrolled of whom 42 samples could be assessed for MSI 

14 mutations identified from 50 patients (7 hMLH1, 7 hMSH2) 

MSI-H 19/50 

As below 

IVSP complemented genomic sequencing by detection of mutations not identified by genomic analysis....An appreciable proportion of young colon cancer probands carry a germ-line mutation in a DNA mismatchrepair gene 

Sensitivity of genomsequencing 80% anIVSP 64%; IVSP angenomic sequencing complementary. 
C - 176

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 X 

Amsterdam R Other (specify) 

3/6 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 X Amsterdam R Other (specify) 

5/5 (one did not have tissue for MS

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

 ND 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased?

X 

If yes, which clinical criteria (check all that apply)? Am 1 + Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) CRC <30 

X 

Other (specify) 
C - 177

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 12 (B ) 7 

Am R + - (C) 2 (D) 19 Beth 1 + Beth R + MSI-H* X MSI-L* Age <50 IHC (no staining) Other (specify)** 

Patients with CRC <30 *Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number 

MMR

Am 1 + + (A) 4 (B) 10 Am R + - (C) 3 (D) 8 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** X 

Patients with CRC < 30 

Family history 1 vs family history 0 as noted above 
C - 178

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number 

MMR

Am 1 + + (A) 7 (B) 3 Am R + - (C) 3 (D) 8 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** X 

Patients with CRC < 30 

Family history 2 vs family history 0 as noted above

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Numb

M

Am 1 + + (A) 3 (B) 3 Am R + - (C) 3 (D) 8 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** X 

Patients with CRC < 30 

Family history 3 vs family history 0 as noted above (i.e. Amsterdam 1 positive)
C - 179

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 180

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 181

Data Extraction Form For Benefits/Harms 

Fujita, 1996 (RefID 2533) UI 8895676 

Japan 

Single center 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

3356 patients with CRC underwent surgery and follow up between 1968 and 1993. They were classified into 4 categories: Amsterdam 14 Japanese A 31 Japanese B 100 Sporadic 1604 Kaplan-Meier was performed 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl x 

Subjects who died within 30 days of surgery were excluded from calculation 

Outcomes Dropouts, explanations, other comments Study Conclusions 

5-year survival rate: Amsterdam 92.3% Japanese A 81.2% Japanese B 66.5% Sporadic 60% The first 2 groups were better than the latter 2 groups (P<0.05). 

Not all patient with CRCs were accounted for? “Prognosis of HNPCC is better tha

sporadic CRC, and Japanese A critused to select putative HNPCC patamong those with sporadic CRC.” 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 182

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely 

X 

Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 ND 

X 

NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated Yes No If the method of random allocation is stated, is it appropriate Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes No Were data collection tools shown or are they known to be reliable? Yes No Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 ND NA 

Analysis A (stroIs there a sample size calculation or power calculation Yes Partially No Is there a statistically significant difference between groups? Yes No ND Are the statistical methods appropriate? Yes No ND Indicate the unit of allocation Community Organization/group Provider Client Institution Indicate the unit of analysis Community Organization/group Provider Client Institution If the unit of allocation and analysis differed, was the cluster analysis Yes No NA 
C - 183

Domain/question Place an “X” in one done? Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No Can’t 

tell 
C - 184

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Graff 2005; Ref ID 125 Single center Australia 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Investigated experiences of 12 individuals with HNPCC in a semi structured telephone interview. All patients had been asked to inform family members. 

Pathogenic mutation causing HNPCC 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl X 

Pathogenic mutation, English speaking, excluded those with inconclusive test results. All had received genetic counseling. All had an HNPCC-related cancer. 

Semi-structu
C - 185

Outcomes Dropouts, 

explanations, 

other comments 

Study Conclusions 

12 subjects (4 men and 3 women) Respondents older than nonrespondents (55 versus 49), had more cancer diagnoses per individual and were more likely to be female (no statistical comparisons made). All had told some (mainly immediate family) at-risk family members about predictive testing. Family reactions ranged form interest to disinterest. Men expressed a need for guidance and support in communicating to relatives more than women. Letters and booklets were thought to enhance the quality of information but further aids were unlikely to increase the number of relatives made aware of predictive testing by the probands. 

19 invited, 2 declined and five failed to respond 

Respondents informed their immediate family about the availability of genetic testing but more distant relatives were not directly informed. Men expressed a need for guidance or support in communicating to relatives more than women. Letters and booklets were thought to enhance the quality of information. 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 186

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely X 

Somewhat likely 

Not 

likely 

What % of selected individuals agreed to participate? 80-100 X 

60-79 

<60 ND NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? Yes 

X 

No 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No X 

NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND X 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts X A 

(stro

Indicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

X 

80-100 

60-79 <60 ND X 

NA 

Analysis A (stro
C - 187

Domain/question Place an “X” in one Is there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? Yes No X 

ND 

Are the statistical methods appropriate? Yes No X 

ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

X 

80-100 

60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

X 

Yes 

No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No X 

Can’t 

tell 
C - 188

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Green S 1995, Ref ID 2706 Single center UK. 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Data on all screening colonoscopies performed in 61 HNPCC family members from start of a screening program in 1990-1994 analyzed. 

Amsterdam I criteria 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

Member of family fulfilling Amsterdam I criteria and undergoing a colonoscopy. 

Analysis of c

Outcomes Dropouts, explanations, other comments Study Conclusions 

2 patients (3 %) had a malignant neoplasm (one Dukes B and the other dukes C) at ages 66 and 41, respectively. Another patient presented with an obstructing sigmoid carcinoma. Seven patients had adenomas (mean age 45.3, range 26-63). 

ND These findings support the hypothe

adenomas do not occur in large numHNPCC families but, because of the malignant conversion rate, biennial colonoscopy with removal of polyps recommended. 
C - 189

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 5 9 8a 8b,8c 

Public health or policy 

2,3 5 9 8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 190

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely X 

Somewhat likely 

Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 X 

ND 

NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No X 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No X 

NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND X 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 X 

ND 

NA 

Analysis A (stro
C - 191

Domain/question Place an “X” in one Is there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? Yes X 

No 

ND 

Are the statistical methods appropriate? Yes No X 

ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No X 

Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 X 

ND 

NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND X 

NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No X 

Can’t 

tell 
C - 192

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Gritz E 2005, Ref ID 227 Single center US. Note, same patients as Vernon, SW: participation in colorectal cancer screening: a review. J Natl Cancer Inst 1997; 89:1406. 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

155 affected and unaffected persons with HNCC completed psychological tests before genetic testing and at 2 weeks, 6 and 12 months afterward. Among 89 affected participants, 33 were HNPCC predisposing mutation carriers. Among unaffected participants, 19 were HNPCC mutation carriers and 47 were noncarriers who received definitive negative test results.

Amsterdam or suggestive family history followed by mutation testing. (Not clear if original or revised Amsterdam). 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

≥18 Probands: fulfilled Amsterdam or suggestive family history: Family of mutation positive probands at 25 to 50% risk of carrying a mutation. “Affected” included any index patients or relatives with a prior diagnosis of any cancer excluding non-melanoma skin cancer; unaffected included relatives with no personal history of cancer. 

Short form oQuestionnairCenter for EpDepression sState anxiety Anxiety inveFerrans and P(QQLI) Cancer worriPerceived ris
C - 193

Outcomes Dropouts, 

explanations, other 

comments 

Study Conclusions 

Affected participants: Nonwhites had significantly higher mean scores on the CES-D, STAI-S and RIES over time on all measures compared with whites; Those with lower educational levels had significantly higher scores than those who had higher educational levels. Carriers had significantly higher mean RIES scores at 2 weeks and 6 months 

postdisclosure compared with affected participants and uninformative results. Scores decreased significantly form 2 weeks to 12 months postdisclosure except for those with uninformative results, which did not changed. Unaffected participants: Race or ethnicity were significantly associated with mean CES-D and STAI-S scores over time with nonwhites having significantly higher means scores compared with whites and those with lower educational levels having significantly higher scores compared with those who had higher educational levels. Nonwhites also had significantly higher mean RIES scores compared with whites. Carriers had significantly higher mean RIES scores compared with noncarriers at the 2 week and 6 month postdisclosure time points. Post hoc comparisons showed a significant reduction in mean RIES scores for both unaffected carriers and noncarriers form 2 weeks to 6 months post disclosure. Effect of baseline distress on postdisclosure outcomes: Cluster analysis identified two well-defined subgroups for affected and unaffected participants using the mean baseline scores on the CES-D, STAIS-S, QLI, and SSQ scales: a high, and low-distress cluster. The proportion comprising the high-and lowdistress groups was the same among affected and unaffected participants (30.3%). The high-distress groups had significantly higher scores (regardless of mutation status) at two weeks, 6 and 12 months postdisclosure. Cancer worries and perceived risk scores were not affected by distress, and changes in these scores were primarily related to mutation status. 

126 affected participated (79 families) but 15 (12%) did not receive their results. 178 unaffected relatives of mutationpositive index patients invited; 68 (38%) refused and 24 (13%) completed the questionnaire only. Of 86 unaffected who completed the questionnaire and gave blood samples for testing, 4 (5%) did not receive their results. Individuals who declined their genetic test results did not differ from those who received results on demographic or distress variables. 

Although HNPCC genetic testing dresult in long-term adverse psycholooutcomes, unaffected mutation carrexperience increased distress during immediate postdisclosure time perioFurthermore, those with higher levebaseline mood disturbance lower quand lower social support may be at short and long-term increased distre
C - 194

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 X X 8c 

Family member/other high risk individual 

2,3 5 X 

9 

X 

8a 

8b,8c 

Public health or policy 

2,3 5 X 

9 

X 

8a 

8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence 
C - 195

Domain/question Place an “X” in one Selection Bias X 

A 

(stro

Are individuals selected to participate likely to be representative of target population? 

X 

Very likely 

Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 X 

60-79 

<60 ND NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders X A 

(stro

Prior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes X 

No 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No X 

NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes X 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND X 

NA 

Data Collection methods X 

A 

(stro

Were data collection tools shown or are they known to be valid? X 

Yes 

No 

Were data collection tools shown or are they known to be reliable? X 

Yes 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 X 

60-79 

<60 ND NA 

Analysis X A 
C - 196

Domain/question Place an “X” in one 

(stro

Is there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? X 

Yes 

No ND 

Are the statistical methods appropriate? X 

Yes 

No ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 X 

60-79 

<60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? X 

Yes 

No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No X 

Can’t 

tell 
C - 197

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Hadley, DW 2003, Ref ID 946 Multicenter, US (one author from Ireland but patients US). Same patients as Hadley Ref ID 657) 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Elucidate factors affecting decisions regarding genetic testing in individuals form families with newly identified HNPCC. 165 adult men and women from 15 families identified with HNPCC mutations invited to participate; 104 agreed to participate. 

Mutations 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

Members of families with newly identified HNPCC with any of the following features: HNPCC-associated cancer demonstrating MSI or a family history suggestive of HNPCC and 1stdegree relatives at 50% risk of inheriting 

the mutation. Had to agree to fill out questionnaires, undergo counseling and participate in telephone interviews 6 and 12 months later. 

Individuals cout baseline qgenetic educa6 and 12 mon
C - 198

Outcomes Dropouts, explanations, other comments Study Conclusions 

104/165 (63%) eligible patients (including index cases and 1st degree relatives agreed to 

participate. This included 54 probands (of whom 87% agreed to participate) and 111 family members of whom 51% agreed to participate. Awareness of genetic testing: those at higher household income levels were more aware of genetic testing for cancer (p=0.001) and colon cancer (p=0.009) than those at lower household income levels. There was no significant association between participants’ awareness and age, sex, personal cancer history or number of 1sdegree relatives. Perceived risk of being a carrier: Participant’s feelings about their chances of getting colon cancer were significantly associated with their beliefs about the likelihood that they carry a mutation (p<0.001) Intentions toward genetic testing: The intention to pursue genetic testing was found to have a positive association with participants beliefs that cancer may be explained by family heredity (p=0.006). Concern about the psychosocial effect of genetic testing on the family demonstrated a negative association with their intention to pursue testing (p=0.001). Participant’s concerns about their ability to handle the emotional aspects of genetic test results demonstrated a negative association with their intentions to pursue testing (p<0.001). No association between age, sex or cancer status in regard to their intentions toward genetic testing. Reasons for pursuing genetic tests: 50% of respondents believe that the most important 

ND Genetic counseling and testing offe

the potential to focus cancer screening resources in individuals truly at increased risk, thereby reducing mortality and morbidity. Fears of discrimination and concerabout psychological and psychosocissues may present barriers to the uof current cancer prevention strategies, including genetic counseling and testing. 
C - 199

Outcomes Dropouts, explanations, other comments Study Conclusions 

reason for undergoing genetic testing was to learn about their children’s risk. Second most important reason (17%) was to guide cancer screening; third (13%) as to confirm their belief that they carry a mutation. With respect to the importance of genetic testing for reproductive decision making, a statistically significant difference was detected between those younger than the median age of 43 or older (p=0.002). Reasons for not pursuing testing: Worry about losing health insurance (39%), concerns about how it might affect the family (27%), concerns about handling the results emotionally (10%). A statistically significant difference was detected between those younger than the median age of 43 compared with those older with respect to concerns about handling the emotional aspects of genetic testing (p=0.006). Testing decisions: 44/54 (81%) of eligible probands eventually chose to undergo genetic testing for HNPCC. 56/111 eligible first-degree relatives chose to pursue genetic testing (intention-to-treat analysis). Of those agreeing to participate in the study, 44/47 (94%) probands agreed to undergo genetic testing and 56/57 (98%) of family members agreed. 
C - 200

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 X 8c 

Family member/other high risk individual 

2,3 5 9 8a 

X 

8b,8c 

Public health or policy 

2,3 5 9 8a 

X 

8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence 
C - 201

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

X 

Very likely 

Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 X 

<60 

ND NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes X 

No 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

X 

Yes 

No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND X 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 X 

<60 

ND NA 

Analysis A (stro
C - 202

Domain/question Place an “X” in one Is there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? X 

Yes 

No ND 

Are the statistical methods appropriate? X 

Yes 

No ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 X 

<60 

ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

X 

Yes 

No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No X 

Can’t 

tell 
C - 203

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Hadley, DW 2004, Ref ID 657 (same patients as Hadley Ref ID 946)Multicenter, United States 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Assess impact of genetic counseling and mutation testing on endoscopic screening procedures and adherence to recommended endoscopic screening guidelines in 56 at-risk individuals from families known to carry an HNPCC mutation. 

Deleterious mutation 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

≥18, no personal history of cancer, 50% risk of carrying their families previously identified deleterious HNPCC mutation who chose to undergo genetic testing and who completed questionnaires at baseline, 6 and 12 months. Excluded those simultaneously participating in clinical trials involving colonoscopy 

Genetic coundescribed in 
C - 204

Outcomes Dropouts, explanations, other comments Study Conclusions 

30/56 patients had at least one colonoscopy before genetic counseling and testing. 12/56 had at least one colonoscopy within 12 months after testing. Mutation-positive individuals were more likely to undergo colonoscopy. Among mutation-negative individuals, use of colonoscopy and flexible sigmoidoscopy decreased significantly between pre and post testing. Among mutation-positive individuals, a nonsignificant increase in use was noted. On logistic regression, colonoscopy was significantly more likely in mutationpositive individuals (OR 61, 95% CI 5.8-652, p=0.006), and increasing age (OR 1.1, 95% CI 1.008-1.2, p=.03). Employment, income, sex or colonoscopy status before testing did not predict use. 11/56 were classified as nonadherent with colonoscopy recommendations 12 months after testing . Mutation positive (OR 7.5, 95% CI 1.3-42.2, p=0.02) and unemployed individuals (OR 8.6, 95% CI 1.3-56.6, p=0.025) were significantly less likely to adhere to guidelines for endoscopic screening. Age, sex, income, or pre-testing colonoscopy status were not associated with adherence. 

ND. Genetic counseling and testing for H

significantly influences the use of cendoscopy and adherence to recomfor colon cancer screening. 
C - 205

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 X 

5 

X 

9 

8a 8b,8c 

Public health or policy 

2,3 X 

5 

X 

9 

8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 206

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

X 

Very likely 

Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 X 

ND 

NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes X 

No 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

X 

Yes 

No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND X 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 X 

ND 

NA 

Analysis A (stro
C - 207

Domain/question Place an “X” in one Is there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? X 

Yes 

No ND 

Are the statistical methods appropriate? Yes No ND Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

X 

80-100 

60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

X 

Yes 

No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No X 

Can’t 

tell 
C - 208

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Halbert, CH 2004, Ref ID 405 Multicenter, United States 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Evaluate whether genetic testing for HNPCC mutations and receipt of positive test results have an effect on the use of colonoscopy. Identify factors associated with adherence to identify potential barriers to patient compliance. 222 eligible, 98 in final sample. 

Mutations. 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

25% risk of having HNPCC identified in family. Excluded individuals with colon cancer or a history of colectomy. 

All eligible inthose who agtelephone. Thparticipate in session. Subtesting were mutation resuFollowing diby telephone behaviors at 

Outcomes Dropouts, explanations, other comments Study Conclusions 

134/222 participated but after exclusions, 98 were available for analysis. Men and subjects who were younger were significantly more likely to be lost to follow-up or not participate. Colonoscopy use before genetic counseling was not significantly different between test result groups. During the 12 months following genetic testing, 73% (16/22) of mutation carriers and 16% (8/49) noncarriers and 22% (6/27) decliners reported having a colonoscopy (p<.001). Mutation carriers were significantly more likely than noncarriers and decliners to have a colonoscopy. No clinical or sociodemographic factors were significantly associated with colonoscopy use during the 12 months followinggenetic counseling. 

Excluded 25/14 due to a prior history of colectomy or colon cancer and 2/134 who developed colon cancer during the study, and 9/134 who received genetic test results during follow-up. 

Genetic testing may motivate increcolonoscopic screening among HNmutation carriers. Increased efforts needed to assess patients’ family hcolon cancer and provide appropriareferrals for genetic counseling and target colonoscopic screening to hiindividuals. 
C - 209

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 X 

5 

X 

9 

8a 8b,8c 

Public health or policy 

2,3 X 

5 

X 

9 

8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 210

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

X 

Very likely 

Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 X 

<60 

ND NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes X 

No 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

X 

Yes 

No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND X 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 X 

<60 

ND NA 

Analysis A (stro
C - 211

Domain/question Place an “X” in one Is there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? X 

Yes 

No ND 

Are the statistical methods appropriate? X 

Yes 

No ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes X 

No 

Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 X 

<60 

ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

X 

Yes 

No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No X 

Can’t 

tell 
C - 212

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Hampel 2005, Ref ID 29 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

70 HNPCC families including 88 probands and 373 mutation-positive family members evaluated for risk of CRC and endometrial cancer. 

Mutations and clinical criteria, mostly Amsterdam I or II 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

Excluded if mutation unknown diagnosis or data incomplete 

Cohort 1, 45 mutation for Probands had Cohort 2, famHNPCC famMLH1 or MSprobands had 

Outcomes Dropouts, 

explanations, 

other 

comments 

Study Conclusions 

Mean age of diagnosis of CRC 55.1 (95% CI 52.6-57.6)) for men and 60.3 (95% CI 58.0-62.6) for women; approximately 10-15 years older than previous estimates of age at onset for CRC among HNPCC patients. Lifetime risk of CRC was 68.7% for men and 52.2% for women. Mean age of onset for endometrial cancer in women was 58.6 (95% CI 56.9-60.3). Lifetime risk of endometrial cancer was approximately 54% Mean age of ovarian cancer was 54.1 (95% CI 53.5-54.7). Women had a 13.5% lifetime risk for ovarian cancer. Among probands, average age at diagnosis of CRC in men was 44.8 (95% CI 41.6-48.0) and for women was 47.0 (95% CI 43.0-51.0) Among probands, average age of diagnosis of endometrial cancer was 53.1 (95% CI 50.5-55.7) 

ND A markedly later age of onset of CRC at 61 year than 

previously reported (approximately 44 years) is suggested, resulting mainly from a more rigorous method of analysis in which all gene-positive individuals (both affected and unaffected with cancer) are considered. Lifetime cancer risk may be lower for CRC and endometrial cancer than presently assumed. If confirmed, these data suggest a need to alter counseling practices, and to consider HNPCC in older individuals than before. 
C - 213

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC-related 

cancers/factors such as race, 

age sex, comorbidities that 

may be related to 

accuracy/penetrance 

Be

o

mu

dete

de

fam

Proband with CRC 

2,3 5 

X 

8c 

Family member/other high risk individual 

2,3 5 9 8a 

X 

8b,8c 

Public health or policy 

2,3 5 9 8a 

X 

8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 214

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

X 

Very likely 

Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 X 

ND 

NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No X 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No X 

NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No X 

ND 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 X 

ND 

NA 
C - 215

Domain/question Place an “X” in one Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? Yes No X 

ND 

Are the statistical methods appropriate? X 

Yes 

No ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND X 

NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND X 

NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No X 

Can’t 

tell 
C - 216

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Jarvinen, H 2000, Ref ID 5 Multicenter, Finland. Same patients (and includes) Jarvinen HJ. Gastroenterology 1995; 108:1405 Ref ID 2746 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Incidence of CRC and survival compared in 2 cohorts of at-risk members of 22 families with HNPCC. Colonic screening at 3-year intervals arranged for 133 subjects while 119 had no screening. 

Amsterdam I criteria or other suggestive family history (some mutation positive). 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

Asymptomatic member of an HNPCC family at 50% risk of. Other inclusion/exclusion criteria as per Jarvinen 1995; 108:1405 as follows: 

Initial colonobarium enemthen repeated intervals. The was 56% on the third rounround. 

Outcomes Dropouts, explanations, 

other comments 

Study Conclusion

Cancer morbidity data, survival and causes of death checked at the Finnish Cancer Registry and the Population Registry of Finland. Significantly more patients developed CRC in control group (19 (16%) versus 8 (6%), p=0.014; RR CRC 0.377 (95% CI 0.171-0.829). Among mutation-positive subjects, the frequency of CRC was 18% (8 of 44) in the study group and 41% (19 of 46) in the control group , p=0.02; RR 0.440 (95% CI 0.215-0.900). Mortality was greater in study group (9 of 119 (8%) versus 0/133 in study group (p<0.001).Overall there we re 10 deaths (8%) in the study group compared with 26 deaths (22%) in the control group during a 15-year period (p<0.001). Relative risk of deaths for the screened group was 0.344 (95% CI 0.173-0.683) overlap and 0.348 (95% CI 0.122-0.999) for mutation positive subjects. 

252 asymptomatic individuals belonging to 22 HNPCC families and being at 50% risk. Of those invited to participate, 78 eligible declined and 40 could not be traced; these patients constituted the control group. 

Colonoscopic screeniat 3-eyar intervals mothan halves the risk oCRC, prevents CRC deaths, and decreases overall mortality by about 65% in HNPCC families. 
C - 217

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 5 9 8a 8b,8c 

Public health or policy 

2,3 5 9 8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 218

Domain/question Place an “X” in one Selection Bias X 

A 

(stro

Are individuals selected to participate likely to be representative of target population? 

X 

Very likely 

Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? X 

80-100 

60-79 <60 ND NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? X 

Yes 

No 

If the method of random allocation is stated, is it appropriate? Yes X 

No 

Was the method of random allocation reported as concealed? Yes X 

No 

Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No X 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No X 

NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND X 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts X A 

(stro
C - 219

Domain/question Place an “X” in one Indicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

X 

80-100 

60-79 <60 ND NA 

Analysis X 

A 

(stro

Is there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? X 

Yes 

No ND 

Are the statistical methods appropriate? X 

Yes 

No ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

X 

80-100 

60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes X 

No 

ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No X 

Can’t 

tell 
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 Study: Katballe Gut 2002 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? X Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? X Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of strategy 

used for testing patients 

with CRC)

Which MMR 

mutations 

were sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply

Consecutive Danish patients with CRC in 4 counties (11/1995 to 10/1998) Inclusion criteria: Amsterdam criteria I or II or on extended pedigree; Amsterdam criteria with HNPCC related cancers; Amsterdam criteria except youngest was 50-55yrs; pts ≤40 with at least one CRC among family members; Proband and one first degree relative with CRC before 55 yrs 

Prospective population study; if a family met criteria for suspected HNPCC, screening for hMSH2 or hMLH1 were done and tumor were analyzed for MSI; HNPCC defined as Amsterdam I or II; or suspected HNPCC based on history plus pathogenic hMLH1 or hMSH2 mutation. 

MLH1 and MSH2 

 Y N ? 

≥5 MSI markers used? 

X 

MSI-H defined by ≥ 2 markers? 

X (>=2 bands, but OK) 

Microdissection? X Gene screening? X Deletion analysis? 

 X 

Conversion analysis? 

 X 

Am 1 

Am R Beth 1 Beth R MSI-H MSI-L IHC Age <50 X Suggestive family history 

X 

Specify Other 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 221

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

1514 with 1° CRC 

(median age 71 yrs) 1328 eligible, 1200 in study, excluded pts who died shortly after dx, psychological problems, ethical reasons, did not complete questionnaires 

10 pts were found with MMR among the 41 HNPCC who were sequenced Æ or 

among the 1200 CRC who were included. 

11/1200 (0.9%, 0.5-1.6%) fulfilled Amsterdam I 18/1200 (1.5%, 95% CI 0.8-2.25%) fulfilled Amsterdam II 

The sensitivity of 

MSI is only 61% in 

terms of detecting 

families that 

satisfied the 

Amsterdam criteria I 

or II . 

“The sensitivity of MSI is only 61% in terms of detecting families that satisfied the Amsterdam criteria I or II Æ MSI is not as appropriate to identify the families with suspected HNPCC.” “The majority of mutation carriers present with a strong family Hx of HNPCC-related Cancers.” 

No 
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 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 X 

Amsterdam R X Other (specify) 

5/11 Am1 people; (4/10 independeclustering) 8/18 Am2 (including AM1) people

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 X Amsterdam R X Other (specify) 

Am1 Æ7/10 analyzable had MSI-H Am1 Æ0/10 analyzable had MSI-L Am2 Æ10/16 analyzable has MSI-Am2 Æ1/16 analyzable has MSI-L 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

X 

If yes, which clinical criteria (check all that apply)? Am 1 + X Am R + X Beth 1 + Beth R + Age <50 X Suggestive family history (specify) X Other (specify) 
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COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test 

Number with

MMR+ 

Number wi

MMRAm 1 + X + 5 6 

Am R + - 5 25 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

All 45 Patients with CRC and S-HNPCC as defined above (actually 41, because 4 were not analyzed)*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test 

Number with

MMR+ 

Number wi

MMRAm 1 + + 8 10 

Am R + X - 2 21 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

All 45 Patients with CRC and S-HNPCC as defined above (actually 41, because 4 were not analyzed) *Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
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COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with 

MMR+ 

Number with

MMRAm 1 + + 10 5 

Am R + - 0 20 Beth 1 + Beth R + MSI-H* X MSI-L* X Age <50 IHC (no staining) Other (specify)** 

MSI-H+MSI-L vs MSI-S among all people analyzable out of the 45 *Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with 

MMR+ 

Number with

MMRAm 1 + + 10 3 

Am R + - 0 20 Beth 1 + Beth R + MSI-H* X MSI-L* Age <50 IHC (no staining) Other (specify)** 

MSI-H vs MSI-S among all people analyzable out of the 45 *Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 225

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MSI-H or L 

Number with

MSI-S 

Am 1 + + 11 7 Am R + X - 4 18 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 226

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
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Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
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Data Extraction Form For Benefits/Harms 

Keller, 2002 Ref ID 984 Germany 

Single center 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Longitudinal study, data reported come from self-completed questionnaires preand post-counseling (4-6 weeks after counseling) 94 individuals received counseling between 7/2000 and 8/2001 73/82 completed a post-counseling questionnaire; 8 of those did not have a precounseling questionnaire; Final sample size: 65, 31 patients and 34 unaffected individuals 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

>18 year olds, patients with HNPCCrelated cancer or unaffected individuals from families at risk for HNPCC; referred by physician or self-referred 

Standardized team counselpathologists, surgeons, and comprised of psychosocial 

Outcomes Dropouts, explanations, other comments Study Conclusions 

Distress and worries related to HNPCC declined after counseling. An increase was found in personal belief in control of cancer risk. A trend was observed toward greater anticipated ability to cope with a positive gene test after counseling. Changes after counseling were generally more pronounced for persons at risk, as compared to patients with cancer. 1/3 of the sample reported enhanced communication specific to hereditary disease within the family after counseling. A substantial minority, however, experienced increased worry and physical symptoms after counseling. 

 “Counselees demonstrated less stre

perceived cancer threat as well as ebeliefs regarding personal control osuggesting an overall beneficial imcomprehensive counseling. Further needed to identify those individualfor increased fear and worry relateso that they may be most appropriacounseled.” 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 229

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 ND NA Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated Yes No If the method of random allocation is stated, is it appropriate Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes No Were data collection tools shown or are they known to be reliable? Yes No Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 ND NA 

Analysis A (stroIs there a sample size calculation or power calculation Yes Partially No Is there a statistically significant difference between groups? Yes No ND Are the statistical methods appropriate? Yes No ND Indicate the unit of allocation Community Organization/group Provider Client Institution Indicate the unit of analysis Community Organization/group Provider Client Institution If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No NA 

Is the analysis performed by intervention allocation status (i.e. Yes No Can’t 
C - 230

Domain/question Place an “X” in one intention to treat) rather than the actual intervention received? tell Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No Can’t 

tell 
C - 231

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Keller, 2004 RefID 585 Germany Single center 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Of 140 eligible patients and their families, 36 families participated in the information session, 25 participants were patients, and 11 were spouses or relatives of patients who had died or were too ill to attend. Participation rate 26%. Questionnaire completed by spouses or relatives were not included in the analysis. Of the 104 nonparticipating patients addressed by mail, 61 responded (59%), 12 died , 48 returned a completed questionnaire. Sample here consisted of 73 patients: 25 participants and 48 nonparticipants 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl 

>18 year olds, had surgery for CRCassociated or HNPCC-associated cancer between 1994 and 1998, met Amsterdam or Bethesda or FH suggestive of HNPCC FAP were excluded 

Eligible patieannouncing tmutations angenetic counand their fampartcipate in session on Hinformation sanswering a 
C - 232

Outcomes Dropouts, explanations, 

other comments 

Study Conclusions 

“Among participants, 60% reported being very worry because of familial cancer; among non-participants 35% reported being very worry because of familial cancer (P<0.05). 

28% of participants reported impairment of daily life because of these worries 

compared to 19% of nonparticipants (difference not significant). Similarly, perceived distress of spouse and family from patient’s point of view was higher in participants than in nonparticipants (difference not significant). Regardless of whether patients participated in the information session, most were in favor of genetic testing. Twice as many nonparticipants (25%) as participants (13%) were undecided about whether to accept genetic testing. All participants definitely expressed the wish to know whether a mutation is present in the family, compared to 81% of nonparticipants.” 

No actual genetic testing was done. 

“Expressed intention and attitude ttesting do not reliably predict actuacounseling or testing. Thorough incounseling should be provided to ewith clinical criteria suggestive of considerable distress related to the disorder should be adequately addrshould be communication issues.” 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 233

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 ND NA Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated Yes No If the method of random allocation is stated, is it appropriate Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes No Were data collection tools shown or are they known to be reliable? Yes No Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 ND NA 

Analysis A (stroIs there a sample size calculation or power calculation Yes Partially No Is there a statistically significant difference between groups? Yes No ND Are the statistical methods appropriate? Yes No ND Indicate the unit of allocation Community Organization/group Provider Client Institution Indicate the unit of analysis Community Organization/group Provider Client Institution If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No NA 
C - 234

Domain/question Place an “X” in one Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No Can’t 

tell 
C - 235

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Kruhoffer, 2005, RefID 129 Country: Denmark and Finland Center: 15 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion 

criteria 

Colorectal cancer tissue from a total of 151 patients was collected from 15 different clinics in Denmark and Finland. A sample set consisting of 101 stages II and III cancers (34 MSI-H; 67 MSI-L and MSS) were used. MSI classification data were correlated to the overall survival of the patients for stage II (in 36 patients) and III (in 65 patients) tumors separately. 

Among the 101 stages II and III cancer patients, 19 had sporadic MSI tumors, 15 had hereditary MSI tumors, and 67 had MSS tumors. MSI tumors with HNPCC origin were classified by a maximum likelihood MSI classifier with a “leave-one-out” cross validation scheme basically as described (Dyrskjot et al, 2003). All HNPCC cases included in this study carry MLH1 or MSH2 mutations identified by sequencing. 

Did all patients have a personal history 

of an HNPCC-related cancer? 

Check one 

Yes No Uncl  x 

A sample set consisting of 101 stages II and III cancers (34 MSI-H; 67 MSI-L and MSS) was included in the analyses for the relation between microsatellite status, cancer stage, and patient survival. 

65 patients wreceiving adj
C - 236

Outcomes Dropouts, explanations, other 

comments 

Study Conclusions 

Kaplan-Meier survival analyses were performed for stage II and III tumors 

separately. Note that MSS in this study includes MSI-L. 

The overall survival was highly significantly related to the classification in 

36 Stage II patients, as 10 out of 11 patients that died within 5 years belonged to the MSS group (p=0.0014). 

Among 65 patients with Stage III tumors receiving adjuvant chemotherapy, 

16 were classified as MSI tumors and 49 as MSS tumors. As 6 MSI and 30 MSS patients died within 5 years of follow-up, there was no significant difference in overall survival between these group (p=0.55) 

No “dropouts” due to retrospective design. 

“Our findings are in accordance witrecent publications, the MSI classifMSI-H) clearly proved to be a stronof survival in stage II disease, and trelation between MSI classification prognosis in stage II patients.” 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 237

Domain/question Place an “X” in one Selection Bias A 

(stron

Are individuals selected to participate likely to be representative of target population? 

Very likely Somewhat likely Not 

likely x 

What % of selected individuals agreed to participate? 80-100 60-79 <60 ND x NA Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stron

Is the method of random allocation stated Yes No If the method of random allocation is stated, is it appropriate Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stronPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No Can’t tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes No 

Blinding A (stronWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND NA 

Data Collection methods A 

(stron

Were data collection tools shown or are they known to be valid? Yes No Were data collection tools shown or are they known to be reliable? Yes No Withdrawals and Dropouts A (stronIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 ND NA 

Analysis A (stronIs there a sample size calculation or power calculation Yes Partially No Is there a statistically significant difference between groups? Yes No ND Are the statistical methods appropriate? Yes No ND Indicate the unit of allocation Community Organization/group Provider Client Institution Indicate the unit of analysis Community Organization/group Provider Client Institution If the unit of allocation and analysis differed, was the cluster analysis Yes No NA 
C - 238

Domain/question Place an “X” in one done? Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No Can’t tell 

Intervention Integrity A (stronWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No Can’t tell 
C - 239

Data Extraction for Analytic Validity 

Study: (Author, year, UI) Kurzawki, G, 2002, Ref ID 1253 Inclusion criteria (all must be yes) Did study evaluate biological material from patients considered to be at risk for HNPCC? Did the study report ANY of the following? (check which one below) 

5) Proportion MSI-H with NIH markers versus other markers 6) Sensitivity or specificity of MSI-H using NIH markers compared with a reference standard that the study claims is bet7) Sensitivity or specificity of IHC compared with an immunohistochemical standard that study claims is better 8) Sensitivity or specificity of a genetic technique compared with a reference standard (or combination of standards) 

Are data (proportions or 2 X 2 tables) extractable? Exclusion criteria (exclude if yes) 

 Yes No 

Did the study include the index test in the reference standard? X 

Describe source of biological materials (and whether patients were known 

to have an HNPCC phenotype) 

Summarize how materials were 

processed and analyzed 

46 unrelated patients with CRC: nine families fulfilled Amsterdam II and in 37 there was a strong suspicion of HNPCC. In 19, MLH1 or MSH2 mutations or polymorphisms had been identified by DNA sequencing 

Genomic DNA analyzed using DHPLC and sequencing 
C - 240

MSI Proportion (add additional 2 X 2 tables where relevant) 

Proportion MSI-H using NIH markers (≥2 markers) Proportion MSI-H using other markers 

MSI with a reference standard 

MSI-H using another 

reference standard 

Describe 

 Positive Negative MSI-H using NIH markers 

MSI-H (≥2 markers) 

 MSI-S or MSI-L 

IHC with a reference standard 

IHC using another 

immunohistochemical 

reference standard 

Describe 

 Positive Negative IHC Positive Describe Negative 
C - 241

Genetic technique with a reference standard 

Reference standard 

genetic technique 

Describe 

Sequencing 

 Positive Negative Index genetic technique Positive 

42 

NR 

Describe: DHPLC 

Negative 42 

 Study Quality 

1 Was the description of how MSI or IHC and other genetic techniques described in sufficient detail that others could replicate it (e.

either a full description or relevant references)? 

2 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

3 Was MSI, IHC, other genetic testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

4 Was there a clear description of which mismatch repair mutations were being tested for? 5 Were quality control methods described for the molecular and genetic tests? 6 Did the authors attempt to address the reproducibility of results (reliability of tests)? 7 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or tho

did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 

8 Was microdissection performed? 9 Did the study specify whether the biological tissues were from patients known to have HNPCC clinically? 

10 Did the study include a control group in which biological material was obtained from patients known not to have HNPCC clinical11 Overall rating (A B C) 
C - 242

Study: Lamberti 1999 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? X Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? X Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and 

analytic strategy 

(brief description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory testing 

(see definitions below) 

Predictors analyze

(check all that 

apply) 

I do not know from which population these index pts come from (!) 160 index pts who were GR1 Am1 (57); 

GR2 (Loose criteria with relatives with extracolonic HNPCC malignancies Æ) Am2 NOT Am1 (12); GR3

CRC aged <50 AND with relatives with nonCRC HNPCC related cancer (n=45); GR4 Apparently sporadic CRC <50y OR sporadic CRC with synchronous or metachronous CRC tumor (no age limit; n=46) German Study, 

Blood sample ant tumor tissues from all index pts (when possible); All Gr1 and Gr2 pts were screened for mutations irrespectively of MSI status; Gr3 and GR4 screened only if MSI was positive 

hMLH1 and hMSH2 

 Y N ? 

≥5 MSI markers used? 

X 

MSI-H defined by ≥ 2 markers? 

X (By >40% of loci being unstable) 

Microdissection? X Gene screening? X Deletion analysis? 

 X 

Conversion analysis? 

 X 

D2S123, D2S136, D3S1298, D3S1611, D5S346, D6S470, D18S35, D18S37, BAT25, HBAP1 

Am 1 X 

Am R Beth 1 Beth R MSI-H MSI-L IHC Age <50 Suggestive family history Specify Other 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 243

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

Overall N=160 index pts; Am1 N=57; Am2 N=69 (57 + 12); % male unknown; dropouts not described [[57 Am I mean age at dx of CRC = 44 (11.8); 12 non-Am I mean age at dx of CRC = 37 (10.8)]] 

17 with MMR/69 patients with CRC and Am2; 11 MLH1; 6 MSH2 

[Of the enrolled population, which is not described.] 57/160 are Am1 and 69/160 essentially AM2 

ND 

A positive MSI is an efficient indicator of a germline mutation in the mismatch repair genes MSH2 and MLH1, both in patients meeting the clinical criteria for HNPCC and pts with sporadic CRC. It is a matter of debate whether the age cutoff should be used at all for the MSI test. Pts with CRC <50 y should be examined anyhow, even if F Hx is negative. 

No 
C - 244

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 X 

Amsterdam R X Other (specify) 

Am1: 15/57 Am2 (INCLUDING AM1): 17/69 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 X Amsterdam R X Other (specify) 

MSI-H: Am1: 35/49 MSI-H Am2: 39/57 [Not all tumors assessed for MSI] 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

 X (But, again, the 

population is not really defined) 

If yes, which clinical criteria (check all that apply)? Am 1 + Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) Other (specify) 
C - 245

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + X + 15 42 

Am R + - 2 10 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Among people with Am2, *Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests 

CheckONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + 15 24 

Am R + - 2 16 Beth 1 + Beth R + MSI-H* X MSI-L* Age <50 IHC (no staining) Other (specify)** 

Contrast BETWEEN MSI-H vs MSI-S & MSI-? AMONG PEOPLE with AMI2 In total 52 that could be assessed This is the only contrast you can extract with certainty; You cannot compare against MSI-S only, because of the way the data are reported. *Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 246

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 247

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 248

Study: Lee, 2005 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? X Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? X Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population inclusion/exclusion 

criteria; country, single or 

multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which 

MMR 

mutations 

were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

Inclusion: CRC with Am I or Am II family history; or CRC ≤ 40; or 

CRC with 1st degree relative with 

CRC or HNPCC-like cancer (cancer of endometrium, small bowel, biliary tract, pancreas, stomach, ureter, ovary, renal pelvis or glioblast); or proband ≥

2 primary cancers classical of HNPCC (CRC or HNPCC-like cancer) Exclusion: familial adenomatous polyposis Singapore Single center 

Blood sample for genotyping and paraffin-embedded colon cancer tissue blocks for MSI testing 

MLH1 and MSH2 

 Y N ? 

≥5 MSI markers used? 

X 

MSI-H defined by ≥ 2 markers? 

X 

Microdissection? X Gene screening? X Deletion analysis? 

 X 

Conversion analysis? 

 X 

Am 1 

Am R x Beth 1 Beth R MSI-H x MSI-L IHC Age <50 x Suggestive family history 

x 

Specify Other Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 249

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

N=46 Median 39 yr (18-82) 30 (65%) men 

7/46 WITH DELETERIOUS MUTATIONS 

5/46 Am2 23/46 >=2 first degree relatives 5 multiple tumors 13 apparently sporadic aged <40 

ND 

Singapore patients may have the same key characteristics for HNPCC as those described widely in the western families. 

ND 

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 

Amsterdam R X Other (specify) 

3/5 AM2 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 Amsterdam R Other (specify) 

ND 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

ND 
C - 250

 MMR+ MMRAm2 3 2 3 1st degree family members CRC 2 7 2 1st or 2nd degree family members CRC 1 13 CRC with Multiple cancers (not adenomas) 

1 4 

Sporadic aged <40 0 13  MMR+ MMRMSI-H 4 7 MSI-L 0 2 MSI-S 1 21  Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

 X 

If yes, which clinical criteria (check all that apply)? Am 1 + Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) Other (specify) 
C - 251

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) (B) 

Am R + - (C) (D) Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 252

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 253

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 254

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Lerman, 1996 RefID 2617, US Single Center 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

24 consecutive index colon cancer patients (who did not fulfill the Amsterdam criteria) identified 61 first degree relatives for the study (index patient consent rate = 83%). Of these first degree relatives, 44 (72%) completed the interview, 2 (2%) declined, 16 (26%) could not be reached. This was a pilot study in preparation for a prospective study of genetic testing for HNPCC-associated mutations. 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  x 

Person who had a prior diagnosis of cancer were excluded. 

15-minute te

Outcomes Dropouts, explanations, other comments Study Conclusions 

“Anticipated reactions to positive and negative results of a genetic test: Over 1⁄2 of respondents indicated that they would become depressed or very anxious if they tested positive. Most subjects reported that they would react by changing their health habits, including diet and screening behaviors. In response to negative test result, about 1/3 of respondents anticipated they would feel guilty and 1⁄2 would still worry because the test might be wrong. About 1⁄2 of respondents expected that they would decrease their use of screening tests and make fewer attempts to reduce dietary fat if they tested negative.” 

Pilot study, no actual genetic tests were done. 

“These preliminary results underscimportance of educating patients abpotential risks, benefits, and limitagenetic testing, with particular emppossibility of adverse psychologicaimplications for health insurance. Tfor false reassurance following a nresult should be addressed by emphresidual risks of cancer among nonpredisposing mutations.” 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 255

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 ND NA Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated Yes No If the method of random allocation is stated, is it appropriate Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes No Were data collection tools shown or are they known to be reliable? Yes No Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 ND NA 

Analysis A (stroIs there a sample size calculation or power calculation Yes Partially No Is there a statistically significant difference between groups? Yes No ND Are the statistical methods appropriate? Yes No ND Indicate the unit of allocation Community Organization/group Provider Client Institution Indicate the unit of analysis Community Organization/group Provider Client Institution If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No NA 

Is the analysis performed by intervention allocation status (i.e. Yes No Can’t 
C - 256

Domain/question Place an “X” in one intention to treat) rather than the actual intervention received? tell Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No Can’t 

tell 
C - 257

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Liljegren 2004, Ref ID 541 SAME PATIENTS AS Lindgren G. Gut 2002; 50:228-234. Single center Sweden 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Evaluated psychological aspects of colorectal cancer screening in 240 individuals at high risk for CRC. 

Patients categorized into: 1) mutation positive; 2) suggestive family history but not fulfill Amsterdam I; 3) two close relatives affected by CRC. 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

Subjects from Cancer Family Clinic at Karolinska Hospital form 1990-1999 who fell into three categories described. 

Colonoscopieyears in patieperiod betweall eligible paquestionnaire colonoscopy Anxiety and Medical Outc(SF-36). 
C - 258

Outcomes Dropouts, explanations, 

other comments 

Study Conclusions 

Perceived benefit of colonoscopy was rated an 8/10 on a visual analogue scale where 0 = no benefit and 10 =great benefit. Risk perception: Among 28 mutation-positive subjects 17 (61%) reported their personal risk as being 40% or less and 36% perceived a 1 to 20% lifetime risk of CRC. In risk groups 2 and 3 (129 and 83 individuals), approximately 50% either under-or over-estimated their personal lifetime risk of colorectal. Cancer. HAD scores we within the normative data range. SF-36 scores were significantly lower on mental health (p=.013) and vitality (p=0.16) compared with the reference population. Women and significantly lower scores on vitality (p=0.48) while men had lower scores on mental health (p=0.47). 88% of subjects gave a correct answer to the questions “Why have you been offered regular colonoscopies”. 

304 included of whom 23 later proven to be mutation negative and were excluded. 16 excluded because address could not be found. 265 sent questionnaire of 91% returned usable data. 

A majority of the study sample understood why they were surveillance and regular colonoscopies were well-toleratedwide range of risk perception as well as low-risk perceptiomutation positive subjects is acceptable as long as these individuals adhere to surveillance programs and do not demonstrate increased levels of anxiety or depression. 
C - 259

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 5 X 

9 

X 

8a 

8b,8c 

Public health or policy 

2,3 5 X 

9 

X 

8a 

8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 260

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely X 

Somewhat likely 

Not 

likely 

What % of selected individuals agreed to participate? 80-100 X 

60-79 

<60 ND NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes X 

No 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No X 

NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No X 

ND 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? X 

Yes 

No 

Were data collection tools shown or are they known to be reliable? X 

Yes 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 X 

60-79 

<60 ND NA 

Analysis A 
C - 261

Domain/question Place an “X” in one 

(stro

Is there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? X 

Yes 

No ND 

Are the statistical methods appropriate? X 

Yes 

No ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 X 

60-79 

<60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

X 

Yes 

No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No X 

Can’t 

tell 
C - 262

Data Extraction Form For Benefits/Harms

Au, Year, UI, Country Single or Multicenter? 

Lindgren, G 2002, Ref ID 1300 SAME PATIENTS AS Lilegren A 2004, Ref ID 541 Single Center, Sweden 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Evaluate outcomes in 304 patients at increased risk for CRC including HNPCC who were undergoing surveillance colonoscopy. 

Patients categorized into: 1) mutation positive; 2) suggestive family history but not fulfill Amsterdam I; 3) two close relatives affected by CRC. 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

Subjects from Cancer Family Clinic at Karolinska Hospital form 1990-1999 who fell into three categories described. 

Screening coevery two yeadenoma in wyear. Adenoma prepopulation esstudies. 

Outcomes Dropouts, explanations, other 

comments 

Study Conclusions Quality sco

304 subjects underwent 765 colonoscopies 10 colorectal cancers found in 9 individuals before recruitment into the surveillance program. 4 had CRC at first colonoscopy and two developed Metachronous cancers during surveillance. RR of adenoma before age 54 compared with general population 2.6 (p=0.001) RR for tested gene carriers in HNPCC families 4.5. 

ND There was clear overrepresentation of 

adenomas in all three family types compared with the reference population. In HNPCC, we found earlier onset of adenomas and faster progression to cancer. 

Non-uniform protocol, 

and thus may have been 

in these high risk patien

effects were occurring 

accounted for in the an

based upon autopsy s

detection rates ba
C - 263

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 5 9 8a 8b,8c 

Public health or policy 

2,3 5 9 8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 264

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely X 

Somewhat likely 

Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 X 

ND 

NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No X 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No X 

NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No X 

ND 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 X 

ND 

NA 
C - 265

Domain/question Place an “X” in one Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? X 

Yes 

No ND 

Are the statistical methods appropriate? Yes X 

No 

ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 X 

ND 

NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No X 

ND 

NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No X 

Can’t 

tell 
C - 266

Study: Liu 2004 Ref ID 445 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? x Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? x Was a minimum of hMLH1 and hMSH2 sequencing performed? x 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

28 independent Chinese families: 15 fulfill Am1 criteria; 13 not Am1 but fulfill Japanese Clinical Diagnosis Criteria for HNPCC. 

DHPLC analysis was performed 

HMSH2 and hMLH1 Y N ? 

≥5 MSI markers used? 

 x 

MSI-H defined by ≥ 2 markers? 

 x 

Microdissection? x Gene screening? x Deletion analysis? 

 x 

Conversion analysis? 

 x 

Am 1 x 

Am R Beth 1 Beth R MSI-H MSI-L IHC Age <50 Suggestive family history Specify Other Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 267

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

28 probands from 28 families are described onwards. They all meet the Japanese HNPCC criteria; they are assumed to be with CRC – not clearly stated though. Overall, 128 pts among all family members has CRC. Among these 128: 45.9 yrs mean age onset of CRC, 55% male 

7 patients with MMR/28 patients with CRC; 2 with hMSH2 and 5 with hMLH1 

15/28 probands from AM1 families ND 

“Three novel germline mutations were found, the germline G204X nonsense mutation in the 3rd exon of 

hMSH2 is the first MMR gene mutation found in Chinese Mongolian people.” 

 
C - 268

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 X 

Amsterdam R Other (specify) 

5/15 7/28 Japanese HNPCC criteria 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 Amsterdam R Other (specify) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

 x 

If yes, which clinical criteria (check all that apply)? Am 1 + Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) Other (specify) 
C - 269

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with 

MMR+ 

Number with

MMRAm 1 + x + 5 10 

Am R + - 2 11 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Japanese Criteria for HNPCC; Sample seems to be a convenience Sample; unknown selection. *Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 270

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 271

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 272

Study: Luce, 1995 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? x Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? x Was a minimum of hMLH1 and hMSH2 sequencing performed? x 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

Consecutive cases of CRC referred to a tertiary hospital. Patients reporting 2 or more cases of colorectal or endometrial cancer in first-degree relatives or only 1 affected firstdegree relative younger than 50 years old were interviewed, and detailed pedigrees were obtained. Country: US Center: Single 

Genetic testing was done in all enrolled patients with CRC 

hMLH1 & hMSH2 Y N ? 

≥5 MSI markers used? MSI-H defined by ≥ 2 markers? Microdissection? Gene screening? Deletion analysis? Conversion analysis? 

None of these testing was done 

Am 1 x 

Am R Beth 1 Beth R MSI-H MSI-L IHC Age <50 Suggestive family history Specify Other Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 273

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

N=19 cases/families Mean age=40.3 %male=ND Dropouts=0 

6 cases/families with MMR / 19 cases/families with CRC 4 MLH1 2 MSH2 

19 cases/families with CRC, 63% positive AM 

ND ND 

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 x 

Amsterdam R Other (specify) 

6/12 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 Amsterdam R Other (specify) 

ND 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

ND 
C - 274

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

 x 

If yes, which clinical criteria (check all that apply)? Am 1 + Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) Other (specify) COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test 

Number with

MMR+ 

Number with

MMRAm 1 + x + (A) 6 (B) 6 

Am R + - (C) 0 (D) 7 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

See definition above; Essentially they are not Am1 Selected Familial CRCs (proband and 1 or 2 1st degree) *Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 275

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 276

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 277

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Lynch, HT 1996, Ref ID 2661 Single center USA. 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Evaluated reaction of 51 members of Navaho Indian CRC patients who received MLH1 genetic testing underwent genetic counseling. 

Mutation 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

As per study description. Family reserv

with intercedcounseling. 

Outcomes Dropouts, 

explanations, other 

comments 

Study Conclusions Quality 

Many family members believed the family’s fate was influenced by factors such as fear, a taboo, or a curse. Four family members denied their heightened cancer genetic risk. Several who had not undergone gene testing wanted their children testing. 

ND DNA-based genetic counseling requires compassion and empathy 

coupled with intensive pre-education regarding potential penalties and advantages that might emanate from this knowledge. Special care must be given to patients’ culture. 

No formal instru

reactions. Im

educational le

canc
C - 278

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 5 9 X 

8a 

8b,8c 

Public health or policy 

2,3 5 9 X 

8a 

8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 279

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely Somewhat likely X 

Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 X 

ND 

NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No X 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No X 

NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND X 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 X 

ND 

NA 
C - 280

Domain/question Place an “X” in one Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? Yes No X 

ND 

Are the statistical methods appropriate? Yes No X 

ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No X 

Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 X 

ND 

NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

X 

Yes 

No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No X 

Can’t 

tell 
C - 281

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Lynch 1997 (RefID 2355) US; also see Lynch 1996 for similar information Single Center 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

High-risk members from 4 extended HNPCC families that were selected from Creighton’s HNPCC resource, which comprised about 100 HNPCC extended kindredsEmotional responses to receiving genetic test results in 130 individuals from 4 extended families with HNPCC. 36% had positive test 64% had negative test 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

Of the 89 members did not enroll in the study, 67% did not respond to the invitation, 22% had their counseling postponed, and 10% refused, citing fear of knowing, fear of insurance discrimination, surviving guilt and disinterest. 

Genetic coun

Outcomes Dropouts, explanations, other comments Study Conclusions 

1. When mutation positive, 20% exhibited sadness, particularly because of concern of the potential risk 

of transmission of the deleterious mutation to their children. Others were concerned about their personal cancer destiny, particularly the possibility of an unfavorable prognosis that could lead to their early death and may put their children in jeopardy of not having a biological parent to care for them. 

2. Following a positive test result, 67% were considering prophylactic colectomy. 3. Following a positive test result, 87% were considering prophylactic total abdominal hysterectomy and 

bilateral salpingo-oophorectomy. 

4. 18/130 were concerned with sharing information with their insurance company. 

“DNA testing should be restricted verified candidate families and accgenetic counseling...patients exhibrange of emotional responses when genetic testing status...Whether... results, has a long-term effect on bquality of life, or on cancer morbidmortality remains to be seen....” 
C - 282

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 5 9 8a 8b,8c 

Public health or policy 

2,3 5 9 8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 283

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 ND NA Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes No Were data collection tools shown or are they known to be reliable? Yes No Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 ND NA 

Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially No Is there a statistically significant difference between groups? Yes No ND Are the statistical methods appropriate? Yes No ND Indicate the unit of allocation Community Organization/group Provider Client Institution Indicate the unit of analysis Community Organization/group Provider Client Institution If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No NA 
C - 284

Domain/question Place an “X” in one Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No Can’t 

tell 
C - 285

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Meiser 2004 Australia Multicenter 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Psychological impact of predictive genetic testing in 114 individuals (32 carriers and 82 non-carriers) were assessed after disclosure of carrier status. 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl 

Only individuals in whose families a known HNPCC mutation had already been identified, who chose to receive a predictive genetic testing result and had never had CRC or any of HNPCC-related cancers were included in the study. 

Self-answerepost-test at 2 carrier status 

Outcomes Dropouts, explanations, other comments Study Conclusions 

1. Compared to baseline, carriers showed a significant increase in mean scores for intrusive and avoidant 

thoughts about CRC and a significant decrease in mean depression scores 2 wk and 4 mo post notification of results. 

2. Carriers had significantly higher intrusive and avoidant thoughts 2 wk and 12 mo post-notification 

compared to non-carriers. 

3. Compared to baseline, non-carriers showed a significant decrease in mean scores for intrusive and 

avoidant thoughts, and mean depression scores at all follow up time points. 

Predictive genetic testing for HNPpsychological benefits amongst noand no adverse psychological outcoobserved amongst carriers. 
C - 286

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 5 9 8a 8b,8c 

Public health or policy 

2,3 5 9 8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence 
C - 287

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 ND NA Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes No Were data collection tools shown or are they known to be reliable? Yes No Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 ND NA 

Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially No Is there a statistically significant difference between groups? Yes No ND Are the statistical methods appropriate? Yes No ND Indicate the unit of allocation Community Organization/group Provider Client Institution Indicate the unit of analysis Community Organization/group Provider Client Institution If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No NA 
C - 288

Domain/question Place an “X” in one Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No Can’t 

tell 
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Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Mesters 2005 Netherlands Single 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

30 persons from the database of the Netherlands Foundation for the Detection of Hereditary Tumors (the article did not explicitly state who these subjects were but 37% had confirmed carrier status) were interviewed at home. 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

Outcomes Dropouts, explanations, other comments Study Conclusions 

1. Disclosure was stimulated if people felt morally obliged to do so or when they anticipated regret if 

something happened because it is preventable. Presence of external cues (eg, professionals) appeared important for disclosure as well. 

2. Disrupted and tense family relations were reasons not to disclose, as well as young age of the message 

recipients and negative experience at their first attempt to disclose. 

3. Disclosure was restricted to the nuclear family. A personal approach was preferred. 

4. Participant disclosed only the presence of the hereditary defect and the possibility of testing. It was 

mostly considered the recipients’ responsibility and own choice to obtain further information. 

5. Limitations of this study include that findings might be affected by recall bias since the interviews 

focused on prior experiences about HNPCC disclosure. 

See paper concerning practice imp
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Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 5 9 8a 8b,8c 

Public health or policy 

2,3 5 9 8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
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Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 ND NA Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes No Were data collection tools shown or are they known to be reliable? Yes No Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 ND NA 

Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially No Is there a statistically significant difference between groups? Yes No ND Are the statistical methods appropriate? Yes No ND Indicate the unit of allocation Community Organization/group Provider Client Institution Indicate the unit of analysis Community Organization/group Provider Client Institution If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No NA 
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Domain/question Place an “X” in one Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No Can’t 

tell 
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Study: Moslein, 1996Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? XWas genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? X Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

*although not all study patients had CRC but data on those patients with CRC are extractable 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

Patients having sporadic, familial colorectal carcinoma, and HNPCC meeting the Amsterdam criteria Convenience samples Country: Germany Center: multi 

The DNA sequence of hMSH2 and hMLH1 was evaluated in all patients 

HMSH2 and hMLH1 Y N ? 

≥5 MSI markers used? 

X 

MSI-H defined by ≥ 2 markers? 

X 

Microdissection? X Gene screening? X Deletion analysis? 

 X 

Conversion analysis? 

 X 

Am 1 x 

Am R Beth 1 Beth R MSI-H X MSI-L IHC Age <50 Suggestive family history 

x 

“Familial CRC” Other 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
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Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

N=36 Mean age=51 %Male=ND Dropouts=0 

13 patients with MMR/ 36 patients with CRC 7 MLH1 4 MSH2; 1 MLH1&MSH2 

NA 5/9 patients who met 

Amsterdam criteria (HNPCC) had positive MSI 8/12 patients who had familial CRC had positive MSI 

“In this study, MSI was present in 100% (10/10) tumors (whether from familial or HNPCC cases) in which mutations in either hMLH1 or HMSH2 were subsequently found. These results suggest that MSI may be a very sensitive method for distinguishing a subset of HNPCC colon cancers (i.e., those resulting from defective mismatch repair) from sporadic colon cancers.” 

ND 
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 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 x 

Amsterdam R Other (specify) 

6/14 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 x Amsterdam R Other (specify) 

5/9 positive MSI (≥ 2 markers show

Note: 5 (14-9) patients who met Amnot have tissue available for MSI t

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

ND 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

x 

If yes, which clinical criteria (check all that apply)? Am 1 + x Am R + Beth 1 + Beth R + Age <50 Suggestive family history (Familial CRC) x Other (MSI+) x 
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What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 5 (B) 0 

Am R + - (C) 0 (D) 4 Beth 1 + Beth R + MSI-H* X MSI-L* Age <50 IHC (no staining) Other (specify)** 

Patients with CRC who met Amsterdam criteria (n=14) Note: 5 patients did not have tissue for MSI testing, so that they are not in the 2x2 table What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 1 (B) 6 

Am R + - (C) 0 (D) 0 Beth 1 + Beth R + MSI-H* X MSI-L* Age <50 IHC (no staining) Other (specify)** 

Patients with sporadic CRC (n=7) Note: 5 patients did not have tissue for MSI testing, so that they are not in the 2x2 table 
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What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 4 (B) 4 

Am R + - (C) 0 (D) 4 Beth 1 + Beth R + MSI-H* x MSI-L* Age <50 IHC (no staining) Other (specify)** 

Patients with familial CRC (n=15) Note: 3 patients did not have tissue for MSI testing, so that they are not in the 2x2 table 

“MSI” 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
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Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
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Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
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Data Extraction for Analytic Validity 

Study: (Author, year, UI) Muller 2001, Ref ID 731 Inclusion criteria (all must be yes) Did study evaluate biological material from patients considered to be at risk for HNPCC? Did the study report ANY of the following? (check which one below) 

9) Proportion MSI-H with NIH markers versus other markers 10) Sensitivity or specificity of MSI-H using NIH markers compared with a reference standard that the study claims is bet11) Sensitivity or specificity of IHC compared with an immunohistochemical standard that study claims is better 12) Sensitivity or specificity of a genetic technique compared with a reference standard (or combination of standards) 

 5) Reliability of MSI/IHC/genetic method across laboratories or within a laboratory Are data (proportions or 2 X 2 tables) extractable? *NIH markers are BAT-25, BAT-26 D2S123, DS346 and D17S250 Exclusion criteria (exclude if yes) 

 Yes No 

Did the study include the index test in the reference standard? X 

Describe source of biological materials 

(and whether patients were known to 

have an HNPCC phenotype) 

Summarize how materials were processed and 

analyzed 

20 colorectal cancers from different institutions including those known to be MSI-H or have DNA mismatch repair mutations of hMLH1 (n=6) or hMSH2 (n=11) or negative controls (n=3) 

A set of two unstained slides from each case were sent to participating medical centers with capability of performing immunoperoxidase assays for hMLH1 and hMSH2 
C - 301

MSI Proportion (add additional 2 X 2 tables where relevant) 

Proportion MSI-H using NIH markers (≥2 markers) Proportion MSI-H using other markers 

MSI with a reference standard 

MSI-H using another 

reference standard 

Describe 

 Positive Negative MSI-H using NIH markers 

MSI-H (≥2 markers) 

 MSI-S or MSI-L 

IHC with a reference standard 

IHC using another 

immunohistochemical 

reference standard 

Describe 

 Positive Negative IHC Positive Describe Negative 

Genetic technique with a reference standard 

Reference standard 

genetic technique 

Describe 

 Positive Negative Index genetic technique Positive Describe Negative 
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Intra or inter-hospital reliability 

data 

Describe 

Of 18 participating centers 2 were excluded: one because slides were damaged in transit and the other because Sensitivity for detecting loss of hMSH2 2 expression ranged from 84 to 100%; 10 centers identified all six. 5/6 

were in the same case suggesting that staining or interpretation were not random. 14/16 laboratories showed 100% specificity (one laboratory had 93% specificity due to staining failure on one s

demonstrated 45% specificity due to weak or absent staining in most cases. Re-review of returned hMSH2 slides shoed lack of internal positive control staining in at least 2 of the 6 hMSH

8 of 16 centers. The other 8 centers had 100% sensitivity and 93-100% specificity on re-review. The slides that 

positive control staining were largely accounted for by two cases suggesting the possibility of fixation or proceVariation of staining quality and interpretation was much greater for hMLH1 than for hMSH2. individual cente

sensitivity and 40 to 100% specificity. Re-review of the returned slides resulted in sensitivities of 0-90%. 12 centers experienced difficulty with lack ocontrol or high background. Overall, four laboratories performed relatively well with both hMLH1 and hMSH2 staining protocols. The key these and distinguishing them from the rest was a heated antigen retrieval step. Steam treatment in the presence the best results although steam and citrate buffer also provided acceptable results.

 Study Quality 

1 Was the description of how MSI or IHC and other genetic techniques described in sufficient detail that others could replicate it (e.

either a full description or relevant references)? 

2 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

3 Was MSI, IHC, other genetic testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

4 Was there a clear description of which mismatch repair mutations were being tested for? 5 Were quality control methods described for the molecular and genetic tests? 6 Did the authors attempt to address the reproducibility of results (reliability of tests)? 7 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or tho

did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 

8 Was microdissection performed? 9 Did the study specify whether the biological tissues were from patients known to have HNPCC clinically? 

10 Did the study include a control group in which biological material was obtained from patients known not to have HNPCC clinical11 Overall rating (A B C) 
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Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Murakami 2004 Japan Single 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

47 participants who had genetic testings.42 completed 1-month psychologic follow up 

6/27 probands had positive genetic test 21/27 probands received uninformative test result 5/15 relatives received positive test 10/15 received negative test 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

Probands or unaffected relatives whose family members had been identified as carrying the hMSH2/hMLH1 mutation before or during this psychologic study procedure, and age ≥ 20 yrs. 

Structured CDSMIIIR or 

Outcomes Dropouts, explanations, other comments Study Conclusions 

3/27 probands had minor depression at 1 month (1 had minor depression at baseline). 2/42 had posttraumatic stress symptoms at 1 month (both had positive test results). 5/42 had feelings of guilt. The only predictor of psychologic distress found was the presence of a major or minor depression (OR 19.41; 95%CI, 1.42-264.95). 

See Figure 1 in original paper. Disclosure of genetic test results fo

may not cause significant psycholoJapanese probands or relatives, but providers should not neglect to ass

individuals for psychologic responminor depression or posttraumatic symptoms. 
C - 304

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 5 9 8a 8b,8c 

Public health or policy 

2,3 5 9 8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 305

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 ND NA Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes No Were data collection tools shown or are they known to be reliable? Yes No Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 ND NA 

Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially No Is there a statistically significant difference between groups? Yes No ND Are the statistical methods appropriate? Yes No ND Indicate the unit of allocation Community Organization/group Provider Client Institution Indicate the unit of analysis Community Organization/group Provider Client Institution If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No NA 
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Domain/question Place an “X” in one Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No Can’t 

tell 
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Study: Nakahara, 1997 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? X Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? X Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

Patients who met the Japanese registry’s clinical diagnostic criteria for HNPCC proposed at the 34th

Annual Meeting of the JSCCR Country: Japan Center: Single 

MSI was performed in all 32 patients with (multiple) colonic and/or extracolonic cancers. PCR-SSCP analysis of hMSK2 and hMLH1 mutations was performed in DNA samples from 11 MSI phenotype cases (of the 18 MSI+ cases). 

hMSK2 and hMLH1 Y N ? 

≥5 MSI markers used? 

x 

MSI-H defined by ≥ 2 markers? 

x 

Microdissection? X Gene screening? X Deletion analysis? 

 X 

Conversion analysis? 

 X 

Not the NCI marker set. 

Am 1 x 

Am R Beth 1 Beth R MSI-H MSI-L IHC Age <50 Suggestive family history Specify Other x MSI phenotype, defined as “MSI+ in cancer lesions at most of the microsatellite loci examined.”

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 308

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

N=32 Mean age=59.3 %Male=41 Exclusions=66%, MSI- (n=14) or non-MSI phenotype cases (n=7) For the 11 patients with CRC and MSI phenotype: Mean Age: 45 Male: 45%

7 patients with MMR / 11 patients with CRC and MSI phenotype 1 MLH1, 2 MSH2; 4 MLH1&MSH2 

32 patients with CRC, 22% positive AM, 56% positive MSI 

5 (28%) of the 18 MSI+ patients fulfilled the Amsterdam criteria 2 (14%) of the 14 MSI- patients fulfilled the Amsterdam criteria 

“The ‘Japanese criteria’ have the advantage of being able to detect more HNPCC kindreds from borderline HNPCC kindreds, compared to the Amsterdam criteria. We propose that a microsatellite assay is necessary to detect more “true-HNPCC kindreds” from borderline HNPCC kindreds, and molecular findings of MSI phenotype should be added to the criteria for HNPCC.” 

ND 
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 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 

Amsterdam R Other (specify) 

ND 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 X Amsterdam R Other (specify) 

5/7 MSI high 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

ND 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

x 

If yes, which clinical criteria (check all that apply)? Am 1 + Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) Other (Japanese registry’s clinical diagnostic criteria for HNPCC) 

x 
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What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index 

test 

Am 1 + x + Am R + - Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Patients with CRC who fulfilled the Japanese registry’s clinical diagnostic criteria for HNPCC and had MSI phenotype (MSI+ in cancer lesions at most of the microsatellite loci examined) 

NOTE THAT WE DO NOT KNOW HOW MANY OF THE MSI-L&S WERE AM1 SO WE CANNOT ESTIMATE THE SAME TABLE ASSUMING THAT ALL MSI-S&L ARE NEGATIVE FOR MUTATIONS 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 311

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 312

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 313

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Norum 2000 Norway Single Center 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

11 health insurers in Norway were mailed a questionnaire to evaluate two hypothetical individuals’ requests for insurance; one has HNPCC mutation, the other has BRCA1/BRCA2 Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl 

Outcomes Dropouts, explanations, other comments Study Conclusions 

9/11 insurers responded. 2 companies reported results on personal indemnity insurance. There were no restrictions for individuals with a genetic risk of breast CA when seeking life insurance or disability pension. One company raised premium on the one with genetic risk of CRC. Another company offered standard or higher rate of premium on personal indemnity insurance in the person with genetic risk of CRC. 

 In Norway: 

“According to the survey, individuhereditary risk for breast or CRC mexperience insurance discriminatiobased on family history of cancer. testing results are generally not incdetermination of health insurance. 

based on the present state, Norweghave no need for hesitation in undegenetic testing out of fear of insuradiscrimination.” 
C - 314

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 5 9 8a 8b,8c 

Public health or policy 

2,3 5 9 8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 315

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 ND NA Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes No Were data collection tools shown or are they known to be reliable? Yes No Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 ND NA 

Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially No Is there a statistically significant difference between groups? Yes No ND Are the statistical methods appropriate? Yes No ND Indicate the unit of allocation Community Organization/group Provider Client Institution Indicate the unit of analysis Community Organization/group Provider Client Institution If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No NA 
C - 316

Domain/question Place an “X” in one Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No Can’t 

tell 
C - 317

Study: Park, 1999, 2002, & 2007 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? X Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? X Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population inclusion/exclusion 

criteria; country, single or 

multicenter, 

Design and analytic 

strategy (brief 

description of strategy 

used for testing patients 

with CRC)

Which 

MMR 

mutations 

were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors anal

(check all tha

apply) 

123 pts (123 families) with Suspected-HNPCC which were not Am1; from 8/58 centers (7 countries) of ICG-HNPCC. Criteria: Cat1: (vertical CRC transmission OR 2 CRC siblings in family) AND (multiple CR tumors including adenoma OR >=1 CRC before 50y OR extracolonic cancer endometrium/urinary tract/small intestine/stomach/hepatobiliary /ovary) Cat2 (Sporadic): 1 CRC patient AND (onset<40y OR endometrial, 

Urinary tract, or small intestine cancer in patient or a sibling <50y OR 2 siblings with other integral colonic cancers, one aged <50y ) They compare them against 154 Am1 families which have received genetic testing

All 123 S-HNPCC received genetic testing. All 154 excluded Am1 have received genetic testing (Discussion) All detected mutations were verified by sequencing 

HMLH1 (n=123) HMSH2 (n=122) HPMS1 (n=27) HPMS2 (n=24) 

 Y N ? 

≥5 MSI markers used? 

 X 

MSI-H defined by ≥ 2 markers? 

 X 

Microdissection? X Gene screening? X Deletion analysis? 

X 

Conversion analysis? 

 X 

Am 1 

Am R Beth 1 Beth R MSI-H MSI-L IHC Age <50 Suggestive family history Specify Other Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 318

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

N=123 S-HNPCC; ND on %M; dropouts not pertinent 

24 with MMR/123 with CRC; 16 with hMLH1 / 123 ; 8 with hMSH2 /122 

N=123 S-HNPCCNOT-AM1 and n=154 AM1 pts 

NA Criteria I detected a 

mutation rate of 28% which is sufficiently high to recommend genetic testing in such families; useful in clinical setting; should be offered genetic testing. Criteria II have been ineffective as a means of identifying individuals with mutations; should not be offered genetic testing. 

NO 
C - 319

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 X 

Amsterdam R Other (specify) X 

19/67 for Cat1; 5/56 for Cat2 Æ 2

77/154 for Am1 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 Amsterdam R Other (specify) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

X 

If yes, which clinical criteria (check all that apply)? Am 1 + X Am R + Beth 1 + Beth R + Age <50 Suggestive family history (see the description of the population)

X 

Other (Discussion: 50 centers didn’t test because the PIs are clinicians; testing is expensive; as of that paper, it was unclear whether non Am1 pts need testing) 

X 
C - 320

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + X + 77 77 

Am R + - 24 99 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Defined above. Am1 vs Cat1 & Cat2 S-HNPCC *Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 321

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 322

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 323

Study: Peel, 2000 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? X Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? X Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

Referral HNPCC case families (Amsterdam criteria) These were from the ICG HNPCC group database (but they give MSI-HL vs MSI which is not covered in the other papers) 

MSI were performed in 10 families; MSH2 and MLH1 were done in 11 families 

MSH2 and MLH1 Y N ? 

≥5 MSI markers used? 

X 

MSI-H defined by ≥ 2 markers? 

 X 

Microdissection? X Gene screening? X Deletion analysis? 

 X 

Conversion analysis? 

 X 

Am 1 X 

Am R Beth 1 Beth R MSI-H MSI-L IHC Age <50 Suggestive family history Specify Other Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 324

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

11 families 5 families/11 

families have MMR 3 MSH2 and 2 MLH1; 2 mutations were previously identified; 3 were novel mutations 

5/556 had Am1 (.9% ; 0.3 to 2.1%) NO comments 

“The prevalence of HNPCC in the general population is most likely closer to 1% rather than 5% Previously reported characteristics of HNPCC like proximal tumor location may not always hold true in a population-based sample.” 

No 
C - 325

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 X 

Amsterdam R Other (specify) 

5/11 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 X Amsterdam R Other (specify) 

4/9 MSI H or Low 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

ND 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

X 

If yes, which clinical criteria (check all that apply)? Am 1 + X? Am R + X? Beth 1 + Beth R + Age <50 Suggestive family history (specify) Other (specify) 
C - 326

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + 3 1 

Am R + - 0 5 Beth 1 + Beth R + MSI-H* X MSI-L* X Age <50 IHC (no staining) Other (specify)** 

MSI only X 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 327

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 328

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 329

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Perrin, 2001, RefID 1361 Country: France Center: single 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

A retrospective series of 225 patients diagnosed with sporadic CRC. 208 tumors were tested for IHC, and correlations with survival were performed by log-rank test and Cox model. 

ND 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  x 

A retrospective series of 225 patients diagnosed between 1990 and 1998 with sporadic colorectal cancers underwent surgery. There were 121 men and 104 women. 46% were >65 years old. 

ND 

Outcomes Dropouts, explanations, other comments Study Conclusions 

27 of 208 (13%) of the tumors tested showed abnormal 

MSH2 and/or MLH1 expression. 

For the whole population, there was no association of 

MSH2 and/or MLH1 status (by IHC) with either overall or disease-free survival. 

56/225 tumors were located in the proximal colon. 15/56 

(31%) tumors were negative for either protein or both. Negativity of MLH1, and “MSH2 and/or MLH1” were correlated with a localization in the proximal colon (p=0.02 and p<0.01, respectively). Negativity of MSH2 was close to being significantly associated to this localization (p=0.07); the absence of significance may be due to a low number of cases (n=10). 

For the subpopulation of proximal tumors, MSH2 and/or 

MLH1 negativity was associated with a longer diseasefree survival. 

17/225 (7.5%), no IHC testing Abnormal IHC measurement of ML

MSH2 protein were frequently tumoproximal colon; in this subpopulatioproximal tumors, MSH2 and/or MLnegativity was associated with a lonfree survival. 

Place an X in boxes that the study is relevant 
C - 330

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 5 9 8a 8b,8c 

Public health or policy 

2,3 5 9 8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 331

Domain/question Place an “X” in one Selection Bias A 

(stron

Are individuals selected to participate likely to be representative of target population? 

Very likely Somewhat likely 

x 

Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 ND x NA Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stron

Is the method of random allocation stated Yes No If the method of random allocation is stated, is it appropriate Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stronPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes No 

Blinding A (stronWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND NA 

Data Collection methods A 

(stron

Were data collection tools shown or are they known to be valid? Yes No Were data collection tools shown or are they known to be reliable? Yes No Withdrawals and Dropouts A (stronIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 ND NA 

Analysis A (stronIs there a sample size calculation or power calculation Yes Partially No Is there a statistically significant difference between groups? Yes No ND Are the statistical methods appropriate? Yes No ND Indicate the unit of allocation Community Organization/group Provider Client Institution Indicate the unit of analysis Community Organization/group Provider Client Institution If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No NA 
C - 332

Domain/question Place an “X” in one Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No Can’t 

tell 

Intervention Integrity A (stronWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No Can’t 

tell 
C - 333

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country 

Single or Multicenter? 

Peterson 2003 (RefID 833) US Single Center 

Study description (N enrolled) How was HNPCC 

defined? 

Inclusion/exclusion criteria 

39 members of 5 families, representing 30 separate households were interviewed by telephone. Each family included a proband with HNPCC mutation who had genetic counseling and testing through clinical research protocol, each family included at least 5 members estimated to be at 50% risk of carrying a mutation (regardless if they have had actual genetic testing) 

Did all patients 

have a personal 

history of an 

HNPCC-related 

cancer? 

Check one 

Yes No Uncl  x 

Outcomes Dropouts, explanations, other comments Study Conclusions 

1. The identification of a cancer-predisposing gene mutation was new information that confirmed their 

beliefs, and families were not shocked or surprised by the news. This information was not viewed as a medical crisis. 

2. No one reported that the news of the mutation was intentionally withheld from any family members. 

They willingly shared this information with other relatives, including those who did not participate in the study. 

3. Compared with mutation carriers, those who tested negative for a mutation and those who did not test 

perceived those topics as less personally relevant and were less involved in discussing the mutation and the need for genetic testing. Spouse’s reactions were similar. 

4. Probands assumed that each adult who was told about the mutation would be responsible for telling 

his or her children; however, probands did not follow up with these adults to find out if news about the mutation had been actually conveyed to those children. Participants (other than probands) viewed themselves as primarily responsible for notifying their nuclear family and encouraging them to seek counseling or testing. 

“Family members who were persuthose services by the proband were to have counseling and testing and likely to seek those services soonercounseling should attempt to identiexisting communication norms witand ways that family members can active role in encouraging others to their cancer risk and options for tesInterventions may also need to targimmediate family and to unaffected members who may be central to thcommunication process (eg, spouscarriers).” 
C - 334

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 5 9 8a 8b,8c 

Public health or policy 

2,3 5 9 8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 335

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 ND NA Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes No Were data collection tools shown or are they known to be reliable? Yes No Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 ND NA 

Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially No Is there a statistically significant difference between groups? Yes No ND Are the statistical methods appropriate? Yes No ND Indicate the unit of allocation Community Organization/group Provider Client Institution Indicate the unit of analysis Community Organization/group Provider Client Institution If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No NA 
C - 336

Domain/question Place an “X” in one Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No Can’t 

tell 
C - 337

Pinol, 2005 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? X Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? X Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

Newly diagnosed CRC; Spain, multi-center Excluded FAP, IBD, patient or family refusal to participate 

MSI and IHC performed in all patients with CRC; testing centralized in 2 centers; pts with MSI and/or IHC of MSH2 or MLH1 underwent germline genetic testing for MSH2 and MLH1 by probe amplification and sequencing 

MSH2 and MLH1 Y N ? 

≥5 MSI markers used? 

 X* 

MSI-H defined by ≥ 2 markers? Microdissection? X Gene screening? X Deletion analysis? 

X 

Conversion analysis? 

 X 

Pre screen with BAT26 and if non-conclusive the remaining battery of the 5 marker set 

Am 1 

Am R Beth 1 X Beth R X MSI-H MSI-L IHC X Age <50 X Suggestive family history 

X 

Specify Other Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 338

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

1222/1872 eligible 11 among the 91 

tumor samples that were genotyped (MSI OR IHC abnormalities) [To clarify: 91 were MSI OR IHC abnormal; 73 were abnormal in MSI AND IHC; 10 ONLY in MSI and 8 ONLY in IHC] 

24% Beth revised 18% Beth original 1.8% (22)Am2 

See tables for various data 

MSI testing and IHC testing are equivalent in terms of costeffectiveness to screening for the MMR. 

Unclear 
C - 339

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 

Amsterdam R Other (specify) 

4/22 = 18% (but 4 MSI-S and IHCgenetically tested) AMONG THE GENETICALLY T4/18 = 22% 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 Amsterdam R Other (specify) 

ND 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

ND 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

 X, on MSI or IHC results 

If yes, which clinical criteria (check all that apply)? Am 1 + Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) Other (specify) 
C - 340

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) (B) 

Am R + - (C) (D) Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 341

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 342

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 343

Study: Pistorius, 2000 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? X Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? X Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

106 pts with Fam Hx or early Hx of Cancer were referred 72 patients met Bethesda, but not all of them CRC. Patients from Germany and Czech Republic (multicenter?) 

MSI was performed; sequencing was done in patients with MSIH IHC only in those with mutations (negative stain in all) 

hMLH1 and hMSH2 and hmsh6 

 Y N ? 

≥5 MSI markers used? 

X 

MSI-H defined by ≥ 2 markers? 

X 

Microdissection? x Gene screening? x Deletion analysis? 

 x 

Conversion analysis? 

 x 

The NCI marker set was used 

Am 1 X 

Am R x Beth 1 x Beth R MSI-H MSI-L IHC Age <50 Suggestive family history Specify Other Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 344

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

72 index patients fulfilled Bethesda; Not all 72 CRC 19 Am2 and they are all CRC 

15 patients with MMR in 38 MSI-H patients, 6 MLH1 8 MSH2 1 MSH6 Among 19 Am2, 17 were MSI-H and 12/17 had mutations 

72/106=68% had Beth Among Beth: 50/72 Beth1-3 19/72 Am1 

Family history and MSI-H are both strong indicators of germline mutations in MSH2, MLH1 and MSH6 genes. Identification of mutation status allows clear-cut decisions on whether or not inclusion in surveillance programs is indicated. 

Of the 15 families with mutations, 60 patients and family members were analyzed, 26 were affected and 8 were asymptomatic. Thewere included in the HNPCC surveillancprogram, where as 2noncarriers were excluded from this program. But 3 of 2noncarriers presentewith colon CA at ag35, testicular CA at age 30, and 2 colon polyps at age 58, respectively. 
C - 345

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 x 

Amsterdam R X Other (specify) 

12/17 (Amsterdam and MSI-H) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 Amsterdam R Bethesda x 

38/72 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

ND 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

X 

If yes, which clinical criteria (check all that apply)? Am 1 + Am R + Beth 1 + x Beth R + X Age <50 Suggestive family history (specify) Other (specify) 
C - 346

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test 

Number with

MMR+ 

Number wi

MMRAm 1 + + 12 5 

Am R + x - 3 18 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Patients with CRC who fulfilled Bethesda and had MSI-H 

MSI only 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 347

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 348

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 349

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Plaschke 2004, Ref ID 382, Multicenter Germany 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Clinical and molecular data on families with an established mutation in MSH6 were compared with families with MLH1 and MSH2 mutations 

Fulfillment of Amsterdam 1 or 2 plus Bethesda plus absence of MSH6 expression by protein staining plus normal expression of MLH1 and MSH2 by protein staining Patients selected from a registry based upon Amsterdam I and II criteria without age restriction and Bethesda guidelines A total of 706 families had been registered. Available tumors were analyzed for MLH1 and MSH 2 protein expression and selected patients in whom these proteins were expressed underwent additional testing for MSH6 expression. 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

1) Patients had developed tumors 

that were either low or high MSI 

2) MLH1 and MSH2 were 

expressed in the tumor cells 

Tumor samples from selected patients were studied for MSH6 expression with IHC and by sequencing. 

Observation underwent an
C - 350

Outcomes Dropouts, explanations, other comments Study Conclusions 

Among 396 enrolled members of families with pathogenic MSH6 mutations, 115/29%) were affected by malignant disease. This rate was lower than for families with MLH1 or MSH2 mutations (37.5%). Colorectal cancer was statistically less frequent among all tumors (42.4 versus 66 percent). Such a difference was not observed for any other HNPCC tumors. Median age of onset of colorectal cancer in putative mutation carriers was 10- years higher than for MLH1 and MSH2 (54 versus 44 percent). Median age of onset of any tumor was 8 years higher for MSH6 (51 versus 43). Cumulative risk by age to develop CRC or any tumor was statistically lower for MSH6 mutation carriers compared with MLH1 and MSH2. 

ND “Later age of disease onset and lower 

incidence of colorectal cancer may contto lower proportion of identified MSH6 mutations in families suspected of HNPHowever, in approximately half of thesfamilies, at least one patient developed colorectal or endometrial cancer in the fdecade of life. Therefore, a surveillance program as stringent as that for families MLH1 or MSH2 mutations is recomme

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence 
C - 351

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely Somewhat likely X 

Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 X 

ND 

NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes X 

No 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No X 

NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No X 

ND 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 X 

ND 

NA 
C - 352

Domain/question Place an “X” in one Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? X 

Yes 

No ND 

Are the statistical methods appropriate? Yes No ND Indicate the unit of allocation Community X 

Organization/group 

Provider Client Institution 

Indicate the unit of analysis Community X 

Organization/group 

Provider Client Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND X 

NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No X 

ND 

NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

X 

Yes 

No Can’t 

tell 
C - 353

Study: Ponz de Leon 1999 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

1899 colorectal malignancies and 1831 patients registered of whom a family history could be obtained in 1721 Cancer registry in Modena, Italy and surrounding communities 

Patients and families classified into five categories to a more or less likely genetic basis: 1) HNPCC (Amsterdam 1) 2) suspected HNPCC (2 generations with CRC or other HNPCC tumor plus at least one before 50 plus 50% or more of siblings affected by cancer), 3) juvenile cases 4) A specific cancer aggregation; 5) Sporadic cases. MSI and mutation analysis performed in 18 families with HNPCC and 18 with suspected HNPCC 

hMLH1 and hMSH2 Y N ? 

≥5 MSI markers used? 

X 

MSI-H defined by ≥ 2 markers? 

X 

Microdissection? X Gene screening? X Deletion analysis? 

 X 

Conversion analysis? 

 X 

Am 1 X 

Am R Beth 1 Beth R MSI-H X MSI-L IHC Age <50 Suggestive family history 

X 

Specify Family history categories 1-5 as in column 2 Other Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 354

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

18 in HNPCC and 18 suspected 

3 mutations detected of 36 patients analyzed 2 hMSH2 1 MLH1 

Of 1721 in whom a family history could be obtained, 47 individuals fulfilled Am-1 and 84 suspected HNPCC 

ND 

 Incidence of mutations using a population-based approach lower than previously reported and limited to families with fullblown HNPCC. 

ND 

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 X 

Amsterdam R Other (specify) 

3/18 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 X Amsterdam R Other (specify) 

11/18 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

 ND 
C - 355

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

X 

If yes, which clinical criteria (check all that apply)? Am 1 + X Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) Other (specify) “Suspected HNPCC as above” X COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with 

MMR+ 

Number with

MMRAm 1 + + (A) DATA CANNOT BE 

EXRACTED 

(B) 

Am R + - (C) (D) Beth 1 + Beth R + MSI-H* X MSI-L* Age <50 IHC (no staining) Other (specify)** 

Amsterdam criteria 1 positive *Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 356

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 357

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 358

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country 

Single or Multicenter? 

Porteous, 2003, RefID 736, UK, Single 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criter

A total of 160 patients who were approached for the Colorectal Cancer Genetic Susceptibility (COGS) study between February 1999 and July 2000, were eligible to participate in the patient feedback 

study. Of the 160 questionnaires given to patients, 111 (69%) were returned completed. Only one of the 111 patients who completed the questionnaire had declined to participate in the COGS study. 

Newly diagnosed CRC patients under the age of 55 years are offered genetic testing for 3 mutations in MMR genes (i.e. MLH1, MSH2, MSH6) 

Did all patients have a 

personal history of an 

HNPCC-related 

cancer? 

Check one 

Yes No Uncl x 

Patients who were approached for Colorectal Cancer Genetic Suscept(COGS) study between February 1July 2000, were eligible to particippatient feedback study. 

Outcomes Dropouts, 

explanations, 

other comments 

Study Conclusions 

“Of the 111 participants, 61 (55%) were male and 50 (45%) were female ranging in age from 31 to 55 years (mean=48.6, 

s.d.=5.5). The risk of HNPCC for their relatives was low for 60% of participants (n=65), moderate for 39% (n=42) and high for 2% (n=2). The majority were married or living with a partner (n=90, 83%), and had been educated up to age 16 years only (n=56, 60%). In all, 81% (n=89) had at least one child.

After receiving the information sheet about the COGS study, but prior to discussing participation with a member of staff, 91% (n=101) reported that they had decided to take the genetic test, one individual (1%) had decided not to take the test and nine (8%) were undecided.Most participants (n=97, 87%) reported some degree of difficulty coming to terms with the diagnosis of cancer, with 15% (n=17) overall reporting that it had been very difficult. 

Similarly, the majority of participants (n=89, 80%) indicated that 

it had been difficult to cope physically with their illness and treatment, with eight participants (7%) overall reporting that it 

 “The majority of participants found it highly acceptable to 

have information about HNPCC brought to their attention at time when they were coping with a new diagnosis of colorectal cancer, despite a lack of prior awareness that the disease could run in families. The vast majority had decided to accept the offer of a genetic test and reported high levels osubjective understanding concerning genetic testing. The results suggest that although receiving information about HNPCC did not cause most participants undue worry, a minority of participants rated this worry at the top end of the scale. This was despite the fact that most had been informed that their relatives were at low risk of HNPCC. Likewise, these participants tended to have rated worry about their cancer and its treatment at the top end of the scale.”
C - 359

Outcomes Dropouts, 

explanations, 

other comments 

Study Conclusions 

had been very difficult. A total of 45% of participants (n=48) rated their current worry about their cancer and its treatment at or above the midpoint of 4 on a 1 (not at all) to 7 (all the time) scale.In total, 19% of participants (n=21) rated their current level of 

worry caused by the genetics information at or above the midpoint of 4 on a 1 (not at all) to 7 (all the time) scale. Of these 

21 participants, 80% (n=16) had rated their current worry about their cancer and its treatment at or above the midpoint, 57% (n=12) and 43% (n=9) had been informed that their relatives 

were at low or moderate risk of HNPCC, respectively.” 
C - 360

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 x 5 x 8c 

Family member/other high risk individual 

2,3 5 9 8a 8b,8c 

Public health or policy 

2,3 5 9 8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence 
C - 361

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 ND NA Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes No Were data collection tools shown or are they known to be reliable? Yes No Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 ND NA 

Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially No Is there a statistically significant difference between groups? Yes No ND Are the statistical methods appropriate? Yes No ND Indicate the unit of allocation Community Organization/group Provider Client Institution Indicate the unit of analysis Community Organization/group Provider Client Institution If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No NA 
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Domain/question Place an “X” in one Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No Can’t 

tell 
C - 363

Study: Raedle 2001 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

Single center, Germany 125 consecutive, unrelated patients with CRC 

Family history obtained from all, MSI performed on all MLH1 promoter methylation analysis performed on all MSI Genomic sequencing performed in all patients with MSI 

MLH1 and MSH2 Y N ? 

≥5 MSI markers used? 

X 

MSI-H defined by ≥ 2 markers? 

X 

Microdissection? X Gene screening? X Deletion analysis? 

 X 

Conversion analysis? 

 X 

Am 1 X 

Am R X Beth 1 X Beth R MSI-H MSI-L IHC Age <50 Suggestive family history 

X 

Specify Analyzed each component of Bethesda 1 Other Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 364

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion (What did 

the authors conclude about 

the testing strategy or other 

major findings).

Implicatio

to family 

/other findi

or commen

from autho

relevant t

any key 

question 

125 no dropouts 56 women 69 men Mean age 52 +/-14.4 (range 22-95) 

11 had pathogenic mutation in MLH1 (4) MSH2 (8) or both (1) NB. Doesn’t add up to 11, because they double count the common. 

58/125 fulfilled at least one of seven Bethesda criteria (31 met one, 12 met 2, 8 met 3 and 7 met 4). Criteria B4 mostly commonly satisfied (i.e. CRC or endometrial cancer diagnosed age <45) MSI 22/125 

ND “Bethesda guideline useful 

for selecting patients for MSI. MLH1 and MSH2 should be recommended in all patients with CRC and MSI who fulfill at least one Bethesda criteria. MLH1 promoter methylation may accompany rather than initiate carcinogenesis in patients with CRC who have mismatch repair gene defects.” Authors present diagram suggesting that patients with CRC who fulfill AM II should undergo genetic testing for MLH1 and MSH2. Those who do not should be asked Bethesda. If positive, MSI testing should be done and genetic testing done if MSI positive. 

ND 
C - 365

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 X 

Amsterdam R X Other (specify) 

ND [CANNOT ANSWER THIS QUEWE HAVE MMR AMONG MSI-H

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 X Amsterdam R X Other (specify) 

Based on the proportions and the Cpaper: Sens of Am1 is 6/22 Sens of Am-R is 10/22 Spec of Am1 is 97/103 Spec of Am-R is 93/103 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

ND 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

 X 

If yes, which clinical criteria (check all that apply)? Am 1 + Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) Other (specify) 
C - 366

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + X + (A) 6 (B) 0 

Am R + - (C) 5 (D) 11 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Nonselected patients  with CRC who were MSI-H *Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 8 (B) 2 

Am R + X - (C) 3 (D) 9 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Nonselected patients  with CRC who were MSI-H 
C - 367

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 11 (B) 6 

Am R + - (C) 0 (D) 5 Beth 1 + X Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Nonselected patients  with CRC who were MSI-H What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 9 (B) 6 

Am R + - (C) 2 (D) 5 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Nonselected patients  with CRC who were MSI-H 

BG, items 1-3 
C - 368

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 2 (B) 2 

Am R + - (C) 10 (D) 8 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** X 

Nonselected patients  with CRC who were MSI-H 

BG, item #2 

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 8 (B) 1 

Am R + - (C) 4 (D) 9 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** X 

Nonselected patients  with CRC who were MSI-H 

BG, Item #3: 
C - 369

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test 

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 8 (B) 3 

Am R + - (C) 4 (D) 7 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** X 

Nonselected patients  with CRC who were MSI-H 

Individuals with CRC or endometrial Cancer <45 

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number w

MMR+ 

Am 1 + + (A) 1 Am R + - (C) 11 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** X 

Nonselected patients  with CRC who were MSI-H 

Individuals with right-sided CRC with an undifferentiated pattern (solid, Cribriform, defined as poorly differentiated or undifferentiated Carcinoma composed of irregular, solid sheets of Large eosinophilic cells and containing small gland-like Spaces). 
C - 370

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check 

ONE 

Index test

Number with

MMR+ 

Nu

Am 1 + + (A) 3 (BAm R + - (C) 9 (DBeth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** X 

Nonselected patients  with CRC who were MSI-H 

Individuals with signet-ring cell type CRC diagnosed At age <45 

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with 

MMR+ 

Number with

MMRAm 1 + + (A) 0 (B) 0 

Am R + - (C) 12 (D) 10 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** X 

Nonselected patients  with CRC who were MSI-H 

Individuals with adenomas Dx <40 
C - 371

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 372

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 373

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Renkonen-Sinisalo, 2000, RefID 1701 Finland, Single 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

The patients came from 57 different families (49 families with a known mutation, 8 families that met the Amsterdam criteria). There were 137 CRC patients. They were divided into 2 groups, depending on how CRC had been diagnosed. 35 cases of CRC in 33 patients had been diagnosed via the surveillance program. Of these, 13 patients in whom CRC had been detected during very first surveillance examination (age range, 28-59 years). Among the others, 20 patients had been aware of their inherited susceptibility to cancer and had undergone thorough colonic examination at least once. 115 carcinomas had been found in 104 patients merely because of symptoms. These patients had undergone colonic examination and mostly not been aware of their susceptibility to cancer nor had they taken part in any surveillance program. 

Known mutation or Amsterdam criteria 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl x 

All new CRC cases occurring in known HNPCC families in Finland from 1983 to the end of 1997. 

Surveillance colonoscopy enema and si
C - 374

Outcomes Dropouts, 

explanations, other 

comments 

Study Conclusions 

No significant difference in age or gender distribution between the 2 groups of patients. The stage distribution of the 34 tumors in the surveillance group was significantly more favorable then that of the 114 tumors in the nonsurveillance group. In the nonsurveillance group, CRC was considered disseminated or nonoperable in 19 cases (17%). Synchronous CRC occurred in 2 patients in the surveillance group and in 10 in the nonsurveillance group. During this 15-year period, metachronous CRC was observed in 2 and 11 patients in the surveillance and nonsurveillance group, respectively. Five of 33 patients (15%) in the surveillance group had died, compare to 40 of 104 patents (38%) in the nonsurveillance group. CRC resulted in 2 deaths in the surveillance group and 33 deaths in the nonsurveillance group. The cumulative CRC-specific survival was 93% in the surveillance group, significantly higher than the 68% in the nonsurveillance group (p<0.02) 10 years after surgery. Similar results were shown for patients’ overall survival. 

Retrospective analysis; some data was missing 

“Colonoscopic surveillance is beneficial in at-risk members o

families. Even if CRC cannot be prevented by polypectomies, found during surveillance usually are detected at least so early expectation of survival is excellent and significantly better thahad been no surveillance.” 
C - 375

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 5 9 8a 8b,8c 

Public health or policy 

2,3 5 9 8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 376

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 ND NA Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes No Were data collection tools shown or are they known to be reliable? Yes No Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 ND NA 

Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially No Is there a statistically significant difference between groups? Yes No ND Are the statistical methods appropriate? Yes No ND Indicate the unit of allocation Community Organization/group Provider Client Institution Indicate the unit of analysis Community Organization/group Provider Client Institution If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No NA 
C - 377

Domain/question Place an “X” in one Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No Can’t 

tell 
C - 378

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Rijcken, 2003 The Netherlands Single center 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Females from HNPCC families identified by physicians and national registry. Annual screening performed and results treated accordingly. Women encouraged to report symptoms Women included in the program had a known MMR gene mutation and/or belonged to a family fulfilling the Amsterdam criteria II 41 enrolled and followed for 5 year (median, 5 months to 11 years) At enrollment:  85% premenopausal  Genetic status known for following:  8 MLH1  2 MSH2  1 MSH6 

MMR gene mutation or Amsterdam criteria II 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

MMR gene mutation or Amsterdam criteria II, 30-35 years 

Annual screegynecologicaultrasound (T(normal ≤35 as indicated 
C - 379

Outcomes Dropouts, explanations, other comments Study Conclusions 

179 TVU performed over 197 patient years, Endometrial sampling for 11 from 17 TVU results, 4 from clinical symptoms 14/17 endometrial samplings showed no severe pathology, 3 premalignant lesions detected resulting in 1 hysterectomy for premenopausal woman and 2 hysteroscopy and curettage procedures with normal histology for 1 pre- and 1 postmenopausal women. Another postmenopausal woman reporting symptoms was diagnosed with interval endometrial cancer outside regular screening program resulting in hysterectomy and bilateral salpingo-oophorectomy 115 serum CA125 levels ranged from 1–24 kU/L with median of 7 kU/L No ovarian cancers detected by screening or outside of annual screening 

None “No asymptomatic malignant lesions detected 

asymptomatic premalignant lesions detected aappropriately. Annual gynecologic screening was triage for endometrial sampling remains juswomen motivated for it but also that recognitireporting of clinical symptoms by the women importance.” 
C - 380

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 

x 

5 9 8a 8b,8c 

Public health or policy 

2,3 5 9 8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 ND NA Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stro
C - 381

Domain/question Place an “X” in one Prior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes No Were data collection tools shown or are they known to be reliable? Yes No Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 ND NA 

Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially No Is there a statistically significant difference between groups? Yes No ND Are the statistical methods appropriate? Yes No ND Indicate the unit of allocation Community Organization/group Provider Client Institution Indicate the unit of analysis Community Organization/group Provider Client Institution If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No Can’t 

tell 
C - 382

Study: Rossi, 2002Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? x Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? x Was a minimum of hMLH1 and hMSH2 sequencing performed? x 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

25 different Brazilian families with suspected hereditary CRC were consecutively enrolled from 1/1995 to 7/1999 Country: Brazil Center: Single 

• Am2 criteria, or • A relative (degree?) with CRC or HNPCC-related cancer, or • Proband aged <50y with or without affected relatives, or 

Multiple neoplasms in a proband 

All patients underwent gene sequencing. Note: MSI was not considered as a standard preliminary test in this study 

HMSH2 and hMLH1 Y N ? 

≥5 MSI markers used? MSI-H defined by ≥ 2 markers? Microdissection? Gene screening? x Deletion analysis? 

 x 

Conversion analysis? 

 x 

No MSI 

Am 1 x 

Am R x Beth 1 Beth R MSI-H MSI-L IHC Age <50 X Suggestive family history 

X 

Familial CRC without fulfilling Amsterdam criteria Other 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 383

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

N=25 Mean age=46.16 Dropout=0 Probands 60% males 

10 patients with MMR / 25 patients with CRC 8 MLH1 2 MSH2 

25 patients with CRC, 20% positive AM-I, 4% AM-II, 28% familial CRC w/o fulfilling AM, 44% probands <50 years 

ND “In addition to the 

Amsterdam criteria I and II, the physician in Brazil should also consider HNPCC when a patient is young and has CRC or when a patient has CRC and a family history of HNPCC, even if it is nontypical, with specific attention being given to gastric tumors.” 

ND 
C - 384

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 x 

Amsterdam R Other (specify) 

1/4 (4 have Am1) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 

Amsterdam R x Other (specify) 

2/5 (5 have Am2= 4with Am1 an

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 Amsterdam R Other (specify) 

ND 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

ND 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

x 

If yes, which clinical criteria (check all that apply)? Am 1 + x Am R + x Beth 1 + Beth R + Age <50 x Suggestive family history (Familial CRC without fulfilling Amsterdam criteria) 

x 

Other (specify) 
C - 385

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index 

test 

Am 1 + + Am R + X - Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Patients with CRC who had suspected hereditary CRC (fulfilled AM-1 or AM-II, Familial CRC without fulfilling Amsterdam criteria, probands presented at an early age (<50years) or a proband presented with multiple CRCs) *Note comparator to MSI-H or MSI-L is MSI-stable COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with 

MMR+ 

Number with

MMRAm 1 + X + (A) 1 (B) 3 

Am R + - (C) 1 (D) 0 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Of the 5 pts with Am2, 4 had Am1! So among those with AM2 NB very small table *Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 386

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

a suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

full description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 387

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 388

Study: Salovaara, 2000 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? X Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? X Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

535 consecutive patients with CRC from 9 hospitals in Finland mean age 67 

MSI wad done in all patients, founder mutations 1 & 2 in MLH1 were looked for in all patients; MLH1 and MLH2 sequencing was done in MSI positive and founder mutations negative patients 

Founder mutations 1 and 2 in MLH1 and other mutations in MLH1 and MLH1 

 Y N ? 

≥5 MSI markers used? 

 x 

MSI-H defined by ≥ 2 markers? 

 x 

Microdissection? x Gene screening? x Deletion analysis? 

 x 

Conversion analysis? 

 x 

Am 1 x 

Am R x Beth 1 Beth R MSI-H MSI-L IHC Age <50 Suggestive family history Specify Other x MSI 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 389

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

535 mean age 67 

18 MMR/535 patients with CRC 17 MLH1 and 1 MSH2 

3/535 (0.6%) Am I 2/535 (0.4%) Am R 

Large scale molecular screening for HNPCC can be done by a 2-stage procedure: screening for founders mutations, then MSI, then sequencing for those with + MSI. The authors also proposed that a strategy that screened for MSI based on age<50, familial disease or multiple tumor, would identify most tumors. 

 
C - 390

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 X 

Amsterdam R X Other (specify) 

Combined 5/535 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 X Amsterdam R X Other (specify) 

Combined 4/5 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? If yes, which clinical criteria (check all that apply)? 

X 

Am 1 + Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) Other (specify) 
C - 391

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + 4 1 

Am R + - 14 516 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Consecutive CRCs 

Combined Am1&R X 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + 18 48 

Am R + - 0 469 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Consecutive CRCs 

MSI X 
C - 392

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 393

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 394

Study: Samowitz, 2001 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? X Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? X Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or 

multicenter, 

Design and 

analytic strategy 

(brief description of 

strategy used for 

testing patients with 

CRC)

Which 

MMR 

mutations 

were 

sought? 

Characteristics of laboratory testing (see 

definitions below) 

Predictors 

analyzed (check al

that apply) 

Population based sample from the Keiser Permanente Medical care program of Northern California or a 8 county area in Utah. Eligible were incident CRC cases aged between 30 and 79 who were mentally competent 

1917 people were contacted, but complete questionnaire data and MSI screening in 1066; Then the 130/171 MSI-H who could be sequenced/had good quality DNA in paraffin blocks Æ MMR mutations 

MLH1 & MSH2 

 Y N ? 

≥5 MSI markers used? 

X 

MSI-H defined by ≥ 2 markers? 

 X** 

Microdissection? X Gene screening? X Deletion analysis? 

X only for Finnish mutation 

Conversion analysis? 

 X 

3 measures of instability 1: BAT 26; 2: TGFbetaRII 126-128; 3: 10 tetranucleotide repair markers used previously (ref); Measures 1 and 3 are reported to be are highly correlated with the 5 2nucleotide marker set. ** with >=one measure of instability. 

Am 1 

Am R Beth 1 Beth R MSI-H MSI-L IHC Age <50 Suggestive family history 

X 

CRC in Fam Other X Endom Ca in Fam Age <55 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 395

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

1917 people were contacted, but complete questionnaire data and MSI screening in 1066; Then the 130/171 MSI-H who could be sequenced/had good quality DNA in paraffin blocks Æ MMR mutations 

Among the 130 MSHI-H 7 MMR mutations (5 ML1; 3 MSH2 – one person had both mutations) 

Of 130 MSI-H: 18% (23/129)with CRC in family 5% (6/129) with endometrial ca in family 16% (21/130) aged<55y 

ND 

The clinical criteria notwithstanding, MSI testing is important on its own. Ought to be done in all CRC. Incorporation of MSI testing in clinical practice might lessen the need for clinical criteria. The estimated prevalence of HNPCC in the CRC population (0.86%) might be an underestimate. 

ND 
C - 396

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 

Amsterdam R Other (specify) 

ND 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 Amsterdam R Other (specify) 

ND 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

ND 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

X 

If yes, which clinical criteria (check all that apply)? Am 1 + Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) Other (specify) MSI-H X 
C - 397

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + 4 17 

Am R + - 3 107 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** X 

Among 130 CRC with MSI-H 

Age <55y 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + 4 19 

Am R + - 3 103 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** X 

Among 130 CRC with MSI-H 

Familial CRC 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 398

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 399

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 400

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Schmeler, K, 2006, US, Multicenter 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

315 women with HNPCC identified. Those who had undergone prophylactic TAH (14) compared with those who had undergone prophylactic TAH/BSO (47) compared with age-matched, mutation positive women who had not undergone either procedure (n=210 for endometrial cancer and n=223 for ovarian cancer) 

Documented MLH1, MSH2 or MSH6 germline mutation. Did all patients have a personal history of an HNPCC-related cancer Check one: 

Yes No Uncl 

 X 

Women with genetic variants of unknown functional significance excluded. 

TAH versus 

Outcomes Dropouts, explanations, other comments Study Conclusion

0/61 who had undergone a hysterectomy developed endometrial cancer compared with 69/210 controls (p<0.001) 0/47 women who had undergone oophorectomy developed ovarian cancer compared with 12/223 controls (p=0.09) Surgical complications in 1/61 women who underwent prophylactic surgery (ureteral injury, ureterovaginal and ureteroeneteric and then rectovaginal fistula)) 

Patients selected from a registry, and thus presumably all had a 

family history of HNPCC. ? selection bias for groups with 

increased penetrance? Did not describe how patients were assigned to surgery or no surgery ? selection bias. Did not describe what type of screening was performed on women who did not undergo the procedures. Other risk factors for gynecologic malignancy (eg, BMI) not assessed. Effects on survival or death not assessed. Quality of 

life not assessed 

Findings support consideration of TAH/BSO in women with Lynch the age of 35, or once childbearincompleted. (Median age of diagnendometrial cancer was 46, ovaria
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Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Scott, 2001 RefID 1607 Australia, single 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Index patients from 95 families with HNPCC. Each pedigree was classified as either Amsterdam-criteria positive or Bethesda-criteria positive. Genetic analysis was performed on a fresh blood specimen from the youngest living affected proband in each family. Disease verification in affected individuals was based on either examination of pathology reports or death certificates. 

Amsterdam-criteria positive or Bethesdacriteria positive 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl x 

ND ND 

Outcomes Dropouts, explanations, other 

comments 

Study Conclusions 

Of the 95 families, 12 harbored a genetic change in the hMSH2 gene, 22 harbored a change in the hMLH1gene, and 61 were found not to harbor any change in either gene. When mutation status based on the Amsterdam criteria was compared with that based on the Bethesda criteria, differences between the two groups were observed. As expected, 60% (20/33) of Amsterdam-criteria families were mutation positive, compared with 20% of Bethesda-criteria families (12/32). There appeared to be no relationship, between the sites of mutations, that could be used to establish a genotype-phenotype correlation, for either hMLH1 or hMSH2. Within each of the two groups, a comparison between the age at onset of CRC, the spectrum of extracolonic disease, and how this related to the mutation-negative group was made. Cancer Occurrence The overall percentage of tumors observed—in the total population, in the mutation-negative group, and in the hMSH2 mutation–positive and hMLH1 mutation– positive groups—is shown in table 3 of the paper. Briefly, the 9.86% of the total population had CRC and 1.37% had endometrial or ovarian carcinoma. Other types of malignancy were less prevalent. 

ND “In conclusion, use of the Bethesda 

the selection of families for gene anin a reduced probability of mutation Nevertheless, families or patients thidentified on the basis of these criteoffered genetic testing for genes assHNPCC, since this will result in an rate of identification of gene-mutatTo improve the probability of mutadetection if the Bethesda criteria arwe would suggest that mutation anaperformed in conjunction with DNA microsatellite testing and, posimmunohistochemical staining for Dmismatch-repair proteins. Subdivision of the mutation-positivmutation-negative groups makes it tease out subtle differences between populations, such as similar ages at disease in the mutation-negative anmutation–positive groups and disea
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Outcomes Dropouts, explanations, other 

comments 

Study Conclusions 

The relative standardized incidence rates (SIRs), including 95% confidence intervals (95% CIs), for all cancers identified in each group are shown in table 4 of the paper. Among all patients the SIR for CRC was 159.5 (139 – 182.9). This was of the same order among MLH1 and MSH2 carriers (196.8 [143-270.7] and 134.2 [99.1-181.8], respectively), as well as in the mutation negative part of the population (158.6 [132.8 – 189.4]). Data on other malignancies were more sparse, but the estimated SIRs were of similar order across all subgroups (see Table 4 of the paper). II. CRC The age distributions of the hMSH2 mutation–positive group and the hMLH1 mutation–positive group were essentially identical, but there was an about 5-year shift in the distribution seen in the mutation-negative group. The average age at diagnosis in hMSH2 mutation-positive families was 45.77 years, and that in hMLH1 mutation-positive families was 47.16 years. When the authors adjusted for familial clustering there was no statistical difference between the two groups. The average age at diagnosis in the mutation-negative group was 52.7 years. The average age at diagnosis was significantly different from that in the hMSH2 mutation-positive group but was not statistically different from that in the hMLH1 mutation-positive group. III. Extracolonic cancers: The frequencies of the following cancer types were found not to differ between the hMSH2 mutation–positive group, the hMLH1 mutation–positive group, and mutation-negative group: lymphoproliferative disease, renal/renal tract cancers, endometrial/ovarian cancers, and stomach cancer. The authors of the paper note that all these malignancies were overrepresented in all three groups, compared with the expected frequency in the general population. 

differences within each mutation-poBetter knowledge of the disease speassociated with mutation status will management of these families. Bettclassification of the mutationnegatiaid in identification of additional ge

associated with this disorder.” 
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Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 5 9 8a 8b,8c 

Public health or policy 

2,3 5 9 8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
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Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely Somewhat likely x Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 ND x NA Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes No Were data collection tools shown or are they known to be reliable? Yes No Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 ND x NA 

Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially No Is there a statistically significant difference between groups? Yes No ND Are the statistical methods appropriate? Yes No ND Indicate the unit of allocation Community Organization/group Provider Client Institution Indicate the unit of analysis Community Organization/group Provider Client Institution If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No NA 
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Domain/question Place an “X” in one Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No Can’t 

tell 
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Study: Shia, 2005 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? x Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? x Was a minimum of hMLH1 and hMSH2 sequencing performed? x 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which 

MMR 

mutations 

were 

sought? 

Characteristics of laboratory testing (see 

definitions below) 

Predictors analyzed 

(check all that apply) 

A group of 112 colorectal adenocarcinomas (n=83) or adenomas (n=29) obtained from 110 patients treated at the cancer center was assembled. These cases had a family history that fulfilled one of the following criteria: 1) Amsterdam criteria I or II, 2) a set of relaxed AC that we referred to as “HNPCC-like”, and 3) Bethesda criteria Country: US Center: single 

All patients started with MSI testing, followed by mutation analysis 

MLH1 and MSH2 

 Y N ? 

≥5 MSI markers used? 

x 

MSI-H defined by ≥ 2 markers? 

 x 

Microdissection? Gene screening? x Deletion analysis? Conversion analysis? 

*MSI markers were scored for the appearance of novel locus-specific bands in the tumor but not in the DNA from normal tissue. A tumor was scored “MSI present” when the proportion of successfully typed loci exhibiting instability was ≥0.3. 

Am 1 

Am R Beth 1 Beth R MSI-H MSI-L IHC Age <50 Suggestive family history Specify Other Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
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Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with a 

mismatch repair 

mutation (describe 

which MMR gene). 

(e.g. 23 patients with 

MMR/1000 patients 

with CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall 

population 

positive for 

each predictor 

(e.g. of 1000 

patients with 

CRC, 5% 

positive AM, 

10% positive 

Beth, 12% suggestive 

family history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

110 patients derived from 84 kindreds., 48/110 males, 50.5 (range 23-78) years old Total number of tumors assessed was 112, including 83 primary colorectal adenocarcinomas and 29 colorectal adenomas 

49 patients with MMR / 92 patients with CRC analyzed. 13 MLH1, 31 MSH2, 5 MSH6 36 patients (from 32 families) with disease-causing MMR / 92 patients with CRC analyzed. 9 MLH1, 23 MSH2, 4 MSH6 13 patients with variants of uncertain clinical significant MMR / 92 patients with CRC analyzed. 4 MLH1, 8 MSH2, 1 MSH6 

83 colorectal carcinomas, 51% AM I or II, 39% HNPCC-like patients, and 11% Beth 29 colorectal adenomas, 45% AM I or II, 55% HNPCC-like patients 

Abnormal IHC was detected in 31 of 41 tumors that exhibited MSI, whereas all 63 tumors that did not exhibit MSI showed presence of expression of all three proteins. 

Even though the authors found that IHC could not replace MSI testing, the authors recommended the use of IHC as a first line screening method to identify patients for mutation analysis because of the simplicity and ready availability. 

ND 
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 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 

Amsterdam R Other (specify) 

ND 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 x Amsterdam R x Other (specify) 

31/48 tumors MSI present 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 Amsterdam R Other (HNPCC-like) x 

4/43 tumors MSI present 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 x 

Amsterdam R x Other (specify) 

29/55 tumors 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 Amsterdam R Other (HNPCC-like) x 

4/44 tumors 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

x 

If yes, which clinical criteria (check all that apply)? Am 1 + x Am R + x Beth 1 + x Beth R + Age <50 Suggestive family history (“HNPCC-like”, defined as 3 or more colorectal cancers among the first- and second-degree relatives) 

x 

Other (specify) 
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COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests 

CheckONE 

Index 

test 

Am 1 + + Am R + - Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) 

x 

Colorectal adenomas (n=17) and carcinomas (n=64), fulfilled one of the inclusion criteria: 1) Amsterdam criteria I or II, 2) a set of relaxed AC that we referred to as “HNPCC-like”, and 3) Bethesda criteria

Other (specify)** 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index 

test 

Am 1 + + Am R + - Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (MSI present)** 

x 

Colorectal adenomas (n=12) and carcinomas (n=61), fulfilled one of the inclusion criteria: 1) Amsterdam criteria I or II, 2) a set of relaxed AC that we referred to as “HNPCC-like”, and 3) Bethesda criteria
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Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
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Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
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Study: (Southey, 2005, RefID 3071) Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? X Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? X Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief description of 

strategy used for testing 

patients with CRC)

Which 

MMR 

mutations 

were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

Men and women from Victorian Colorectal Cancer Family Study who were younger than age 45 years when diagnosed with a histologically confirmed, first primary adenocarcinoma of the colon or rectum from July 1, 1992 to September 30, 1996. A random selection of 222 patients were asked to participate. Country: Australia Single center 

Patients answered a risk factor questionnaire, received IHC and MSI screening. Germline MMR mutation testing was conducted for all patients with one or more of the following characteristics: 

a family history that fulfilled the Amsterdam Criteria for hereditary nonpolyposis colorectal cancer (HNPCC); having a tumor that was high MSI, low MSI, or that lacked expression of at least one MMR protein; and presence in a random sample of 23 patients selected from those who had tumors that were MS stable and did not lack expression of any MMR protein.

hMLH1, hMSH2, hMSH6, and hPMS2 

 Y N ? 

≥5 MSI markers used? 

x 

MSI-H defined by ≥ 2 markers? 

x 

Microdissection? x Gene screening? x Deletion analysis? 

X* 

Conversion analysis? 

 X 

* Only in 10 people with IHC abnormality and no MMR mutation detected 

Am 1 

Am R x Beth 1 Beth R MSI-H x MSI-L IHC x Age <50 Suggestive family history Specify Other Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 413

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion 

with a 

mismatch 

repair 

mutation (describe 

which MMR 

gene). (e.g. 

23 patients 

with 

MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportio

n of 

overall 

populatio

n positive 

for each 

predictor 

(e.g. of 

1000 

patients 

with CRC, 

5% 

positive 

AM, 10% 

positive 

Beth, 12% 

suggestive 

family 

history)

Correlati

on of 

predictor

s to one 

another 

(e.g. IHC 

versus 

MSI) 

Report 

what study 

found 

such as 

correlatio

n 

coefficient

s or other 

measures 

of 

correlatio

n

Study conclusion (What did the authors conclude about the testing 

other major findings).

N=131, ND, ND Attrition due to death (12.6%), refusal (doctor, 10.4%; patient, 12.6%), or because the patient moved and was not located (5.4%) resulted in 131 consenting to participate (59.0% of those eligible). The median time between diagnosis and interview was 9 months. Invasive tumor samples of primary colorectal adenocarcinoma were obtained from 

18 patients with deleterious MMR / 105 patients with CRC 9 hMLH1 4 hMSH2  4 hMSH6 1 hPMS2 

105 patients with CRC, 11% AM II, 17% MSI-H, 15%MSIL, 19% IHC abnormal 

ND “Because we did not conduct germline MMR testing on all patients w

tumors that were MS stable and IHC normal, we could not calculate and specificity directly. The formula used to estimate the sensitivity specificity were in Appendix.” See Table 2 in original paper for detailed results.  Sensitivity Specificity  (95%CI) (95%CI) Loss of MMR protein 1.00 0.91 Expression (0.82-1.00) (0.83-0.96) High or low MSI 0.94 0.80  (0.73 –1.00) (0.71-0.8Any family history 0.67 0.63  (0.41-0.87) (0.52-0.73
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N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion 

with a 

mismatch 

repair 

mutation (describe 

which MMR 

gene). (e.g. 

23 patients 

with 

MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportio

n of 

overall 

populatio

n positive 

for each 

predictor 

(e.g. of 

1000 

patients 

with CRC, 

5% 

positive 

AM, 10% 

positive 

Beth, 12% 

suggestive 

family 

history)

Correlati

on of 

predictor

s to one 

another 

(e.g. IHC 

versus 

MSI) 

Report 

what study 

found 

such as 

correlatio

n 

coefficient

s or other 

measures 

of 

correlatio

n

Study conclusion (What did the authors conclude about the testing 

other major findings).

hospitals and private pathology laboratories for 118 patients (90%). An assessment of MSI was not successful for 13 (12%) tumor samples because of technical reasons related to tumor DNA quality, which left 105 tumors tested.

Conclusion: “Tumor IHC analysis of four MMR proteins and MSI testing provide sensitive strategy for identifying MMR gene mutation–carrying, earl

colorectal cancer patients, half of whom would have been missed usiAmsterdam Criteria alone. Tumor-based approaches for triaging earl

colorectal cancer patients for MMR gene mutation testing, irrespectifamily history, appear to be an efficient screening strategy for HNPC
C - 415

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 

Amsterdam R x Other (specify) 

9/12 [NOT FOR THE PREVALETHESE ARE ALL AGED <45] 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 Amsterdam R x Other (specify) 

7/9 MSI-H; 2/9 MSI-L [ NOT FOR PREVALENCE QUESTION, THEAGED <45] 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R x Other (specify) 

9/9 [NOT FOR THE PREVALENTHESE ARE ALL AGED <45] 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

 x 

If yes, which clinical criteria (check all that apply)? Am 1 + Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) Other (specify) 
C - 416

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test 

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 9 (B) 3 

Am R + (<45y) x - (C) 9 (D) 84 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Patients with CRC (assuming all MSI-S and IHC(-) are MMR(-)) WILL NOT USE THIS IN THE SUMMARY TABLES (AM2 WHO ARE <45y) *Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 417

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 13 (B) 5 

Am R + - (C) 5 (D) 36 Beth 1 + Beth R + MSI-H* x MSI-L* Age <50 IHC (no staining) 

Patients with CRC who got tested for MMR (All Am2, signal in MSI or IHC and 23 randomly selected out of the sporadics) All <45y 

Other (specify)** 

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) 4 (B) 12 

Am R + - (C) 1 (D) 24 Beth 1 + Beth R + MSI-H* MSI-L* x Age <50 IHC (no staining) Other (specify)** 

Patients with CRC (All Am2, signal in MSI or IHC and 23 randomly selected out of the sporadics) all <45y *Note the 18 patients with MSI-H were not  included in this analysis 
C - 418

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test 

Number with

MMR+ 

Number w

MMRAm 1 + + (A) 18 (B) 8 

Am R + - (C) 0 (D) 33/79* Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) x Other (specify)** 

Patients with CRC *”of the 79 patients with tumors that expressed all proteins,  no mutations were detected in the 33 who were tested” 
C - 419

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 420

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 421

Study: Stormorken 2002 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? X Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

Index patients from the first 56 families registered at the Norwegian Radium hospital 

The patients with the mutations have been described in the Wijnen logistic regression model. 

Mlh1 (1) Msh2 (4) Msh6 (2) 

 Y N ? 

≥5 MSI markers used? 

 NA 

MSI-H defined by ≥ 2 markers? 

 NA 

Microdissection? NA Gene screening? X Deletion analysis? 

 X 

Conversion analysis? 

 X 

Am 1 X 

Am R X Beth 1 Beth R MSI-H MSI-L IHC Age <50 Suggestive family history Specify Other Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 422

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

56 index cases not only with CRC, but we focus on 20 AM2 and 12 AM1 who had CRC 

3/12 AM1 7/20 AM2 

12/56 AM1 20/56 AM2 NA 

Should use IHC as a screening 

 
C - 423

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 

Amsterdam R Other (specify) 

3/12 AM1 7/20 AM2 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 Amsterdam R Other (specify) 

3/12 AM1 4/20 AM2 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

[NOT APLPICABLE FOR THE REST OF THE EXTRACTION FORM BECAUSE WE ARE NOT SURPARTICIPANTS HAD CRC]  Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? If yes, which clinical criteria (check all that apply)? Am 1 + Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) Other (specify) 
C - 424

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + (A) (B) 

Am R + - (C) (D) Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) 

Other (specify)** 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 425

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 426

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 427

Study: Syngal, 2000 [see also Wahlberg 2002] Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? X Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? X Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

Families were identified by self or health care provider referral and were enrolled on the basis of multiple cases of CRC, early age of CRC diagnosis, or the familial association of CRC with other HNPCC associated tumors 

Each pedigree were classified into Amsterdam, Amsterdam Mod, Amsterdam II; each proband had genetic analysis, results entered into a 2X2 analysis with respect to clinical classification 

MSH2 or MLH1 Y N ? 

≥5 MSI markers used? MSI-H defined by ≥ 2 markers? Microdissection? Gene screening? X Deletion analysis? 

 X 

Conversion analysis? 

 X 

Am 1 x 

Am R x Beth 1 x Beth R MSI-H MSI-L IHC Age <50 Suggestive family history Specify Am Mod x Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 428

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

70 families were enrolled, representing 297 CRCs and 364 other cancer diagnoses 

18 MMR/70 families 

28/70 (40%) Am I 39/70 (56%) Am M 34/70 (49%) Am II 56/70 (80%) Beth 

The most sensitive clinical criteria for identifying pathogenic MSH2 and MLH1 were Bethesda Guidelines. 

 
C - 429

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 X 

Amsterdam R Other (specify) 

11/28 Am1 14/34 AmII 13/39 Modified Am (this is like Amso that 2 1st degree related CRC, 2 2 1st degree CRC and 3rd with early 

related) 17/56 Beth 14/56 Beth 1to3 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 Amsterdam II X Other (specify) 

See Wahlberg 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Am Mod X 

See Wahlberg 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

 X 

If yes, which clinical criteria (check all that apply)? Am 1 + Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) Other (specify) 
C - 430

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index 

test 

Am 1 + X + Am R + - Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Families were identified by self or health care provider referral and were enrolled on the basis of multiple cases of CRC, early age of CRC diagnosis, or the familial association of CRC with other HNPCC associated tumors 

MSI only 

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index 

test 

Am 1 + + Am II X - Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Families were identified by self or health care provider referral and were enrolled on the basis of multiple cases of CRC, early age of CRC diagnosis, or the familial association of CRC with other HNPCC associated tumors 

MSI only 
C - 431

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index 

test 

Am Mod X + Am R + - Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Families were identified by self or health care provider referral and were enrolled on the basis of multiple cases of CRC, early age of CRC diagnosis, or the familial association of CRC with other HNPCC associated tumors 

MSI only 

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index 

test 

Am 1 + + Am R + - Beth 1 + x Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Families were identified by self or health care provider referral and were enrolled on the basis of multiple cases of CRC, early age of CRC diagnosis, or the familial association of CRC with other HNPCC associated tumors 

MSI only 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 432

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 433

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 434

Study: Terdiman, 2001 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? X Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? X Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, single 

or multicenter, 

Design and analytic 

strategy (brief description 

of strategy used for testing 

patients with CRC)

Which 

MMR 

mutations 

were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

Eligible families had to have 2 or more first-degree relatives with CRC at any age, an individual with CRC diagnosed before 50 years of age, or a single individual with synchronous or metachronous CRCs. Probands were selected based on convenience and age at cancer diagnosis. When multiple family members were available for molecular testing, the individual with cancer diagnosed at the youngest age was selected as proband. Country: US Center: Single 

Paraffin-embedded tumor samples were obtained from all probands for MSI analysis and MSH2/MLH1 immunostaining Æ Subjects found to have tumors demonstrating MSI-H (n=47) were invited for germline genetic testing of MSH2 and MLH1 Æ Gene testing was carried out in 32 of the 47 eligible families. Eight probands refused testing for fear of insurance discrimination. In 7 instances, the proband was deceased (n=4) or could not be recontacted (n=3) 

MSH2 and MLH1 

 Y N ? 

≥5 MSI markers used? 

X 

MSI-H defined by ≥ 2 markers? 

X 

Microdissection? X Gene screening? X Deletion analysis? 

 X 

Conversion analysis? 

 X 

MSI-H was if 3 or more of the 7 marker loci exhibited band size shifts or if 2 or more loci exhibited hand size shifts when 1 of the involved loci was either BAT26or BAT40 

Am 1 X 

Am R Beth 1 X Beth R MSI-H X MSI-L X IHC X Age <50 Suggestive family history Specify Other Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 435

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g., 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

N=114 Mean age=ND %Male=ND Dropouts=5 (4%), no tumor tissue or medical records 

16 patients with deleterious MMR / 32 patients with CRC 7 MLH1 9 MSH2 5 patients with MMR of variants of uncertain significance / 32 patients with CRC 4 MLH1 1 MSH2 

109 patients with CRC, 43% MSI-H, 3% MSI-L, 23% positive AM, 70% positive Beth 

0/40 MSI-L or MSS tumors had positive IHC 45/56 MSI-H tumors had positive IHC; 6/38 MSI-H tumor had ambiguous IHC 21/44 (48%) MSI-H patients met Amsterdam criteria 42/44 (95%) MSI-H patients met Bethesda criteria 3/61 (48%) MSI-L or MSS patients met Amsterdam criteria 30/61 (49%) MSI-L or MSS patients met Bethesda criteria 

“In clinical practice today, detection of germline MSH2 or MLH1 mutations is best approached by a 2-step method in which the tumor of high-risk patients are tested either for MSIH or for the absence of MSH2 or MLH1 protein staining.” 

ND 
C - 436

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 

Amsterdam R Other (specify) 

ND 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 Amsterdam R X Other (specify) 

21/24 (AM2) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

ND 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

X 

If yes, which clinical criteria (check all that apply)? Am 1 + Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) Other (CRC and MSI-H) X 
C - 437

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index 

test 

Number with 

MMR+ 

(deleterious and variants of unce

significance) 

Am 1 + + (A) 16 Am R + - (C) 1 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) 

X 

Other (specify)** 

Patients with CRC who had MSI-H *6 ambiguous IHC were excluded from this analysis What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with 

MMR+ 

(deleterious and variants of uncertain significanc

Am 1 + + (A) 15 Am R + X - (C) 6 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Patients with CRC who had MSI-H *Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 438

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

upon a suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

gene sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

full description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who underwent sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

those who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 439

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 440

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Van Dalen 2003, RefID 851 Country: US Center: Multicenter 

Study description (N enrolled) How was HNPCC 

defined? 

Inclusion/exclusion criteria 

To document the patterns of colorectal surgery performed for cancer-bearing patients who are part of an Amsterdam criteriapositive family, and to compare rates of metachronous cancers that followed each type of index operation. 39 HNPCC families with 93 affected patients. The number of patients per family ranged from 1 to 13. There were 127 CRC. Median age at diagnosis of index cancer was 47 (range, 25-81) years. A total of 16 patients (17%) had metachronous cancers, whereas it was 14 (4%) had synchronous cancer. 

Amsterdam criteria defined by Vasen et al in 1991 

Did all patients 

have a personal 

history of an 

HNPCC-related 

cancer? 

Check one 

Yes No Uncl x 

Family kindreds fulfilling the Amsterdam criteria for HNPCC were identified from the David G. Jagelman 

Inherited Colorectal Cancer registries. All patient for whom surgical and pathology records were available were eligible to be included in the study. The type of surgery and the outcome of subsequent follow-up were abstracted. 

Patients were treated at the (CCF) and thpatients had and 60 had thPrimary surgcolectomy an16 (48%) of surgery at CC7 (12%) of thsurgery elsewThe median underwent su4-49) years, 42) years for elsewhere. 

Outcomes Dropouts, 

explanations, 

other comments 

Study Conclusions 

Incidence of 2nd surgeries for metachronous cancer: only 1 

patient who had surgery at CCF had a second operation, this after an initial right colectomy. Thus, 1 (6%) of 17 patients had a segmental resection. 15 (28%) of 53 patients who had surgery elsewhere needed a second resection (p=0.094). 

 “There is potentially a high risk of metachronous colorectal 

an index cancer in an HNPCC patient defined according to Acriteria is treated by partial colectomy. However, this risk calowered, either by performing a total colectomy at the time osurgery or possibly by effective postoperative surveillance.” 
C - 441

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 x 5 8c 

Family member/other high risk individual 

2,3 5 9 8a 8b,8c 

Public health or policy 

2,3 5 9 8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 442

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 ND NA Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes No Were data collection tools shown or are they known to be reliable? Yes No Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 ND NA 

Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially No Is there a statistically significant difference between groups? Yes No ND Are the statistical methods appropriate? Yes No ND Indicate the unit of allocation Community Organization/group Provider Client Institution 
C - 443

Domain/question Place an “X” in one Indicate the unit of analysis Community Organization/group Provider Client Institution If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No Can’t 

tell 
C - 444

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Vasen, 1995, UI7577010 The Netherlands National registry 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

5 year followup (1-20 yr) of surveillance program from registry of families with CRC including colonoscopy or sigmoidoscopy with barium enema every 2-3 years. 50 families with 238 CRC (control pts), 388 (79%) first-degree relative screened of 493 could be traced from 597 high-risk relatives 

Amsterdam criteria 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl X 

See study description Surveillance/
C - 445

Outcomes Dropouts, explanations, other comments Study Conclusions 

CRCs in index group were at an advanced histopathological stage compared to screened group; 33 (8.5%) subjects with ≥1 adenomas and 11 (2.8%) CRCs; 5 of 11 CRC patients had negative screening exam 11mo – 3.6 yr previously; in addition to 33 patients with adenoma there were adenomas in 26 patients with CRC. Mortality lower in screened versus control group, 9% vs 45%; 5 year survival higher in screened versus control group, 87% vs 63% 1 patient with severe complication due to screening exam (cardiac arrest with good response to resuscitation); 1 patient had inappropriate exam (sigmoidoscopy rather than colonoscopy) 

80% compliance The screened group had high detec

CRC, better 5 year survival rate, eahistopathological stage than the coPeriodic examination of HNPCC fcancer detection at earlier stage thaunder surveillance. Recommend screening interval of because of aggressive nature of poassociated with HNPCC 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely 

X 

Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 ND NA 
C - 446

Domain/question Place an “X” in one 

X 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

NA A 

(stro

Is the method of random allocation stated Yes No If the method of random allocation is stated, is it appropriate Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No Can’t 

tell 

X 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes No 

Blinding NA A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND NA 

Data Collection methods A 

(stroX 

Were data collection tools shown or are they known to be valid? Yes 

X 

No 

Were data collection tools shown or are they known to be reliable? Yes 

X 

No 

Withdrawals and Dropouts A (stro

X

Indicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 ND NA 
C - 447

Domain/question Place an “X” in one 

X 

Analysis A (stroIs there a sample size calculation or power calculation Yes Partially No 

X 

Is there a statistically significant difference between groups? Yes No ND 

X

Are the statistical methods appropriate? Yes No ND 

X

Indicate the unit of allocation Community Organization/group Provider Client 

X

Institution 

Indicate the unit of analysis Community Organization/group Provider Client 

X

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No NA 

X

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No Can’t 

tell 

Intervention Integrity A (stro

X

What % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND NA 

X

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes 

X

No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No Can’t 

tell 

X
C - 448

Data Extraction Form For Benefits/Harms

Au, Year, UI, Country Single or Multicenter? 

Vasen, HF 2001, Ref ID 1364 Multicenter, Netherlands/Norway 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Survival analysis performed in 138 families with HNPCC. Amsterdam I or II or high suspicion +/- 

mutation testing. 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

Family with HNPCC included in Netherlands HNPCC registry or mutation carriers from a Norwegian registry. 

Cumulative cgroups: 

1) “Pu

relaendhad 

2) Ob

therelamu

3) Th

Outcomes Dropouts, 

explanations, 

other 

comments 

Study Conclusions Qua

Pathogenic mutations in 71 Dutch and 8 Norwegian families (34 MLH1 and 40 MSH2 and 5 MSH6). Mean age at diagnosis of CRC higher in carriers of MSH6 compared with MLH1 or MSH2 (50 versus 43 and 44, respectively), statistical significance not reported. Lifetime risk of developing cancer at any age was significantly higher in MSH2 than MLH1 (p<0.01). Risk of CRC higher in MSH2 than MLH1 (p=0.13). Overall, risk of CRC in male mutation carriers was higher than risk of CRC in female mutation carriers but difference was statistically significant only for MSH2 mutation carriers (p<0.01). The risk of developing endometrial cancer was higher in MSH2 mutation carriers than MLH1 (p=.057) MSH2 carriers had significantly higher risk of developing cancer of the urinary tract by age 70 (12%, p<0.05). 

ND Pending large prospective studies, 

the extension of the current surveillance program in MSH2 mutation carriers with the inclusion of the urinary tract should be considered. 

No de

differen

by cluste

on

No descrreceived surveillahave beepathologgrowth e
C - 449

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC-related 

cancers/factors such as race, 

age sex, comorbidities that 

may be related to 

accuracy/penetrance 

Be

o

mu

dete

de

fam

Proband with CRC 

2,3 5 X 

8c 

Family member/other high risk individual 

2,3 5 9 8a X 

8b,8c 

Public health or policy 

2,3 5 9 8a X 

8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 450

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely X 

Somewhat likely 

Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 X 

ND 

NA 

Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No X 

Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No X 

NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

X 

Yes 

No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No X 

ND 

NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes X 

No 

Were data collection tools shown or are they known to be reliable? Yes X 

No 

Withdrawals and Dropouts A (stro
C - 451

Domain/question Place an “X” in one Indicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 X 

ND 

NA 

Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially X 

No 

Is there a statistically significant difference between groups? X 

Yes 

No ND 

Are the statistical methods appropriate? X 

Yes 

No ND 

Indicate the unit of allocation Community Organization/group Provider X 

Client 

Institution 

Indicate the unit of analysis Community Organization/group Provider X 

Client 

Institution 

If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No X 

NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

X 

Yes 

No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 X 

ND 

NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

X 

Yes 

No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No X 

Can’t 

tell 
C - 452

Data Extraction Form For Benefits/Harms 

Au, Year, UI, Country Single or Multicenter? 

Wagner, 2005, #16341806 The Netherlands Single center 

Study description (N enrolled) How was HNPCC defined? Inclusion/exclusion criteria 

Long-term surveillance & counseling to relatives of HNPCC cases. Mutation carriers referred to local specialist for follow-up and surveillance consisting of colonoscopy every 1-2 years from age 20-25 years and on. Female carriers offered gynecological screening by US and CA125-measurements of blood from age 30-35 years and on. Additional screening for stomach, duodenum or urinary tract as advised. 

MMR genetic testing 

Did all patients have a 

personal history of an 

HNPCC-related cancer? 

Check one 

Yes No Uncl  X 

First- and second-degree relatives of patients with HNPCC-related tumor 

Surveillance gynecologciastomach, duopsychologicaprogram 

Outcomes Dropouts, explanations, other comments Study Conclusions 

Long-term satisfaction with screening, counseling procedure Compliance Worry and consequences on daily activities, work, insurance 

21/115 (18%) mutation carriers died or moved away 70 (74%) responded to questionnaires 28 (40%) were already diagnosed with HNPCCrelated tumor at time of testing 5 has had no screening –burdensome of procedure (1), lack of time (1), plan to go (3) 

Colonoscopy satisfaction of health gene carriers: 88% (37) healthy mutation carrier had colonoscopy screyears  colonoscopy unpleasant 57%  fearful 32%  painful 51%  shameful 16% Faith of colonoscopy effectiveness 90% Worry about complications of colonoscopy 14% Preferred another screening method 71% Long-term satisfaction with counseling: 84% approved of how they were informed of HNPCC g97% approved information given during counseling 4/10 had life, disability or mortgage insurance problemshealth insurance or getting job 
C - 453

Place an X in boxes that the study is relevant 

Perspective Benefits/Harms 

screening (e.g. 

family history, 

Amsterdam, 

Bethesda other) 

Benefits/Harms 

genetic testing 

Benefits/Harms 

counseling/informing 

Efficacy of 

counseling (e.g., 

ability to 

accurately 

convey risks and 

options, 

minimize 

anxiety, inform 

decisions to take 

tests) 

Accuracy/penetrance testing 

for HNPCC for HNPCCrelated cancers/factors such 

as race, age sex, 

comorbidities that may be 

related to 

accuracy/penetrance 

Be

o

mu

det

de

fam

Proband with CRC 

2,3 5 8c 

Family member/other high risk individual 

2,3 

x 

5 x 

9 x 

8a 

x 

8b,8c 

Public health or policy 

2,3 5 9 8a 8b,8c 

*Score Overall Quality of Study as Follows 

(A) Most overall quality rating scores are an “A” and the results of the study are considered to provide strong evidence 

(B) Most overall quality rating scores are a “B” and the results of the study are considered to provide moderate evidence 

(C) Most overall quality rating scores are a “C” and the results of the study are considered to provide weak evidence
C - 454

Domain/question Place an “X” in one Selection Bias A 

(stro

Are individuals selected to participate likely to be representative of target population? 

Very likely Somewhat likely Not 

likely 

What % of selected individuals agreed to participate? 80-100 60-79 <60 ND NA Allocation Bias (RCTs only, for quasi-experimental, case-control/before/after, no control group or other skip to “Confounders”)

A 

(stro

Is the method of random allocation stated? Yes No If the method of random allocation is stated, is it appropriate? Yes No Was the method of random allocation reported as concealed? Yes No Confounders A (stroPrior to the intervention, were there between group differences for important confounders reported in the paper? 

Yes No Can’t 

tell 

If there were differences between groups for important confounders, were they adequately managed in the analysis? 

Yes No NA 

Were there important confounders NOT reported in the paper (describe above under quality score)? 

Yes No 

Blinding A (stroWas (were) the outcome assessor(s) blinded to the intervention or exposure status of the participants? 

Yes No ND NA 

Data Collection methods A 

(stro

Were data collection tools shown or are they known to be valid? Yes No Were data collection tools shown or are they known to be reliable? Yes No Withdrawals and Dropouts A (stroIndicate the % of participants completing the study. (If the % differs by groups, record the lowest). 

80-100 60-79 <60 ND NA 

Analysis A (stroIs there a sample size calculation or power calculation? Yes Partially No Is there a statistically significant difference between groups? Yes No ND Are the statistical methods appropriate? Yes No ND 
C - 455

Domain/question Place an “X” in one Indicate the unit of allocation Community Organization/group Provider Client Institution Indicate the unit of analysis Community Organization/group Provider Client Institution If the unit of allocation and analysis differed, was the cluster analysis done? 

Yes No NA 

Is the analysis performed by intervention allocation status (i.e. intention to treat) rather than the actual intervention received? 

Yes No Can’t 

tell 

Intervention Integrity A (stroWhat % of participants received the allocated intervention or exposure of interest? 

80-100 60-79 <60 ND NA 

Was the consistency of the intervention measured (i.e. intervention was provided to all participants in the same way)? 

Yes No ND NA 

Is it likely that subjects received an unintended intervention (contamination or cointervention) that may influence the results? 

Yes No Can’t 

tell 
C - 456

Study: Wahlberg 2002 also see Syngal 2000 for additional details Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? X Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? X Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply

Families were identified by self or health care provider referral and were enrolled on the basis of multiple cases of CRC, early age of CRC diagnosis, or the familial association of CRC with other HNPCC associated tumors 

48 families with tumors available for MSI analysis, 24 of them also had tumor material of sufficient quality for IHC analysis, gene sequencing done previously in Syngal 2000; they were also resequenced using new sequencers 

MSH2 or MLH1 Y N ? 

≥5 MSI markers used? 

X (NCI) 

MSI-H defined by ≥ 2 markers? 

X 

Microdissection? X Gene screening? X Deletion analysis? 

 X 

Conversion analysis? 

 X 

Am 1 X 

Am Mod X Beth 1 X Beth R MSI-H MSI-L IHC Age <50 Suggestive family history Specify Other Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 457

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

48 families with available tumor tissue for MSI analysis were included (original study by Syngal 2000 enrolled 70 families) 

See Syngal 2000 A combination of the 

Bethesda criteria for HNPCC and an MSI-H phenotype defined the smallest number of cases having all of the germ-line MSH2 and MLH1 mutations that could be detected by DNA sequencing. 

 
C - 458

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 

Amsterdam R Other (specify) 

See Syngal 2000 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 Amsterdam R Other (specify) 

 MSI H and 15/19 Am1 and 3/19 18/28 Modif Amsterdam like Syng24/36 Beth1 and 4/36 20/28 Beth 1 to 3 and 4/28 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

5/19 Am1 7/28 Md Am1 like Syngal 8/28 Beth 1 to 3 9/36 Beth 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

 X 

If yes, which clinical criteria (check all that apply)? Am 1 + Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) Other (specify) 
C - 459

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check 

ONE 

Index test

Number with

MSI-H 

 (NCI) 

Nu

M

Am 1 + X + 15 4 Am R + - 13 16 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

48 families with CRCs with available tumor tissue for MSI analysis 

MSI only 

What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check 

ONE 

Index test

Number with

MSI-H 

 (NCI) 

Nu

M

Am 1 + + 24 12 Am R + - 4 8 Beth 1 + X Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

48 families with CRCs with available tumor tissue for MSI analysis 

MSI only 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 460

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 461

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 462

Study: Wang 1999 & 1997 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? x Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? x Was a minimum of hMLH1 and hMSH2 sequencing performed? x 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

75 unrelated families from France, selected from genetic consultations, multicenter 22 Amsterdam 33 not Amsterdam 12 sporadic CRC 8 CRC+extra-colonic tumor 

Combined DNA and RNA-based screening for hMLH1, hMSH2, hPMS1, hPMS2, and hMSH6 

hMLH1, hMSH2 Y N ? 

≥5 MSI markers used? 

 x 

MSI-H defined by ≥ 2 markers? 

 x 

Microdissection? Gene screening? x Deletion analysis? 

x 

Conversion analysis? 

Am 1 x 

Am R Beth 1 Beth R MSI-H MSI-L IHC Age <50 x Suggestive family history Specify Other Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsattelite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 463

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

See above 26 deleterious 

mutations were found; 19 MLH1 and 7 MSH2 in 55 unrelated families 

Data confirmed that hPMS1, hPMS2, and HMSH6 germline mutations are rare in familial aggregation of CRCs. 

 
C - 464

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 x 

Amsterdam R Other (specify) 

14/22 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 Amsterdam R Other (specify) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

x 

If yes, which clinical criteria (check all that apply)? Am 1 + x Am R + Beth 1 + Beth R + Age <50 x Suggestive family history (specify) x Other (specify) 
C - 465

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check 

ONE 

Index test

Number wit

MMR+ 

Am 1 + x + 14 Am R + - 8 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Unrelated kindreds with HNPCC, some fulfilled Amsterdam, some did not 

MSI only 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 466

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 467

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 468

Study: Wolf, 2005 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? X Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? X Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

81 unrelated patients from Austria who met at least the Beth R guidelines Unclear if all are CRCs, 

Pts who met Beth R, Beth 1, Am Mod, or Am II were preselected; DNA sequencing was done in all, and MSI was done in available tumor samples 

MLH1 and MSH2 Y N ? 

≥5 MSI markers used? 

X 

MSI-H defined by ≥ 2 markers? 

 x 

Microdissection? X Gene screening? x Deletion analysis? 

 x 

Conversion analysis? 

 x 

Am 1 

Am R X Beth 1 X Beth R X MSI-H MSI-L IHC Age <50 Suggestive family history Specify Other X Am Mod (2004) 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsatellite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 469

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

81 pts; 48% M 

19 pts with MMR/81 pts with CRC 12 MLH1 7 MSH2 

Not applicable Detection of 

mutation was 

correlated with Am 

R and Am Mod 

(2004) 

 (P=0.011 and 

P=0.038) 

MSI enhanced specificity in screening; recommend the use of Am Mod (2004) to select for patients for primary sequence analysis if MSI is not possible. 

 

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 

Amsterdam R X Other (specify) 

13/35 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 Amsterdam R X Other (specify) 

16/24 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 
C - 470

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

X 

If yes, which clinical criteria (check all that apply)? Am 1 + Am R + X Beth 1 + X Beth R + X Age <50 Suggestive family history (specify) Other (specify) Am Mod (2004) X COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + 13 22 

Am R + X - 6 40 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Patients who fulfilled Beth R 

MSI only 
C - 471

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + 19 53 

Am R + - 0 9 Beth 1 + X Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Patients who fulfilled Beth R 

MSI only 

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + 16 36 

Am R + - 3 26 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) ** Am Mod (2004) X 

Patients who fulfilled Beth R 

MSI only 
C - 472

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Number with

MMRAm 1 + + 13 9 

Am R + - 0 33 Beth 1 + Beth R + MSI-H* X MSI-L* Age <50 IHC (no staining) Other (specify)** 

Patient who fulfilled Beth R 

MSI only 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 473

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 474

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 475

Study: Yuan 1998 [included data from Han 1996] Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? X Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? x Was a minimum of hMLH1 and hMSH2 sequencing performed? x 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of strategy 

used for testing 

patients with CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

31 families (from Korean Hereditary CRC registry) with suspected HNPCC and 45 patients with sporadic early-onset CRC 

One CRC patient from each family had DNA testing except for one family (hyperplastic polyp); sequencing done in patients with altered mobility on SSCP bands only 

hMLH1 and hMSH2 Y N ? 

≥5 MSI markers used? 

 x 

MSI-H defined by ≥ 2 markers? 

 x 

Microdissection? Gene screening? x Deletion analysis? Conversion analysis? 

Am 1 

Am R Beth 1 Beth R MSI-H MSI-L IHC Age <50 Suggestive family history 

X 

Suspect HNPCC Other Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsattelite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 476

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

31 suspected HNPCC; 45 sporadic CRC Dx <40y (table1 range) suspected HNPCC 50y 65%male Sporadic CRC diagnosed <40y 39y 49% male 

7/31 in suspected HNPCC (5 hMLH1, 2 hMSH2); 1/45 in sporadic CRC (hMSH2) 

The Korean definition of suspected HNPCC is useful in the diagnosis of HNPCC and for identifying those families suspected of having HNPCC. 

 
C - 477

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 

Amsterdam R Other (specify) 

ND 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 Amsterdam R Other (specify) 

ND 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

ND 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

X 

If yes, which clinical criteria (check all that apply)? Am 1 + Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) Korean criteria HNPCC Other (specify) 
C - 478

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index 

test 

Number with 

Abnormal PCR-SSCP who had hMLH1 or hMSH2 

pathogenic mutations 

Am 1 + + 7 Am R + - 1 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Korean criteria X 

MSI only 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 479

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 480

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
C - 481

Study: Yuan 2004 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? x Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? x Was a minimum of hMLH1 and hMSH2 sequencing performed? x 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

14 families that fulfilled the Chinese HNPCC criteria; 3 of them fulfilled Amsterdam 

DHPLC was performed, DNA sequencing was performed if DHPLC were abnormal 

HMLH1 and hMSH2 Y N ? 

≥5 MSI markers used? 

 x 

MSI-H defined by ≥ 2 markers? 

 x 

Microdissection? x Gene screening? x Deletion analysis? 

x 

Conversion analysis? 

 x 

Am 1 

Am R Beth 1 Beth R MSI-H MSI-L IHC Age <50 Suggestive family history Chinese criteria Other x Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsattelite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re
C - 482

Results 

N enrolled, Mean 

age, %male 

dropouts, reasons 

for dropouts 

Proportion with 

a mismatch 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

Proportion of 

overall population 

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

Correlation of 

predictors to one 

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

Study conclusion 

(What did the authors 

conclude about the 

testing strategy or 

other major findings).

Implications to 

family /other findi

or comments fro

authors relevant 

any key question 

14 probands 12 abnormal 

DHPLC; all 12 had point mutations in either hMLH1 or hMSH2 3 pathogenic mutations identified (2 mhl1 and 1 msh2) 

3 were Am1 Detection of large 

genomic deletions should be involved in the routine screening of HNPCC families. 

 
C - 483

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 x 

Amsterdam R Other (specify) 

1/3 were Am1 all other were Chine

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 Amsterdam R Other (specify) 

NA 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R Other (specify) 

NA 

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

x 

If yes, which clinical criteria (check all that apply)? Am 1 + Am R + Beth 1 + Beth R + Age <50 Suggestive family history (specify) Chinese HNPCC Other (specify) Chinese HNPCC criteria 
C - 484

COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index 

test 

Number with 

Abnormal DHPLC that had pathogenic 

mutation by sequence analysis 

Nu

Abnormal D

pathogenic m

Am 1 + X 2 1 Am R + 2 9 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

Patients who fulfilled Chinese HNPCC criteria 

MSI only 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis).
C - 485

Study: Zhu, 2005 Inclusion/exclusion criteria for clinical validity (all must be checked “yes” for study to be included) 

Y

Did study enroll patients with CRC? X Was genetic testing compared with an index test (must have at least one of the following: suggestive family history, MSI, or IHC)? X Was a minimum of hMLH1 and hMSH2 sequencing performed? X 

Characteristics of Design 

Population 

inclusion/exclusion 

criteria; country, 

single or multicenter, 

Design and analytic 

strategy (brief 

description of 

strategy used for 

testing patients with 

CRC)

Which MMR 

mutations were 

sought? 

Characteristics of laboratory 

testing (see definitions below) 

Predictors analyzed 

(check all that apply) 

1. 45 unrelated 

patients with familial CRC, 21 fulfilled Amsterdam 

2. 20 patients 

with CRC <50 yr 

3. 13 patients 

with CRC > 50 yr 

China 

Screen for large deletions and duplications in MSH2 and MLH1 by multiplex ligationdependent probe amplification (MLPA_ 

MSH2 and MLH1 Y N ? 

≥5 MSI markers used? 

NA 

MSI-H defined by ≥ 2 markers? 

NA 

Microdissection? NA Gene screening? X Deletion analysis? 

X 

Conversion analysis? 

 X 

Am 1 X 

Am R Beth 1 Beth R MSI-H MSI-L IHC Age <50 X Suggestive family history Specify Other Specify 

Am 1= original Amsterdam criteria; Am 2 = revised Amsterdam criteria; Beth 1= original Bethesda Guidelines; Beth 2 = Revised Beth

MSI-H = Microsattelite instability high; MSI-L = Microsatellite instability low; IHC= immunohistochemistry (staining for mismatch re

Results 

N enrolled, Mean 

age, %male 

Proportion with 

a mismatch 

Proportion of 

overall population 

Correlation of 

predictors to one 

Study conclusion 

(What did the authors 

Implications to 

family /other findi
C - 486

dropouts, reasons 

for dropouts 

repair mutation 

(describe which 

MMR gene). 

(e.g. 23 patients 

with MMR/1000 

patients with 

CRC 

18 MLH1 

5 MSH2)

positive for each 

predictor (e.g. of 

1000 patients with 

CRC, 5% positive AM, 10% positive 

Beth, 12% 

suggestive family 

history)

another (e.g. IHC 

versus MSI) Report 

what study found 

such as correlation 

coefficients or other 

measures of 

correlation

conclude about the 

testing strategy or 

other major findings).

or comments fro

authors relevant 

any key question 

See above Genomic 

aberration was detected in 4/21 patients with Amsterdam 

Genomic aberration was detected in 4/21 patients with Amsterdam; 3/24 patients without Amsterdam; 2/20 patients with CRC < 50 yr 

Genomic aberrations, large-fragment deletions and duplications, in MSH2 and MLH1 play a role in the pathogenesis of Chinese CRC patients with a family history. 

 

 How was Lynch Syndrome 

defined (check all that apply)? 

Specify numerator and denominator a

not described) 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had an MMR gene mutation? 

Amsterdam 1 X 

Amsterdam R Other (specify) 

4/21 has genomic aberration 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had MSI (high or low, please specify)

Amsterdam 1 Amsterdam R Family history 

Among patients with the Lynch syndrome defined clinically (i.e. fulfillment of the Amsterdam criteria) what proportion had abnormal IHC 

Amsterdam 1 

Amsterdam R 
C - 487

 Yes No Did the study perform testing only on patients who fulfilled clinical criteria (i.e. when clinical suspicion for HNPCC was increased? 

X 

If yes, which clinical criteria (check all that apply)? Am 1 + X Am R + Beth 1 + Beth R + Age <50 X Suggestive family history (specify) X Other (specify) COPY A NEW 2 X 2 TABLE FOR EACH INDEX TEST What was the population (i.e. from table above)? “ND” if not a restricted population (e.g. Patients with CRC who had a suggestive family history)

Index tests Check

ONE 

Index test

Number with

MMR+ 

Numb

M

Am 1 + X + 4 17 Am R + - 3 21 Beth 1 + Beth R + MSI-H* MSI-L* Age <50 IHC (no staining) Other (specify)** 

45 unrelated patients with familial CRC, 21 fulfilled Amsterdam 

MSI only 

*Note comparator to MSI-H or MSI-L is MSI-stable **Add combinations of tests under “other” category (eg. MSI-H plus positive IHC for MLH1; MSI-H plus negative results on methylation analysis). 
C - 488

Grade Explanation for Quality Scoring 

A Most or all of the criteria are fulfilled and the conclusions of the study would be very unlikely to be affected by those that ar

B Some of the criteria are fulfilled and the conclusions of the study would be unlikely to be affected by those that are not C Few or no criteria were fulfilled and the conclusions of the study would be thought likely or very likely to be altered by mult

required criteria for an acceptable study 

Item Criteria 

General Quality Criteria 

1 Were unselected patients with CRC included? (i.e. were representative of patients seen in clinical practice {not selected based 

suggestive family history or other criteria that may cause selection bias) 

2 Inclusion criteria clear? 3 Did the whole sample or a random selection of the sample (i.e. total population of patients with CRC) receive verification usin

sequencing? 

4 Were the results of IHC or MSI or other predictors interpreted without knowledge of the results of sequencing? (i.e. was there 

blinding). 

5 Were the results of sequencing interpreted without knowledge of the results of the index test results (i.e. was there blinding) 6 Did authors describe how uninterpretable or intermediate results were analyzed (e.g. badly stained tissues etc)? 7 Were withdrawals from the study explained? 8 Did the authors report AND analyze results for deleterious MMR mutants 

Analytic Validity 

9 Was the description of how MSI or IHC or other predictors described in sufficient detail that others could replicate it (e.g. eith

description or relevant references)? 

10 Did authors describe what specimens were tested (e.g., blood, 

tumor tissue etc.)? 

11 Was MSI, IHC, sequencing or other testing performed at a 

similar time interval between specimen collection and processing for all subjects evaluated? 

12 Was there a clear description of which mismatch repair mutations were being tested for? 13 Was there a clear description of percentage of eligible subjects for whom valid genotypic data were obtained across study grou

(e.g., the proportion of patients who fulfilled and did not fulfill clinical criteria or those who did or did not have MSI who unde

sequencing....i.e. avoid verification bias)? 

14 Were quality control methods described for the molecular and genetic tests? 15 Did the authors attempt to address the reproducibility of results (reliability of tests)? 16 Did the authors specify that samples from each group of subjects (e.g., those who fulfilled and did not fulfill clinical criteria or 

who did or did not have MSI) included in each batch analyzed? (i.e. this helps minimize the effect of random errors). 
C - 489

Genetic and molecular testing methods 

Examples of tests 

Genetic screening methods Single-stranded conformation polymorphism (SSCP) 

Conformation sensitive gel electrophoresis (CSGE) Denaturing gradient gel electrophoresis (DGGE) Denaturing high-pressure liquid chromatography (DHPLC) 

Deletion analysis Southern blotting 

Multiplex Ligation-dependent Probe Amplification (MLPA) 

Conversion analysis Process of converting diploid cells to haploid cells. This is potentially important in HNPCC since the p

can sometimes make it difficult to identify a mutation in the mutant allele 

MSI methods Should have testing for five or more MSI markers. The five markers are “BAT25, BAT26, D2S123, DIHC All methods ok Microdissection Whether or not the tumor tissue used for MSI is microdissected to reduce the amount of DNA contribu

definitely affects the sensitivity of detecting MSI because the non-tumor cells are microsatellite stable 

peak signals as a percentage of the total signal, not allowing detection of MSI at many loci. 
D-1

Appendix D. List of Excluded Studies

Aaltonen LA, Peltomaki P, Leach FS, Sistonen P, 

Pylkkanen L, Mecklin JP, et al. Clues to the pathogenesis 

of familial colorectal cancer.[comment]. Science 1993 

May 7;260(5109):812-6. Linkage analysis 

Aarnio M, Sankila R, Pukkala E, Salovaara R, Aaltonen 

LA, de la CA, et al. Cancer risk in mutation carriers of 

DNA-mismatch-repair genes. Int J Cancer 1999 Apr 

12;81(2):214-8. Not population of interest 

Abdel-Rahman WM, Ollikainen M, Kariola R, Jarvinen 

HJ, Mecklin JP, Nystrom-Lahti M, et al. Comprehensive 

characterization of HNPCC-related colorectal cancers 

reveals striking molecular features in families with no 

germline mismatch repair gene mutations. Oncogene 2005 

Feb 24;24(9):1542-51. Didn't report any of 5 options 

Ainsworth PJ, Koscinski D, Fraser BP, Stuart JA. Family 

cancer histories predictive of a high risk of hereditary 

non-polyposis colorectal cancer associate significantly 

with a genomic rearrangement in hMSH2 or hMLH1. Clin 

Genet 2004 Sep;66(3):183-8. Not population of interest 

Akrami SM, Dunlop MG, Farrington SM, Frayling IM, 

MacDonald F, Harvey JF, et al. Screening for exonic copy 

number mutations at MSH2 and MLH1 by MAPH. Fam 

Cancer 2005;4(2):145-9. Didn't report any of 5 options 

Alexander J, Watanabe T, Wu TT, Rashid A, Li S, 

Hamilton SR. Histopathological identification of colon 

cancer with microsatellite instability. Am J Pathol 

2001;158(2):527-35. Genetic testing not compared with 

an index test 

Andrew SE, Whiteside D, Buzin C, Greenberg C, Spriggs 

E. An intronic polymorphism of the hMLH1 gene 

contributes toward incomplete genetic testing for HNPCC. 

Genet Test 2002;6(4):319-22. Didn't report any of 5 

options

Anonymous. American Gastroenterological Association 

medical position statement: hereditary colorectal cancer 

and genetic testing. Gastroenterology 2001 

Jul;121(1):195-7. Reference 

Anonymous. MSH6 mutations and risk of uterine cancer. 

Cancer Biol Ther 2004 Jun;3(6):491. News article, not 

study 

Anonymous. Practice parameters for the identification and 

testing of patients at risk for dominantly inherited 

colorectal cancer. Dis Colon Rectum 2001 

Oct;44(10):1403. Guideline

Anonymous. Statement of the American Society of 

Clinical Oncology: genetic testing for cancer 

susceptibility, Adopted on February 20, 1996. J Clin 

Oncol 1996;14(5):1730-6. Not relevant 

Apessos A, Mihalatos M, Danielidis I, Kallimanis G, 

Agnantis NJ, Triantafillidis JK, et al. hMSH2 is the most 

commonly mutated MMR gene in a cohort of Greek 

HNPCC patients. Br J Cancer 2005 Jan 31;92(2):396-404. 

Not population of interest 

Arnold CN, Goel A, Compton C, Marcus V, Niedzwiecki 

D, Dowell JM, et al. Evaluation of microsatellite 

instability, hMLH1 expression and hMLH1 promoter 

hypermethylation in defining the MSI phenotype of 

colorectal cancer. Cancer Biol Ther 2004 Jan;3(1):73-8.

Not population of interest 

Ashktorab H, Smoot DT, Carethers JM, Rahmanian M, 

Kittles R, Vosganian G, et al. High incidence of 

microsatellite instability in colorectal cancer from African 

Americans. Clin Cancer Res 2003 Mar;9(3):1112-7. 

Genetic testing not compared with index test

Ashktorab H, Smoot DT, Farzanmehr H, Fidelia-Lambert 

M, Momen B, Hylind L, et al. Clinicopathological 

features and microsatellite instability (MSI) in colorectal 

cancers from African Americans. Int J Cancer 2005 Oct 

10;116(6):914-9. Genetic testing not compared with an 

index test; hMLH1 and hMSH2 sequencing not 

performed

Bacher JW, Flanagan LA, Smalley RL, Nassif NA, 

Burgart LJ, Halberg RB, et al. Development of a 

fluorescent multiplex assay for detection of MSI-High 

tumors. Dis Markers 2004;20(4-5):237-50. Biological 

material not evaluated 

Bai YQ, Akiyama Y, Nagasaki H, Lu SL, Arai T, 

Morisaki T, et al. Predominant germ-line mutation of the 

hMSH2 gene in Japanese hereditary non-polyposis 

colorectal cancer kindreds. Int J Cancer 1999 Aug 

12;82(4):512-5. Not population of interest 
D-2

Bandipalliam P. Syndrome of early onset colon cancers, 

hematologic malignancies & features of 

neurofibromatosis in HNPCC families with homozygous 

mismatch repair gene mutations. Familial Cancer 

2005;4(4):323-33. Case series

Banno K, Susumu N, Hirao T, Yanokura M, Hirasawa A, 

Aoki D, et al. Identification of germline MSH2 gene 

mutations in endometrial cancer not fulfilling the new 

clinical criteria for hereditary nonpolyposis colorectal 

cancer. Cancer Genet Cytogenet 2003 Oct 1;146(1):58-65. 

Not population of interest 

Banno K, Susumu N, Hirao T, Yanokura M, Hirasawa A, 

Aoki D, et al. Two Japanese kindreds occurring 

endometrial cancer meeting new clinical criteria for 

hereditary non-polyposis colorectal cancer (HNPCC), 

Amsterdam Criteria II. J Obstet Gynaecol Res 2004 

Aug;30(4):287-92. Not population of interest 

Banno K, Susumu N, Yanokura M, Hirao T, Iwata T, 

Hirasawa A, et al. Association of HNPCC and 

endometrial cancers. Int J Clin Oncol 2004 Aug;9(4):262-

9. Not population of interest 

Bapat BV, Madlensky L, Temple LK, Hiruki T, Redston 

M, Baron DL, et al. Family history characteristics, tumor 

microsatellite instability and germline MSH2 and MLH1 

mutations in hereditary colorectal cancer. Hum Genet 

1999 Feb;104(2):167-76. Not population of interest 

Bartosova Z, Fridrichova I, Bujalkova M, Wolf B, 

Ilencikova D, Krizan P, et al. Novel MLH1 and MSH2 

germline mutations in the first HNPCC families identified 

in Slovakia. Hum Mutat 2003 Apr;21(4):449. Not 

population of interest 

Batra S, Valdimarsdottir H, McGovern M, Itzkowitz S, 

Brown K. Awareness of genetic testing for colorectal 

cancer predisposition among specialists in 

gastroenterology. Am J Gastroenterol 2002 

Mar;97(3):729-33. Not population of interest 

Baudhuin LM, Burgart LJ, Leontovich O, Thibodeau SN. 

Use of microsatellite instability and 

immunohistochemistry testing for the identification of 

individuals at risk for lynch syndrome. Fam Cancer 

2005;4(3):255-65. Not primary study 

Baudhuin LM, Ferber MJ, Winters JL, Steenblock KJ, 

Swanson RL, French AJ, et al. Characterization of 

hMLH1 and hMSH2 gene dosage alterations in Lynch 

syndrome patients. Gastroenterology 2005 

Sep;129(3):846-54. Didn't report any of 5 options 

Baudhuin LM, Mai M, French AJ, Kruckeberg KE, 

Swanson RL, Winters JL, et al. Analysis of hMLH1 and 

hMSH2 gene dosage alterations in hereditary 

nonpolyposis colorectal cancer patients by novel methods. 

J Mol Diagn 2005 May;7(2):226-35. Didn't report any of 

5 options 

Baudi F, Fersini G, Lavecchia A, Terracciano R, Leone F, 

Quaresima B, et al. A novel missense germline mutation 

in exon 2 of the hMSH2 gene in a HNPCC family from 

Southern Italy. Cancer Lett 2005 Jun 8;223(2):285-91. 

Not population of interest 

Beck NE, Tomlinson IP, Homfray T, Frayling I, Hodgson 

SV, Harocopos C, et al. Use of SSCP analysis to identify 

germline mutations in HNPCC families fulfilling the 

Amsterdam criteria. Hum Genet 1997 Feb;99(2):219-24. 

Didn't report any of 5 options 

Beck NE, Tomlinson IP, Homfray T, Hodgson SV, 

Harocopos CJ, Bodmer WF. Genetic testing is important 

in families with a history suggestive of hereditary nonpolyposis colorectal cancer even if the Amsterdam criteria 

are not fulfilled. Br J Surg 1997 Feb;84(2):233-7. Not 

population of interest 

Beck NE, Tomlinson IP, Homfray TF, Frayling IM, 

Hodgson SV, Bodmer WF. Frequency of germline 

hereditary non-polyposis colorectal cancer gene mutations 

in patients with multiple or early onset colorectal 

adenomas. Gut 1997 Aug;41(2):235-8. Did not meet 

inclusion criteria for population 

Benatti P, Roncucci L, Ganazzi D, Percesepe A, Di 

Gregorio C, Pedroni M, et al. Clinical and biologic 

heterogeneity of hereditary nonpolyposis colorectal 

cancer. Int J Cancer 2001 Sep 20;95(5):323-8. Duplicate 

publication

Benatti P, Sassatelli R, Roncucci L, Pedroni M, Fante R, 

Di Gregorio C, et al. Tumour spectrum in hereditary nonpolyposis colorectal cancer (HNPCC) and in families with 

"suspected HNPCC". A population-based study in 

northern Italy. Colorectal Cancer Study Group. Int J 

Cancer 1993 May 28;54(3):371-7. Genetic testing not 

compared with an index test; hMLH1 and hMSH2 

sequencing not performed 

Berends MJ, Hollema H, Wu Y, van der ST, Mensink RG, 

ten Hoor KA, et al. MLH1 and MSH2 protein expression 

as a pre-screening marker in hereditary and non-hereditary 

endometrial hyperplasia and cancer. Int J Cancer 2001 

May 1;92(3):398-403. Not population of interest 
D-3

Berends MJ, Wu Y, Sijmons RH, Mensink RG, van der 

ST, Hordijk-Hos JM, et al. Molecular and clinical 

characteristics of MSH6 variants: an analysis of 25 index 

carriers of a germline variant. Am J Hum Genet 2002 

Jan;70(1):26-37. Not population of interest 

Berends MJ, Wu Y, Sijmons RH, van der ST, Ek WB, 

Ligtenberg MJ, et al. Toward new strategies to select 

young endometrial cancer patients for mismatch repair 

gene mutation analysis. J Clin Oncol 2003 Dec 

1;21(23):4364-70. Not population of interest 

Bisgaard ML, Jager AC, Myrhoj T, Bernstein I, Nielsen 

FC. Hereditary non-polyposis colorectal cancer (HNPCC): 

phenotype-genotype correlation between patients with and 

without identified mutation. Hum Mutat 2002 

Jul;20(1):20-7. Not population of interest 

Bocker T, Diermann J, Friedl W, Gebert J, Holinski-Feder 

E, Karner-Hanusch J, et al. Microsatellite instability 

analysis: a multicenter study for reliability and quality 

control. Cancer Res 1997 Nov 1;57(21):4739-43. Genetic 

test not compared to index test 

Boland CR, Fishel R. Lynch syndrome: form, function, 

proteins, and basketball. Gastroenterology 

2005;129(2):751-5. Not relevant 

Boland CR, Thibodeau SN, Hamilton SR, Sidransky D, 

Eshleman JR, Burt RW, et al. A National Cancer Institute 

Workshop on Microsatellite Instability for cancer 

detection and familial predisposition: development of 

international criteria for the determination of 

microsatellite instability in colorectal cancer. Cancer Res 

1998 Nov 15;58(22):5248-57. Review

Broaddus RR, Lynch PM, Lu KH, Luthra R, Michelson 

SJ. Unusual tumors associated with the hereditary 

nonpolyposis colorectal cancer syndrome. Mod Pathol 

2004 Aug;17(8):981-9. Case report 

Brown GJ, St John DJ, Macrae FA, Aittomaki K. Cancer 

risk in young women at risk of hereditary nonpolyposis 

colorectal cancer: implications for gynecologic 

surveillance. Gynecol Oncol 2001;80(3):346-9. Not 

relevant 

Buerstedde JM, Alday P, Torhorst J, Weber W, Muller H, 

Scott R. Detection of new mutations in six out of 10 Swiss 

HNPCC families by genomic sequencing of the hMSH2 

and hMLH1 genes. J Med Genet 1995 Nov;32(11):909-

12. Not population of interest 

Burgart LJ. Testing for defective DNA mismatch repair in 

colorectal carcinoma: a practical guide. Arch Pathol Lab 

Med 2005 Nov;129(11):1385-9. Not relevant 

Burke W, Petersen G, Lynch P, Botkin J, Daly M, Garber 

J, et al. Recommendations for follow-up care of 

individuals with an inherited predisposition to cancer. I. 

Hereditary nonpolyposis colon cancer. Cancer Genetics 

Studies Consortium. Jama 1997;277(11):915-9. Not 

primary study

Cai Q, Sun MH, Lu HF, Zhang TM, Mo SJ, Xu Y, et al. 

Clinicopathological and molecular genetic analysis of 4 

typical Chinese HNPCC families. World J Gastroenterol 

2001 Dec;7(6):805-10. Case series 

Cai SJ, Xu Y, Cai GX, Lian P, Guan ZQ, Mo SJ, et al. 

Clinical characteristics and diagnosis of patients with 

hereditary nonpolyposis colorectal cancer. World J 

Gastroenterol 2003 Feb;9(2):284-7. Not relevant 

Caldes T, Godino J, de la HM, Garcia C, I, Perez SP, Eng 

C, et al. Prevalence of germline mutations of MLH1 and 

MSH2 in hereditary nonpolyposis colorectal cancer 

families from Spain. Int J Cancer 2002 Apr 10;98(5):774-

9. Not population of interest 

Cameron BH, Fitzgerald GW, Cox J. Hereditary sitespecific colon cancer in a Canadian kindred. CMAJ 1989 

Jan 1;140(1):41-5. Case series, no relevant data 

Carethers JM, Smith EJ, Behling CA, Nguyen L, Tajima 

A, Doctolero RT, et al. Use of 5-fluorouracil and survival 

in patients with microsatellite-unstable colorectal 

cancer.[see comment]. Gastroenterology 2004 

Feb;126(2):394-401. Not relevant 

Chai SM, Zeps N, Shearwood AM, Grieu F, Charles A, 

Harvey J, et al. Screening for defective DNA mismatch 

repair in stage II and III colorectal cancer patients. Clin 

Gastroenterol Hepatol 2004 Nov;2(11):1017-25. hMLH1 

and hMSH2 sequencing not performed 

Chan TL, Yuen ST, Chung LP, Ho JW, Kwan KY, Chan 

AS, et al. Frequent microsatellite instability and mismatch 

repair gene mutations in young Chinese patients with 

colorectal cancer. J Natl Cancer Inst 1999 Jul 

21;91(14):1221-6. No data 
D-4

Chapusot C, Martin L, Mungra N, Rageot D, Bouvier 

AM, Bonithon KC, et al. Sporadic colorectal cancers with 

defective mismatch repair display a number of specific 

morphological characteristics: relationship between the 

expression of hMLH1 and hMSH2 proteins and 

clinicopathological features of 273 adenocarcinomas. 

Histopathology 2003 Jul;43(1):40-7. Genetic testing not 

compared with an index test; hMLH1 and hMSH2 

sequencing not performed 

Chapusot C, Martin L, Puig PL, Ponnelle T, Cheynel N, 

Bouvier AM, et al. What is the best way to assess 

microsatellite instability status in colorectal cancer? Study 

on a population base of 462 colorectal cancers. Am J Surg 

Pathol 2004 Dec;28(12):1553-9. Genetic test not 

compared to index test 

Chaves P, Cruz C, Lage P, Claro I, Cravo M, Leitao CN, 

et al. Immunohistochemical detection of mismatch repair 

gene proteins as a useful tool for the identification of 

colorectal carcinoma with the mutator phenotype. J Pathol 

2000 Aug;191(4):355-60. Genetic testing not compared 

with index test 

Chen S, Watson P, Parmigiani G. Accuracy of MSI testing 

in predicting germline mutations of MSH2 and MLH1: a 

case study in Bayesian meta-analysis of diagnostic tests 

without a gold standard. Biostatistics 2005 Jul;6(3):450-

64. Meta-analysis 

Chung DC, Rustgi AK. The hereditary nonpolyposis 

colorectal cancer syndrome: genetics and clinical 

implications. Ann Intern Med 2003;138(7):560-70. Not 

primary study

Church J, Simmang C. Practice parameters for the 

treatment of patients with dominantly inherited colorectal 

cancer (familial adenomatous polyposis and hereditary 

nonpolyposis colorectal cancer). Dis Colon Rectum 2003 

Aug;46(8):1001-12. Guideline 

Church J. Hereditary colon cancers can be tiny: a 

cautionary case report of the results of colonoscopic 

surveillance. Am J Gastroenterol 1998 Nov;93(11):2289-

90. Case report 

Claro I, Cravo M, Gloria L, Gaspar C, Albuquerque C, 

Lage P, et al. Colonic cancer in a 34-yr-old woman: 

should it prompt microsatellite instability studies and 

mismatch repair gene testing? Am J Gastroenterol 1998 

Oct;93(10):1991-2. Case report 

Coggins RP, Cawkwell L, Bell SM, Crockford GP, Quirke 

P, Finan PJ, et al. Association between family history and 

mismatch repair in colorectal cancer. Gut 2005 

May;54(5):636-42. Did not meet inclusion criteria

Cravo M, Lage P, Albuquerque C, Chaves P, Claro I, 

Gomes T, et al. BAT-26 identifies sporadic colorectal 

cancers with mutator phenotype: a correlative study with 

clinico-pathological features and mutations in mismatch 

repair genes. J Pathol 1999 Jul;188(3):252-7. Data not 

extractable 

Crijnen TE, Janssen-Heijnen ML, Gelderblom H, Morreau 

J, Nooij MA, Kenter GG, et al. Survival of patients with 

ovarian cancer due to a mismatch repair defect. Familial 

Cancer 2005;4(4):301-5. Not relevant 

De Felice C, Parrini S, Chitano G, Gentile M, Dipaola L, 

Latini G. Fordyce granules and hereditary non-polyposis 

colorectal cancer syndrome. Gut 2005 Sep;54(9):1279-82. 

Not population of interest 

de Jong AE, van Puijenbroek M, Hendriks Y, Tops C, 

Wijnen J, Ausems MG, et al. Microsatellite instability, 

immunohistochemistry, and additional PMS2 staining in 

suspected hereditary nonpolyposis colorectal cancer. Clin 

Cancer Res 2004 Feb 1;10(3):972-80. hMLH1 and 

hMSH2 sequencing not performed 

de la CA. Testing tumors for microsatellite instability. Eur 

J Hum Genet 1999 May;7(4):407-8. Review

Dietmaier W, Hofstadter F. Detection of microsatellite 

instability by real time PCR and hybridization probe 

melting point analysis. Lab Invest 2001 Oct;81(10):1453-

6. Not relevant 

Dietmaier W, Wallinger S, Bocker T, Kullmann F, Fishel 

R, Ruschoff J. Diagnostic microsatellite instability: 

definition and correlation with mismatch repair protein 

expression. Cancer Res 1997 Nov 1;57(21):4749-56. 

Incomplete data 

Domingo E, Laiho P, Ollikainen M, Pinto M, Wang L, 

French AJ, et al. BRAF screening as a low-cost effective 

strategy for simplifying HNPCC genetic testing. J Med 

Genet 2004 Sep;41(9):664-8. hMLH1 and hMSH2 

sequencing not performed 

Domingo E, Niessen RC, Oliveira C, Alhopuro P, 

Moutinho C, Espin E, et al. BRAF-V600E is not involved 

in the colorectal tumorigenesis of HNPCC in patients with 

functional MLH1 and MSH2 genes. Oncogene 2005 Jun 

2;24(24):3995-8. Not relevant, select patients 

Douglas JA, Gruber SB, Meister KA, Bonner J, Watson P, 

Krush AJ, et al. History and molecular genetics of Lynch 

syndrome in family G: a century later. Jama 

2005;294(17):2195-202. Case report 
D-5

Dovrat S, Figer A, Fidder HH, Neophytou P, Fireman Z, 

Geva R, et al. Mutational analysis of hMsh6 in Israeli 

HNPCC and HNPCC-like families. Familial Cancer 

2005;4(4):291-4. Genetic testing not compared with 

index test 

Drobinskaya I, Gabbert HE, Moeslein G, Mueller W. A 

new method for optimizing multiplex DNA microsatellite 

analysis in low quality archival specimens. Anticancer 

Res 2005 Sep;25(5):3251-8. Genetic test not compared 

to index test

Dunlop MG, Farrington SM, Carothers AD, Wyllie AH, 

Sharp L, Burn J, et al. Cancer risk associated with 

germline DNA mismatch repair gene mutations. Hum Mol 

Genet 1997;6(1):105-10. Not relevant

Dunlop MG, Farrington SM, Nicholl I, Aaltonen L, 

Petersen G, Porteous M, et al. Population carrier 

frequency of hMSH2 and hMLH1 mutations. Br J Cancer 

2000 Dec;83(12):1643-5. Not population of interest

Eisen GM, Weinberg DS. Narrative review: screening for 

colorectal cancer in patients with a first-degree relative 

with colonic neoplasia. Ann Intern Med 2005;143(3):190-

8. Review 

Elsaleh H, Joseph D, Grieu F, Zeps N, Spry N, Iacopetta 

B. Association of tumour site and sex with survival 

benefit from adjuvant chemotherapy in colorectal cancer. 

Lancet 2000;355(9217):1745-50. Not population of 

interest 

Engel C, Forberg J, Holinski-Feder E, Pagenstecher C, 

Plaschke J, Kloor M, et al. Novel strategy for optimal 

sequential application of clinical criteria, 

immunohistochemistry and microsatellite analysis in the 

diagnosis of hereditary nonpolyposis colorectal cancer. Int 

J Cancer 2006 Jan 1;118(1):115-22. Not outcomes of 

interest 

Ensenauer RE, Michels VV, Reinke SS. Genetic testing: 

practical, ethical, and counseling considerations. Mayo 

Clin Proc 2005 Jan;80(1):63-73. Not relevant

Ericson K, Nilbert M, Bladstrom A, Anderson H, Olsson 

H, Planck M. Familial risk of tumors associated with 

hereditary non-polyposis colorectal cancer: a Swedish 

population-based study. Scand J Gastroenterol 2004 

Dec;39(12):1259-65. Not relevant

Evans DG, Walsh S, Jeacock J, Robinson C, Hadfield L, 

Davies DR, et al. Incidence of hereditary non-polyposis 

colorectal cancer in a population-based study of 1137 

consecutive cases of colorectal cancer. Br J Surg 1997 

Sep;84(9):1281-5. Genetic testing not compared with 

index test 

Fallik D, Borrini F, Boige V, Viguier J, Jacob S, Miquel 

C, et al. Microsatellite instability is a predictive factor of 

the tumor response to irinotecan in patients with advanced 

colorectal cancer. Cancer Res 2003 Sep 15;63(18):5738-

44. Not population of interest 

Faragher IG, Whitehead RH. Rapid diagnostic test for 

hereditary nonpolyposis colon cancer kindred using 

polymerase chain reaction. Dis Colon Rectum 1998 

Jul;41(7):938-40. Not relevant 

Fidalgo P, Almeida MR, West S, Gaspar C, Maia L, 

Wijnen J, et al. Detection of mutations in mismatch repair 

genes in Portuguese families with hereditary nonpolyposis colorectal cancer (HNPCC) by a multi-method 

approach. Eur J Hum Genet 2000 Jan;8(1):49-53. 

Unknown if patients had CRC 

Frazier ML, Sinicrope FA, Amos CI, Cleary KR, Lynch 

PM, Levin B, et al. Loci for efficient detection of 

microsatellite instability in hereditary non-polyposis 

colorectal cancer. Oncol Rep 1999 May;6(3):497-505. 

Didn't report any of 5 options 

Froggatt NJ, Brassett C, Koch DJ, Evans DG, Hodgson 

SV, Ponder BA, et al. Mutation screening of MSH2 and 

MLH1 mRNA in hereditary non-polyposis colon cancer 

syndrome. J Med Genet 1996 Sep;33(9):726-30. Data not 

extractable 

Furlan D, Tibiletti MG, Taborelli M, Albarello L, 

Cornaggia M, Capella C. The value of microsatellite 

instability in the detection of HNPCC families and of 

sporadic colorectal cancers with special biological 

features: an investigation on a series of 100 consecutive 

cases. Ann Oncol 1998 Aug;9(8):901-6. No genetic 

testing 

Furukawa T, Konishi F, Shitoh K, Kojima M, Nagai H, 

Tsukamoto T. Evaluation of screening strategy for 

detecting hereditary nonpolyposis colorectal carcinoma. 

Cancer 2002 Feb 15;94(4):911-20. Data not extractable

Gaglia P, Atkin WS, Whitelaw S, Talbot IC, Williams 

CB, Northover JM, et al. Variables associated with the 

risk of colorectal adenomas in asymptomatic patients with 

a family history of colorectal cancer. Gut 1995 

Mar;36(3):385-90. Not population of interest
D-6

Geirdal AO, Reichelt JG, Dahl AA, Heimdal K, Maehle 

L, Stormorken A, et al. Psychological distress in women 

at risk of hereditary breast/ovarian or HNPCC cancers in 

the absence of demonstrated mutations. Fam Cancer 

2005;4(2):121-6. Results of FBOC and HNPCC 

combined in analyses 

Genuardi M, Anti M, Capozzi E, Leonardi F, Fornasarig 

M, Novella E, et al. MLH1 and MSH2 constitutional 

mutations in colorectal cancer families not meeting the 

standard criteria for hereditary nonpolyposis colorectal 

cancer. Int J Cancer 1998 Mar 16;75(6):835-9. Data not 

extractable 

Giardiello FM, Brensinger JD, Petersen GM. AGA 

technical review on hereditary colorectal cancer and 

genetic testing. Gastroenterology 2001;121(1):198-213. 

Review 

Gill S, Lindor NM, Burgart LJ, Smalley R, Leontovich O, 

French AJ, et al. Isolated loss of PMS2 expression in 

colorectal cancers: frequency, patient age, and familial 

aggregation. Clin Cancer Res 2005 Sep 15;11(18):6466-

71. Genetic testing not compared with index test, 

hMLH1 and hMSH2 sequencing not performed 

Giraldo A, Gomez A, Salguero G, Garcia H, Aristizabal F, 

Gutierrez O, et al. MLH1 and MSH2 mutations in 

Colombian families with hereditary nonpolyposis 

colorectal cancer (Lynch syndrome)--description of four 

novel mutations. Familial Cancer 2005;4(4):285-90. Not 

population of interest 

Gologan A, Krasinskas A, Hunt J, Thull DL, Farkas L, 

Sepulveda AR. Performance of the revised Bethesda 

guidelines for identification of colorectal carcinomas with 

a high level of microsatellite instability. Arch Pathol Lab 

Med 2005 Nov;129(11):1390-7. Genetic testing not 

compared with index test, hMLH1 and hMSH2 

sequencing not performed 

Grover S, Stoffel EM, Bussone L, Tschoegl E, Syngal S. 

Physician assessment of family cancer history and referral 

for genetic evaluation in colorectal cancer patients. Clin 

Gastroenterol Hepatol 2004;2(9):813-9. Not relevant

Gryfe R, Kim H, Hsieh ET, Aronson MD, Holowaty EJ, 

Bull SB, et al. Tumor microsatellite instability and clinical 

outcome in young patients with colorectal cancer. N Engl 

J Med 2000;342(2):69-77. Not population of interest, 

hMLH1 and hMSH2 sequencing not performed 

Hall NR, Finan PJ, Ward B, Turner G, Bishop DT. 

Genetic susceptibility to colorectal cancer in patients 

under 45 years of age. Br J Surg 1994 Oct;81(10):1485-9. 

Genetic testing not compared with index test, hMLH1 

and hMSH2 sequencing not performed 

Halling KC, French AJ, McDonnell SK, Burgart LJ, 

Schaid DJ, Peterson BJ, et al. Microsatellite instability 

and 8p allelic imbalance in stage B2 and C colorectal 

cancers. J Natl Cancer Inst 1999;91(15):1295-303. Not 

relevant 

Halvarsson B, Lindblom A, Johansson L, Lagerstedt K, 

Nilbert M. Loss of mismatch repair protein 

immunostaining in colorectal adenomas from patients 

with hereditary nonpolyposis colorectal cancer. Mod 

Pathol 2005 Aug;18(8):1095-101. Not outcomes of 

interest

Halvarsson B, Lindblom A, Rambech E, Lagerstedt K, 

Nilbert M. Microsatellite instability analysis and/or 

immunostaining for the diagnosis of hereditary 

nonpolyposis colorectal cancer? Virchows Arch 2004 

Feb;444(2):135-41. ND on germline mutations 

Hamilton SR, Liu B, Parsons RE, Papadopoulos N, Jen J, 

Powell SM, et al. The molecular basis of Turcot's 

syndrome. N Engl J Med 1995 Mar 30;332(13):839-47. 

Not population of interest 

Hampel H, Frankel WL, Martin E, Arnold M, Khanduja 

K, Kuebler P, et al. Screening for the Lynch syndrome 

(hereditary nonpolyposis colorectal cancer). N Engl J Med 

2005;352(18):1851-60. Not outcomes of interest 

Hatch SB, Lightfoot HM, Jr., Garwacki CP, Moore DT, 

Calvo BF, Woosley JT, et al. Microsatellite instability 

testing in colorectal carcinoma: choice of markers affects 

sensitivity of detection of mismatch repair-deficient 

tumors. Clin Cancer Res 2005 Mar 15;11(6):2180-7. 

Genetic test not compared to index test

Hegde M, Blazo M, Chong B, Prior T, Richards C. Assay 

validation for identification of hereditary nonpolyposis 

colon cancer-causing mutations in mismatch repair genes 

MLH1, MSH2, and MSH6. J Mol Diagn 2005 

Oct;7(4):525-34. Didn't report any of 5 options

Heinimann K, Scott RJ, Buerstedde JM, Weber W, 

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criteria on mutation detection in patients with hereditary 

nonpolyposis colorectal cancer. Cancer 1999 Jun 

15;85(12):2512-8. Not population of interest 
D-7

Hendriks YM, Jagmohan-Changur S, van der Klift HM, 

Morreau H, van PM, Tops C, et al. Heterozygous 

Mutations in PMS2 Cause Hereditary Nonpolyposis 

Colorectal Carcinoma (Lynch Syndrome). 

Gastroenterology 2006 Feb;130(2):312-22. 

Hitchins M, Williams R, Cheong K, Halani N, Lin VA, 

Packham D, et al. MLH1 germline epimutations as a 

factor in hereditary nonpolyposis colorectal cancer. 

Gastroenterology 2005 Nov;129(5):1392-9. Genetic 

testing not compared with index test 

Ho JW, Yuen ST, Chung LP, Kwan KY, Chan TL, Leung 

SY, et al. Distinct clinical features associated with 

microsatellite instability in colorectal cancers of young 

patients. Int J Cancer 2000 Jul 20;89(4):356-60. No data 

Hodgson SV, Harocopos C, Gaglia P. Screening results in 

a family cancer clinic: five years experience. Anticancer 

Res 1993 Nov;13(6B):2581-5. 

Huang D, Chen C, Sun W, Strom CM, Bender RA. Highthroughput gene sequencing assay development for 

hereditary nonpolyposis colon cancer. Clin Colorectal 

Cancer 2004 Nov;4(4):275-9. Unknown population 

Hurlstone DP, Karajeh M, Cross SS, McAlindon ME, 

Brown S, Hunter MD, et al. The role of highmagnification-chromoscopic colonoscopy in hereditary 

nonpolyposis colorectal cancer screening: a prospective 

"back-to-back" endoscopic study. Am J Gastroenterol 

2005 Oct;100(10):2167-73. Compares 2 methods of 

conducting colonsocopy in HNPCC patients on 

adenoma detection 

Hutter P, Couturier A, Membrez V, Joris F, Sappino AP, 

Chappuis PO. Excess of hMLH1 germline mutations in 

Swiss families with hereditary non-polyposis colorectal 

cancer. Int J Cancer 1998 Dec 9;78(6):680-4. Not 

population of interest 

Iino H, Simms L, Young J, Arnold J, Winship IM, Webb 

SI, et al. DNA microsatellite instability and mismatch 

repair protein loss in adenomas presenting in hereditary 

non-polyposis colorectal cancer. Gut 2000 Jul;47(1):37-

42. Adenoma data 

Ikenaga M, Tomita N, Sekimoto M, Ohue M, Yamamoto 

H, Miyake Y, et al. Use of microsatellite analysis in 

young patients with colorectal cancer to identify those 

with hereditary nonpolyposis colorectal cancer. J Surg 

Oncol 2002 Mar;79(3):157-65. hMLH1 and hMSH2 

sequencing not performed; genetic testing not 

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Jass JR, Barker M, Fraser L, Walsh MD, Whitehall VL, 

Gabrielli B, et al. APC mutation and tumour budding in 

colorectal cancer. J Clin Pathol 2003 Jan;56(1):69-73. No 

clinical endpoints 

Jass JR, Cottier DS, Jeevaratnam P, Pokos V, Holdaway 

KM, Bowden ML, et al. Diagnostic use of microsatellite 

instability in hereditary non-polyposis colorectal cancer. 

Lancet 1995;346(8984):1200-1. Abstract 

Jemal A, Murray T, Ward E, Samuels A, Tiwari RC, 

Ghafoor A, et al. Cancer statistics, 2005. CA Cancer J 

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Jernvall P, Makinen MJ, Karttunen TJ, Makela J, Vihko P. 

Microsatellite instability: impact on cancer progression in 

proximal and distal colorectal cancers. Eur J Cancer 1999 

Feb;35(2):197-201. HNPCC not defined 

Jo WS, Bandipalliam P, Shannon KM, Niendorf KB, 

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Number and Family History of Colon Cancer With 

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Johns LE, Houlston RS. A systematic review and metaanalysis of familial colorectal cancer risk. Am J 

Gastroenterol 2001;96(10):2992-3003. Review 

Johnson KA, Rosenblum-Vos L, Petersen GM, Brensinger 

JD, Giardiello FM, Griffin CA. Response to genetic 

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J Med Genet 2000 Mar 20;91(3):207-11. Not population 

of interest 

Johnson PM, Gallinger S, McLeod RS. Surveillance 

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Diseases of the Colon & Rectum 2006 Jan 49(1):80-93. 

Review 

Jong A. Shift in mortality in Lynch syndrome families due 

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Jordan J, Evans G, Evers-Kiebooms G, Julian-Reynier C, 

Kash K, Watson M. Congress report of the seventh 

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Meeting proceedings 
D-8

Jourdan F, Sebbagh N, Comperat E, Mourra N, Flahault 

A, Olschwang S, et al. Tissue microarray technology: 

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hMLH1, and hMSH2 immunohistochemical expression. 

Virchows Arch 2003 Aug;443(2):115-21. Genetic test 

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Jover R, Paya A, Alenda C, Poveda MJ, Peiro G, Aranda 

FI, et al. Defective mismatch-repair colorectal cancer: 

clinicopathologic characteristics and usefulness of 

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Pathol 2004 Sep;122(3):389-94. Genetic testing not 

compared with an index test 

Kakar S, Aksoy S, Burgart LJ, Smyrk TC. Mucinous 

carcinoma of the colon: correlation of loss of mismatch 

repair enzymes with clinicopathologic features and 

survival. Mod Pathol 2004 Jun;17(6):696-700. Not 

population of interest 

Kanazawa T, Watanabe T, Kazama S, Tada T, Koketsu S, 

Nagawa H. Poorly differentiated adenocarcinoma and 

mucinous carcinoma of the colon and rectum show higher 

rates of loss of heterozygosity and loss of E-cadherin 

expression due to methylation of promoter region. Int J 

Cancer 2002 Nov 20;102(3):225-9. Not relevant 

Kee F, Collins BJ, Patterson CC. Prognosis in familial 

non-polyposis colorectal cancer. Gut 1991 

May;32(5):513-6. HNPCC not defined 

Khurana V, Stollman N, Rogers AI. Colorectal cancer: 

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immunohistochemistry. Am J Gastroenterol 2000 

Oct;95(10):2981-2. Not population of interest 

Kievit W, de Bruin JH, Adang EM, Ligtenberg MJ, 

Nagengast FM, van Krieken JH, et al. Current clinical 

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Kievit W, de Bruin JH, Adang EM, Severens JL, 

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Jan;54(1):97-102. Not relevant 

Kim H, Piao Z, Kim JW, Choi JS, Kim NK, Lee JM, et al. 

Expression of hMSH2 and hMLH1 in colorectal 

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Pract 1998;194(1):3-9. hMLH1 and hMSH2 sequencing 

not performed 

Kim WH, Lee HW, Park SH, Kim YI, Chi JG. 

Microsatellite instability in young patients with colorectal 

cancer. Pathol Int 1998 Aug;48(8):586-94. hMLH1 and 

hMSH2 sequencing not performed 

Kim YH, Song SY, Kwon YD, Kim DS, Chun HK, Rhee 

JC. Microsatellite instable double primary cancers of the 

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World J Gastroenterol 2005 Jul 14;11(26):3998-4002. Not 

population of interest 

Kinoshita M, Nakamura J, Kusaka H, Hadama T, Bago K, 

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Chim Acta 1999 Jan;279(1-2):15-23. Not relevant 

Klaus K, Herfarth F, Ogunbiyi OA, Moley JF, Kodner IJ, 

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mismatch repair gene MLH1 in colorectal cancers with 

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Online. Hum Mutat 1998;12(1):73. hMLH1 and hMSH2 

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Koehly LM, Peterson SK, Watts BG, Kempf KK, Vernon 

SW, Gritz ER. A social network analysis of 

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Epidemiol Biomarkers Prev 2003 Apr;12(4):304-13. Not 

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Kohonen-Corish M, Ross VL, Doe WF, Kool DA, Edkins 

E, Faragher I, et al. RNA-based mutation screening in 

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Genet 1996 Oct;59(4):818-24. Not relevant

Koike J, Yamada K, Takano S, Kikuchi Y, Hemmi H, Koi 

M, et al. Undectable expression of hMLH1 protein in 

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Kolble K, Ullrich OM, Pidde H, Barthel B, Diermann J, 

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Sep;79(9):1145-50. Not relevant 

Komesaroff PA, Greenberg P, Piterman L. Genetic 

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2004 Jan;34(1-2):61-5. Not study

Kondo E, Suzuki H, Horii A, Fukushige S. A yeast twohybrid assay provides a simple way to evaluate the vast 

majority of hMLH1 germ-line mutations. Cancer Res 

2003 Jun 15;63(12):3302-8. Not relevant
D-9

Koshiji M, Yonekura Y, Saito T, Yoshioka K. 

Microsatellite analysis of fecal DNA for colorectal cancer 

detection. J Surg Oncol 2002 May;80(1):34-40. hMLH1 

and hMSH2 sequencing not performed 

Kotake K, Koyama Y, Nasu J, Fukutomi T, Yamaguchi N. 

Relation of family history of cancer and environmental 

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study. Jpn J Clin Oncol 1995 Oct;25(5):195-202. Not 

relevant 

Kouraklis G, Misiakos EP. Hereditary nonpolyposis 

colorectal cancer (Lynch syndrome): criteria for 

identification and management. Dig Dis Sci 2005 

Feb;50(2):336-44. Review 

Krajinovic M, Richer C, Labuda D, Sinnett D. Detection 

of a mutator phenotype in cancer cells by inter-Alu 

polymerase chain reaction. Cancer Res 1996 Jun 

15;56(12):2733-7. Not relevant 

Kruschewski M, Noske A, Haier J, Runkel N, 

Anagnostopoulos Y, Buhr HJ. Is reduced expression of 

mismatch repair genes MLH1 and MSH2 in patients with 

sporadic colorectal cancer related to their prognosis? Clin 

Exp Metastasis 2002;19(1):71-7. Not population of 

interest 

Lagarde AE, Stern HS. Genetic pre-disposition to 

colorectal cancer: Initiation of a prospective study in 

Eastern Ontario. Int J Cancer 1996 Feb 20;69(1):68-70. 

No data 

Lage PA, Albuquerque C, Sousa RG, Cravo ML, Salazar 

M, Francisco I, et al. Association of colonic and 

endometrial carcinomas in Portuguese families with 

hereditary nonpolyposis colorectal carcinoma significantly 

increases the probability of detecting a pathogenic 

mutation in mismatch repair genes, primarily the MSH2 

gene. Cancer 2004 Jul 1;101(1):172-7. Family level 

reporting/not per proband 

Laiho P, Launonen V, Lahermo P, Esteller M, Guo M, 

Herman JG, et al. Low-level microsatellite instability in 

most colorectal carcinomas. Cancer Res 2002 Feb 

15;62(4):1166-70. hMLH1 and hMSH2 sequencing not 

performed 

Lanza G, Gafa R, Maestri I, Santini A, Matteuzzi M, 

Cavazzini L. Immunohistochemical pattern of 

MLH1/MSH2 expression is related to clinical and 

pathological features in colorectal adenocarcinomas with 

microsatellite instability. Mod Pathol 2002 Jul;15(7):741-

9. hMLH1 and hMSH2 sequencing not performed 

Leach FS, Polyak K, Burrell M, Johnson KA, Hill D, 

Dunlop MG, et al. Expression of the human mismatch 

repair gene hMSH2 in normal and neoplastic tissues. 

Cancer Res 1996 Jan 15;56(2):235-40. Not relevant 

Lecomte T, Cellier C, Meatchi T, Barbier JP, Cugnenc 

PH, Jian R, et al. Chromoendoscopic Colonoscopy for 

Detecting Preneoplastic Lesions in Hereditary 

Nonpolyposis Colorectal Cancer Syndrome. Clin 

Gastroenterol Hepatol 2005;3(9):897-902. Not relevant 

Lee JS, Petrelli NJ, Rodriguez-Bigas MA. Rectal cancer 

in hereditary nonpolyposis colorectal cancer. Am J Surg 

2001 Mar;181(3):207-10. No outcomes of interest 

Lerman C, Hughes C, Trock BJ, Myers RE, Main D, 

Bonney A, et al. Genetic testing in families with 

hereditary nonpolyposis colon cancer. Jama 1999 May 

5;281(17):1618-22. Not relevant 

Leung WK, To KF, Man EP, Chan MW, Bai AH, Hui AJ, 

et al. Quantitative detection of promoter hypermethylation 

in multiple genes in the serum of patients with colorectal 

cancer. Am J Gastroenterol 2005 Oct;100(10):2274-9. Not 

relevant 

Li FP. Translational research on hereditary colon, breast, 

and ovarian cancers. J Natl Cancer Inst Monogr 

1995;(17):1-4. Review 

Liljegren A, Olsson L, Lindblom A. MSI test to 

distinguish between HNPCC and other predisposing 

syndromes -- of value in tailored surveillance. Dis 

Markers 2004;20(4-5):259-67. Review 

Liljestrom B, Aktan-Collan K, Isomaa B, Sarelin L, 

Uutela A, Groop L, et al. Genetic testing for maturity 

onset diabetes of the young: uptake, attitudes and 

comparison with hereditary non-polyposis colorectal 

cancer. Diabetologia 2005 Feb;48(2):242-50. Diabetes 

focus & compared predictive testing to HNPCC 

Lin KM, Shashidharan M, Thorson AG, Ternent CA, 

Blatchford GJ, Christensen MA, et al. Cumulative 

incidence of colorectal and extracolonic cancers in MLH1 

and MSH2 mutation carriers of hereditary nonpolyposis 

colorectal cancer. J Gastrointest Surg 1998 Jan;2(1):67-

71. Not relevant 

Lin X, Choi JH, Lynch P, Xi L, Wu E, Frazier ML. 

Reduction in hMSH2 mRNA levels by premature 

translation termination: implications for mutation 

screening in hereditary nonpolyposis colorectal cancer. 

Dig Dis Sci 1999 Mar;44(3):553-9. Not relevant
D-10

Lindor NM, Burgart LJ, Leontovich O, Goldberg RM, 

Cunningham JM, Sargent DJ, et al. 

Immunohistochemistry versus microsatellite instability 

testing in phenotyping colorectal tumors. J Clin Oncol 

2002;20(4):1043-8. Genetic testing not compared with 

an index test 

Lindor NM, Burgart LJ, Leontovich O, Goldberg RM, 

Cunningham JM, Sargent DJ, et al. 

Immunohistochemistry versus microsatellite instability 

testing in phenotyping colorectal tumors. J Clin Oncol 

2002 Feb 15;20(4):1043-8. Not relevant 

Lindor NM, Greene MH. The concise handbook of family 

cancer syndromes. Mayo Familial Cancer Program. J Natl 

Cancer Inst 1998;90(14):1039-71. Not relevant 

Lindor NM, Rabe K, Petersen GM, Haile R, Casey G, 

Baron J, et al. Lower cancer incidence in Amsterdam-I 

criteria families without mismatch repair deficiency: 

familial colorectal cancer type X. Jama 

2005;293(16):1979-85. Not population of interest, 

hMLH1 and hMSH2 sequencing not performed 

Lipton LR, Johnson V, Cummings C, Fisher S, Risby P, 

Eftekhar Sadat AT, et al. Refining the Amsterdam Criteria 

and Bethesda Guidelines: testing algorithms for the 

prediction of mismatch repair mutation status in the 

familial cancer clinic. J Clin Oncol 2004 Dec 

15;22(24):4934-43. Data not extractable

Liu B, Farrington SM, Petersen GM, Hamilton SR, 

Parsons R, Papadopoulos N, et al. Genetic instability 

occurs in the majority of young patients with colorectal 

cancer. Nat Med 1995 Apr;1(4):348-52. Not population 

of interest 

Liu B, Parsons R, Papadopoulos N, Nicolaides NC, Lynch 

HT, Watson P, et al. Analysis of mismatch repair genes in 

hereditary non-polyposis colorectal cancer patients. Nat 

Med 1996 Feb;2(2):169-74. Not population of interest 

Liu B, Parsons RE, Hamilton SR, Petersen GM, Lynch 

HT, Watson P, et al. hMSH2 mutations in hereditary 

nonpolyposis colorectal cancer kindreds. Cancer Res 1994 

Sep 1;54(17):4590-4. hMLH1 and hMSH2 sequencing 

not performed

Liu HX, Zhou XL, Liu T, Werelius B, Lindmark G, Dahl 

N, et al. The role of hMLH3 in familial colorectal cancer. 

Cancer Res 2003 Apr 15;63(8):1894-9. Not relevant 

Liu T, Tannergard P, Hackman P, Rubio C, Kressner U, 

Lindmark G, et al. Missense mutations in hMLH1 

associated with colorectal cancer. Hum Genet 1999 

Nov;105(5):437-41. Not relevant 

Liu T, Wahlberg S, Burek E, Lindblom P, Rubio C, 

Lindblom A. Microsatellite instability as a predictor of a 

mutation in a DNA mismatch repair gene in familial 

colorectal cancer. Genes Chromosomes Cancer 2000 

Jan;27(1):17-25. Data not extractable 

Liu T, Wahlberg S, Rubio C, Holmberg E, Gronberg H, 

Lindblom A. DGGE screening of mutations in mismatch 

repair genes (hMSH2 and hMLH1) in 34 Swedish families 

with colorectal cancer. Clin Genet 1998 Feb;53(2):131-5. 

Data not extractable 

Liu WH, Kaur M, Wang G, Zhu P, Zhang Y, 

Makrigiorgos GM. Inverse PCR-based RFLP scanning 

identifies low-level mutation signatures in colon cells and 

tumors. Cancer Res 2004 Apr 1;64(7):2544-51. Not 

relevant 

Llor X, Pons E, Xicola RM, Castells A, Alenda C, Pinol 

V, et al. Differential features of colorectal cancers 

fulfilling Amsterdam criteria without involvement of the 

mutator pathway. Clin Cancer Res 2005;11(20):7304-10. 

Genetic testing not compared with an index test; data 

not extractable 

Lorenzo BJ, Buchner FL, Hemminki K. Familial risk of 

endometrial cancer after exclusion of families that 

fulfilled Amsterdam, Japanese or Bethesda criteria for 

HNPCC. Ann Oncol 2004 Apr;15(4):598-604. Not 

relevant 

Losi L, Di Gregorio C, Pedroni M, Ponti G, Roncucci L, 

Scarselli A, et al. Molecular genetic alterations and 

clinical features in early-onset colorectal carcinomas and 

their role for the recognition of hereditary cancer 

syndromes. Am J Gastroenterol 2005 Oct;100(10):2280-7. 

Not all tested 

Losi L, Fante R, Di Gregorio C, Aisoni ML, Lanza G, 

Maestri I, et al. Biologic characterization of hereditary 

non-polyposis colorectal cancer. Nuclear ploidy, AgNOR 

count, microvessel distribution, oncogene expression, and 

grade-related parameters. Am J Clin Pathol 1995 

Mar;103(3):265-70. Not relevant 

Loukola A, de la CA, Aaltonen LA. Strategies for 

screening for hereditary non-polyposis colorectal cancer. J 

Med Genet 1999 Nov;36(11):819-22. Data not 

extractable 
D-11

Loukola A, Eklin K, Laiho P, Salovaara R, Kristo P, 

Jarvinen H, et al. Microsatellite marker analysis in 

screening for hereditary nonpolyposis colorectal cancer 

(HNPCC). Cancer Res 2001 Jun 1;61(11):4545-9. Data 

not extractable 

Loukola A, Salovaara R, Kristo P, Moisio AL, Kaariainen 

H, Ahtola H, et al. Microsatellite instability in adenomas 

as a marker for hereditary nonpolyposis colorectal cancer. 

Am J Pathol 1999 Dec;155(6):1849-53. Not population 

of interest; hMLH1 and hMSH2 sequencing not 

performed 

Lu HK, Broaddus RR. Gynecologic Cancers in Lynch 

Syndrome/HNPCC. Fam Cancer 2005;4(3):249-54. 

Review 

Lu KH, Dinh M, Kohlmann W, Watson P, Green J, 

Syngal S, et al. Gynecologic cancer as a "sentinel cancer" 

for women with hereditary nonpolyposis colorectal cancer 

syndrome. Obstet Gynecol 2005 Mar;105(3):569-74. Not 

relevant 

Lu KH, Dinh M, Kohlmann W, Watson P, Green J, 

Syngal S, et al. Gynecologic cancer as a "sentinel cancer" 

for women with hereditary nonpolyposis colorectal cancer 

syndrome. Obstet Gynecol 2005;105(3):569-74. Not 

relevant 

Lucci-Cordisco E, Rovella V, Carrara S, Percesepe A, 

Pedroni M, Bellacosa A, et al. Mutations of the 'minor' 

mismatch repair gene MSH6 in typical and atypical 

hereditary nonpolyposis colorectal cancer. Fam Cancer 

2001;1(2):93-9. hMLH1 and hMSH2 sequencing not 

performed 

Lucci-Cordisco E, Zollino M, Baglioni S, Mancuso I, 

Lecce R, Gurrieri F, et al. A novel microdeletion 

syndrome with loss of the MSH2 locus and hereditary 

non-polyposis colorectal cancer. Clin Genet 2005 

Feb;67(2):178-82. Case report 

Luce MC, Binnie CG, Cayouette MC, Kam-Morgan LN. 

Identification of DNA mismatch repair gene mutations in 

hereditary nonpolyposis colon cancer patients. Int J 

Cancer 1996 Feb 20;69(1):50-2. Review 

Luo DC, Cai Q, Sun MH, Ni YZ, Ni SC, Chen ZJ, et al. 

Clinicopathological and molecular genetic analysis of 

HNPCC in China. World J Gastroenterol 2005 Mar 

21;11(11):1673-9. Not population of interest 

Lynch HT, Casey MJ, Lynch J, White TE, Godwin AK. 

Genetics and ovarian carcinoma. Semin Oncol 1998 

Jun;25(3):265-80. Review 

Lynch HT, Coronel SM, Okimoto R, Hampel H, Sweet K, 

Lynch JF, et al. A founder mutation of the MSH2 gene 

and hereditary nonpolyposis colorectal cancer in the 

United States. Jama 2004 Feb 11;291(6):718-24. Not 

population of interest; hMLH1 and hMSH2 

sequencing not performed 

Lynch HT, Kimberling W, Albano WA, Lynch JF, 

Biscone K, Schuelke GS, et al. Hereditary nonpolyposis 

colorectal cancer (Lynch syndromes I and II). I. Clinical 

description of resource. Cancer 1985 Aug 15;56(4):934-8. 

Case report 

Lynch HT, Riley BD, Weissman SM, Coronel SM, 

Kinarsky Y, Lynch JF, et al. Hereditary nonpolyposis 

colorectal carcinoma (HNPCC) and HNPCC-like families: 

Problems in diagnosis, surveillance, and management. 

Cancer 2004;100(1):53-64. Case series 

Lynch HT, Schuelke GS, Kimberling WJ, Albano WA, 

Lynch JF, Biscone KA, et al. Hereditary nonpolyposis 

colorectal cancer (Lynch syndromes I and II). II. 

Biomarker studies. Cancer 1985 Aug 15;56(4):939-51. 

Not relevant 

Lynch HT, Shaw MW, Magnuson CW, Larsen AL, Krush 

AJ. Hereditary factors in cancer. Study of two large 

midwestern kindreds. Arch Intern Med 1966;117(2):206-

12. Case series 

Lynch J, Lynch HT. Genetic counseling and HNPCC. 

Anticancer Res 1994 Jul;14(4B):1651-6. Review 

Machin P, Catasus L, Pons C, Munoz J, Conde-Zurita JM, 

Balmana J, et al. Microsatellite instability and 

immunostaining for MSH-2 and MLH-1 in cutaneous and 

internal tumors from patients with the Muir-Torre 

syndrome. J Cutan Pathol 2002 Aug;29(7):415-20. Not 

population of interest 

Maekawa M, Sugano K, Kashiwabara H, Ushiama M, 

Fujita S, Yoshimori M, et al. DNA methylation analysis 

using bisulfite treatment and PCR-single-strand 

conformation polymorphism in colorectal cancer showing 

microsatellite instability. Biochem Biophys Res Commun 

1999 Sep 7;262(3):671-6. Biological material not 

evaluated 

Maekawa M, Sugano K, Ushiama M, Fukayama N, 

Nomoto K, Kashiwabara H, et al. Heterogeneity of DNA 

methylation status analyzed by bisulfite-PCR-SSCP and 

correlation with clinico-pathological characteristics in 

colorectal cancer. Clin Chem Lab Med 2001 

Feb;39(2):121-8. Genetic testing not compared with 

index test, hMLH1 and hMSH2 sequencing not 

performed 
D-12

Maier S, Dahlstroem C, Haefliger C, Plum A, 

Piepenbrock C. Identifying DNA methylation biomarkers 

of cancer drug response. Am J Pharmacogenomics 

2005;5(4):223-32. Review 

Maillet P, Chappuis PO, Vaudan G, Dobbie Z, Muller H, 

Hutter P, et al. A polymorphism in the ATM gene 

modulates the penetrance of hereditary non-polyposis 

colorectal cancer. Int J Cancer 2000 Dec 15;88(6):928-31. 

Not population of interest 

Maliaka YK, Chudina AP, Belev NF, Alday P, Bochkov 

NP, Buerstedde JM. CpG dinucleotides in the hMSH2 and 

hMLH1 genes are hotspots for HNPCC mutations. Hum 

Genet 1996 Feb;97(2):251-5. Genetic testing not 

compared with index test 

Manavis J, Gilham P, Davies R, Ruszkiewicz A. The 

immunohistochemical detection of mismatch repair gene 

proteins (MLH1, MSH2, MSH6, and PMS2): practical 

aspects in antigen retrieval and biotin blocking protocols. 

Appl Immunohistochem Mol Morphol 2003 

Mar;11(1):73-7. Biological material not evaluated 

Mangold E, Pagenstecher C, Friedl W, Fischer HP, 

Merkelbach-Bruse S, Ohlendorf M, et al. Tumours from 

MSH2 mutation carriers show loss of MSH2 expression 

but many tumours from MLH1 mutation carriers exhibit 

weak positive MLH1 staining. J Pathol 2005 

Dec;207(4):385-95. Didn't report any of 5 options 

Mangold E, Pagenstecher C, Friedl W, Mathiak M, 

Buettner R, Engel C, et al. Spectrum and frequencies of 

mutations in MSH2 and MLH1 identified in 1,721 

German families suspected of hereditary nonpolyposis 

colorectal cancer. Int J Cancer 2005 Sep 20;116(5):692-

702. Not population of interest 

Marcus VA, Madlensky L, Gryfe R, Kim H, So K, Millar 

A, et al. Immunohistochemistry for hMLH1 and hMSH2: 

a practical test for DNA mismatch repair-deficient tumors. 

Am J Surg Pathol 1999 Oct;23(10):1248-55. Not 

population of interest 

Maruyama A, Miyamoto S, Saito T, Kondo H, Baba H, 

Tsukamoto N. Clinicopathologic and familial 

characteristics of endometrial carcinoma with multiple 

primary carcinomas in relation to the loss of protein 

expression of MSH2 and MLH1. Cancer 2001 Jun 

1;91(11):2056-64. Not population of interest 

Maruyama A, Saito T, Hachitanda Y, Tsukamoto N. 

Cancer history and loss of MSH2 and MLH1 protein 

expression in patients with endometrial hyperplasia. Int J 

Gynecol Cancer 2003 May;13(3):352-60. Not population 

of interest 

Mathiak M, Rutten A, Mangold E, Fischer HP, Ruzicka T, 

Friedl W, et al. Loss of DNA mismatch repair proteins in 

skin tumors from patients with Muir-Torre syndrome and 

MSH2 or MLH1 germline mutations: establishment of 

immunohistochemical analysis as a screening test. Am J 

Surg Pathol 2002 Mar;26(3):338-43. Not population of 

interest 

Matloff ET, Shappell H, Brierley K, Bernhardt BA, 

McKinnon W, Peshkin BN. What would you do? 

Specialists' perspectives on cancer genetic testing, 

prophylactic surgery, and insurance discrimination. J Clin 

Oncol 2000 Jun;18(12):2484-92. Not relevant-survey of 

genetic counselor's attitudes & prophylactic surgery 

Matsubara N, Yoshitaka T, Matsuno T, Ikeda M, Isozaki 

H, Tanaka N, et al. Multiple tumors and a novel E2F-4 

mutation. A case report. Digestion 2000;62(2-3):213-6. 

Case report 

Mauillon JL, Michel P, Limacher JM, Latouche JB, 

Dechelotte P, Charbonnier F, et al. Identification of novel 

germline hMLH1 mutations including a 22 kb Alumediated deletion in patients with familial colorectal 

cancer. Cancer Res 1996 Dec 15;56(24):5728-33. 

hMLH1 and hMSH2 sequencing not performed 

Mayer F, Gillis AJ, Dinjens W, Oosterhuis JW, 

Bokemeyer C, Looijenga LH. Microsatellite instability of 

germ cell tumors is associated with resistance to systemic 

treatment. Cancer Res 2002 May 15;62(10):2758-60. Not 

population of interest 

McAllister M. Personal theories of inheritance, coping 

strategies, risk perception and engagement in hereditary 

non-polyposis colon cancer families offered genetic 

testing. Clin Genet 2003 Sep;64(3):179-89. Not relevant - 

methods paper 

McDaniel SH. The psychotherapy of genetics. Fam 

Process 2005 Mar;44(1):25-44. Not relevant - 

background material 

McGrath SB, Bounpheng M, Torres L, Calavetta M, Scott 

CB, Suh Y, et al. High-speed, multicolor fluorescent twodimensional gene scanning. Genomics 2001 Nov;78(1-

2):83-90. Biological material not evaluated 

McGregor DK, Wu TT, Rashid A, Luthra R, Hamilton 

SR. Reduced expression of cytokeratin 20 in colorectal 

carcinomas with high levels of microsatellite instability. 

Am J Surg Pathol 2004 Jun;28(6):712-8. Didn't report 

any of 5 options 
D-13

Mecklin JP, Jarvinen HJ, Aukee S, Elomaa I, Karjalainen 

K. Screening for colorectal carcinoma in cancer family 

syndrome kindreds. Scand J Gastroenterol 1987 

May;22(4):449-53. Not relevant 

Mecklin JP, Jarvinen HJ. Tumor spectrum in cancer 

family syndrome (hereditary nonpolyposis colorectal 

cancer). Cancer 1991;68(5):1109-12. Not relevant 

Mecklin JP. Frequency of hereditary colorectal carcinoma. 

Gastroenterology 1987;93(5):1021-5. Not relevant 

Meiser B. Psychological impact of genetic testing for 

cancer susceptibility: an update of the literature. PsychoOncology 2005 Dec;14(12):1060-74. Review 

Menigatti M, Di Gregorio C, Borghi F, Sala E, Scarselli 

A, Pedroni M, et al. Methylation pattern of different 

regions of the MLH1 promoter and silencing of gene 

expression in hereditary and sporadic colorectal cancer. 

Genes Chromosomes Cancer 2001 Aug;31(4):357-61. Not 

relevant 

Michalopoulos NV, Saetta AA, Lazaris AC, Gigelou F, 

Koilakou S, Patsouris E. Microsatellite instability in 

sporadic and inherited colon adenocarcinomas from Greek 

patients: correlation with several clinicopathological 

characteristics. Acta Gastroenterol Belg 2005 

Jul;68(3):294-301. hMLH1 and hMSH2 sequencing not 

performed 

Millar AL, Pal T, Madlensky L, Sherman C, Temple L, 

Mitri A, et al. Mismatch repair gene defects contribute to 

the genetic basis of double primary cancers of the 

colorectum and endometrium. Hum Mol Genet 1999 

May;8(5):823-9. Data not extractable 

Miller M. Colorectal cancer: tackling the genetic aspects 

of the disease. J Natl Cancer Inst 1998 Jul 

15;90(14):1034-5. Not primary study 

Mitchell RJ, Farrington SM, Dunlop MG, Campbell H. 

Mismatch repair genes hMLH1 and hMSH2 and 

colorectal cancer: a HuGE review. Am J Epidemiol 2002 

Nov 15;156(10):885-902. Review 

Miyaki M, Iijima T, Yamaguchi T, Shirahama S, Ito T, 

Yasuno M, et al. Novel germline hMSH2 genomic 

deletion and somatic hMSH2 mutations in a hereditary 

nonpolyposis colorectal cancer family. Mutat Res 2004 

Apr 14;548(1-2):19-25. Not population of interest; 

hMLH1 and hMSH2 sequencing not performed 

Miyaki M, Konishi M, Muraoka M, Kikuchi-Yanoshita R, 

Tanaka K, Iwama T, et al. Germ line mutations of hMSH2 

and hMLH1 genes in Japanese families with hereditary 

nonpolyposis colorectal cancer (HNPCC): usefulness of 

DNA analysis for screening and diagnosis of HNPCC 

patients. J Mol Med 1995 Oct;73(10):515-20. Not 

population of interest 

Mori Y, Yin J, Sato F, Sterian A, Simms LA, Selaru FM, 

et al. Identification of genes uniquely involved in frequent 

microsatellite instability colon carcinogenesis by 

expression profiling combined with epigenetic scanning. 

Cancer Res 2004 Apr 1;64(7):2434-8. Biological 

material not evaluated 

Moriyama H, Sasamoto H, Kambara T, Matsubara N, 

Ikeda M, Baba S, et al. E2F-4 mutation in hereditary nonpolyposis colorectal cancer. J Exp Clin Cancer Res 2002 

Jun;21(2):185-9. hMLH1 and hMSH2 sequencing not 

performed 

Moslein G, Pistorius S, Saeger HD, Schackert HK. 

Preventive surgery for colon cancer in familial 

adenomatous polyposis and hereditary nonpolyposis 

colorectal cancer syndrome. Langenbecks Arch Surg 2003 

Mar;388(1):9-16. Review 

Moslein G. Clinical implications of molecular diagnosis in 

hereditary nonpolyposis colorectal cancer. Recent Results 

Cancer Res 2003;162:73-8. Not primary study 

Muller A, Edmonston TB, Dietmaier W, Buttner R, Fishel 

R, Ruschoff J. MSI-testing in hereditary non-polyposis 

colorectal carcinoma (HNPCC). Dis Markers 2004;20(4-

5):225-36. Review 

Muller A, Giuffre G, Edmonston TB, Mathiak M, 

Roggendorf B, Heinmoller E, et al. Challenges and pitfalls 

in HNPCC screening by microsatellite analysis and 

immunohistochemistry. J Mol Diagn 2004 Nov;6(4):308-

15. Case report 

Muller A, Schackert HK, Lange B, Ruschoff J, Fuzesi L, 

Willert J, et al. A novel MSH2 germline mutation in 

homozygous state in two brothers with colorectal cancers 

diagnosed at the age of 11 and 12 years. American Journal 

of Medical Genetics 2006 Feb 1;Part(3):195-9. Case 

report 
D-14

Muller-Koch Y, Kopp R, Lohse P, Baretton G, Stoetzer A, 

Aust D, et al. Sixteen rare sequence variants of the 

hMLH1 and hMSH2 genes found in a cohort of 254 

suspected HNPCC (hereditary non-polyposis colorectal 

cancer) patients: mutations or polymorphisms? Eur J Med 

Res 2001 Nov 20;6(11):473-82. Genetic testing not 

compared with index test, hMLH1 and hMSH2 

sequencing not performed 

Murff HJ, Spigel DR, Syngal S. Does this patient have a 

family history of cancer? An evidence-based analysis of 

the accuracy of family cancer history. Jama 

2004;292(12):1480-9. Not relevant 

Muta H, Noguchi M, Perucho M, Ushio K, Sugihara K, 

Ochiai A, et al. Clinical implications of microsatellite 

instability in colorectal cancers. Cancer 1996 Jan 

15;77(2):265-70. Genetic testing not compared with 

index test 

Nakagawa H, Hampel H, de la CA. Identification and 

characterization of genomic rearrangements of MSH2 and 

MLH1 in Lynch syndrome (HNPCC) by novel techniques. 

Hum Mutat 2003 Sep;22(3):258. Biological material not 

evaluated 

Nakagawa H, Lockman JC, Frankel WL, Hampel H, 

Steenblock K, Burgart LJ, et al. Mismatch repair gene 

PMS2: disease-causing germline mutations are frequent in 

patients whose tumors stain negative for PMS2 protein, 

but paralogous genes obscure mutation detection and 

interpretation. Cancer Res 2004 Jul 15;64(14):4721-7. Not 

relevant 

Nash GM, Gimbel M, Shia J, Culliford AT, Nathanson 

DR, Ndubuisi M, et al. Automated, multiplex assay for 

high-frequency microsatellite instability in colorectal 

cancer. J Clin Oncol 2003 Aug 15;21(16):3105-12. Not 

relevant 

Nilbert M, Planck M, Fernebro E, Borg A, Johnson A. 

Microsatellite instability is rare in rectal carcinomas and 

signifies hereditary cancer. Eur J Cancer 1999 

Jun;35(6):942-5. Data not extractable 

Nomura S, Sugano K, Kashiwabara H, Taniguchi T, 

Fukayama N, Fujita S, et al. Enhanced detection of 

deleterious and other germline mutations of hMSH2 and 

hMLH1 in Japanese hereditary nonpolyposis colorectal 

cancer kindreds. Biochem Biophys Res Commun 2000 

Apr 29;271(1):120-9. Not relevant 

Nystrom-Lahti M, Kristo P, Nicolaides NC, Chang SY, 

Aaltonen LA, Moisio AL, et al. Founding mutations and 

Alu-mediated recombination in hereditary colon cancer. 

Nat Med 1995 Nov;1(11):1203-6. Data not extractable 

Nystrom-Lahti M, Wu Y, Moisio AL, Hofstra RM, 

Osinga J, Mecklin JP, et al. DNA mismatch repair gene 

mutations in 55 kindreds with verified or putative 

hereditary non-polyposis colorectal cancer. Hum Mol 

Genet 1996 Jun;5(6):763-9. Not population of interest 

Oliveira C, Westra JL, Arango D, Ollikainen M, Domingo 

E, Ferreira A, et al. Distinct patterns of KRAS mutations 

in colorectal carcinomas according to germline mismatch 

repair defects and hMLH1 methylation status. Hum Mol 

Genet 2004 Oct 1;13(19):2303-11. Not population of 

interest 

Ollikainen M, Abdel-Rahman WM, Moisio AL, Lindroos 

A, Kariola R, Jarvela I, et al. Molecular analysis of 

familial endometrial carcinoma: a manifestation of 

hereditary nonpolyposis colorectal cancer or a separate 

syndrome? J Clin Oncol 2005 Jul 20;23(21):4609-16. Not 

population of interest 

Otway R, Tetlow N, Hornby J, Kohonen-Corish M. 

Evaluation of enzymatic mutation detectiontrade mark in 

hereditary nonpolyposis colorectal cancer. Hum Mutat 

2000;16(1):61-7. Not relevant 

Palmer CG, Hadley DW. Evaluating the impact of genetic 

counseling and testing with signal detection methods. J 

Genet Couns 2005 Feb;14(1):17-27. Not relevant - 

methods paper 

Palmirotta R, Matera S, Curia MC, Aceto G, el Zhobi B, 

Verginelli F, et al. Correlations between phenotype and 

microsatellite instability in HNPCC: implications for 

genetic testing. Fam Cancer 2004;3(2):117-21. Genetic 

testing not compared with index test 

Palmirotta R, Veri MC, Curia MC, Aceto G, D'Amico F, 

Esposito DL, et al. Transcripts with splicings of exons 15 

and 16 of the hMLH1 gene in normal lymphocytes: 

implications in RNA-based mutation screening of 

hereditary non-polyposis colorectal cancer. Eur J Cancer 

1998 May;34(6):927-30. Not relevant 

Pan KF, Liu W, Lu YY, Zhang L, Li ZP, Lu WL, et al. 

High throughput detection of microsatellite instability by 

denaturing high-performance liquid chromatography. 

Hum Mutat 2003 Nov;22(5):388-94. Not relevant 

Paraf F, Gilquin M, Longy M, Gilbert B, Gorry P, Petit B, 

et al. MLH1 and MSH2 protein immunohistochemistry is 

useful for detection of hereditary non-polyposis colorectal 

cancer in young patients. Histopathology 2001 

Sep;39(3):250-8. Not relevant 
D-15

Parc YR, Halling KC, Burgart LJ, McDonnell SK, Schaid 

DJ, Thibodeau SN, et al. Microsatellite instability and 

hMLH1/hMSH2 expression in young endometrial 

carcinoma patients: associations with family history and 

histopathology. Int J Cancer 2000 Apr 1;86(1):60-6. Not 

population of interest 

Park JG, Park YJ, Wijnen JT, Vasen HF. Geneenvironment interaction in hereditary nonpolyposis 

colorectal cancer with implications for diagnosis and 

genetic testing. Int J Cancer 1999 Aug 12;82(4):516-9. 

hMLH1 and hMSH2 sequencing not performed 

Park JG, Vasen HF, Park YJ, Park KJ, Peltomaki P, de 

Leon MP, et al. Suspected HNPCC and Amsterdam 

criteria II: evaluation of mutation detection rate, an 

international collaborative study. Int J Colorectal Dis 2002 

Mar;17(2):109-14. Not population of interest 

Patten SJ, Wallace AJ, Elles R. Benchmark for evaluating 

the quality of DNA sequencing: proposal from an 

International External Quality Assessment Scheme. 

Clinical Chemistry 52[4], 728-736. 2006. Biological 

material not evaluated 

Peltomaki P, Vasen H. Mutations associated with HNPCC 

predisposition -- Update of ICG-HNPCC/INSiGHT 

mutation database. Dis Markers 2004;20(4-5):269-76. 

Review

Peltomaki P, Vasen HF. Mutations predisposing to 

hereditary nonpolyposis colorectal cancer: database and 

results of a collaborative study. The International 

Collaborative Group on Hereditary Nonpolyposis 

Colorectal Cancer. Gastroenterology 1997 

Oct;113(4):1146-58. Genetic testing not compared with 

index test 

Pensotti V, Radice P, Presciuttini S, Calistri D, Gazzoli I, 

Grimalt PA, et al. Mean age of tumor onset in hereditary 

nonpolyposis colorectal cancer (HNPCC) families 

correlates with the presence of mutations in DNA 

mismatch repair genes. Genes Chromosomes Cancer 1997 

Jul;19(3):135-42. Genetic testing not compared with 

index test 

Pentz RD, Peterson SK, Watts B, Vernon SW, Lynch PM, 

Koehly LM, et al. Hereditary Nonpolyposis Colorectal 

Cancer Family Members' Perceptions about the Duty to 

Inform and Health Professionals' Role in Disseminating 

Genetic Information. Genet Test 2005;9(3):261-8. Not 

relevant 

Percesepe A, Borghi F, Menigatti M, Losi L, Foroni M, 

Di Gregorio C, et al. Molecular screening for hereditary 

nonpolyposis colorectal cancer: a prospective, populationbased study. J Clin Oncol 2001 Oct 1;19(19):3944-50. 

hMLH1 and hMSH2 sequencing not performed 

Percesepe A, Kristo P, Aaltonen LA, Ponz dL, de la CA, 

Peltomaki P. Mismatch repair genes and mononucleotide 

tracts as mutation targets in colorectal tumors with 

different degrees of microsatellite instability. Oncogene 

1998 Jul 16;17(2):157-63. Data not extractable 

Peterlongo P, Nafa K, Lerman GS, Glogowski E, Shia J, 

Ye TZ, et al. MSH6 germline mutations are rare in 

colorectal cancer families. Int J Cancer 2003 Nov 

20;107(4):571-9. hMLH1 and hMSH2 sequencing not 

performed, Data not extractable 

Pistorius SR, Nagel M, Kruger S, Plaschke J, Kruppa C, 

Wehrmann U, et al. Combined molecular and clinical 

approach for decision making for surgery in HNPCC 

patients: a report on three cases in two families. Int J 

Colorectal Dis 2001 Nov;16(6):402-7. Case report 

Planck M, Koul A, Fernebro E, Borg A, Kristoffersson U, 

Olsson H, et al. hMLH1, hMSH2 and hMSH6 mutations 

in hereditary non-polyposis colorectal cancer families 

from southern Sweden. Int J Cancer 1999 Oct 

8;83(2):197-202. Data not extractable 

Planck M, Rambech E, Moslein G, Muller W, Olsson H, 

Nilbert M. High frequency of microsatellite instability and 

loss of mismatch-repair protein expression in patients with 

double primary tumors of the endometrium and 

colorectum. Cancer 2002 May 1;94(9):2502-10. Data not 

extractable 

Plaschke J, Kruger S, Jeske B, Theissig F, Kreuz FR, 

Pistorius S, et al. Loss of MSH3 protein expression is 

frequent in MLH1-deficient colorectal cancer and is 

associated with disease progression. Cancer Res 2004 Feb 

1;64(3):864-70. Genetic testing not compared with 

index test 

Plaschke J, Kruger S, Pistorius S, Theissig F, Saeger HD, 

Schackert HK. Involvement of hMSH6 in the 

development of hereditary and sporadic colorectal cancer 

revealed by immunostaining is based on germline 

mutations, but rarely on somatic inactivation. Int J Cancer 

2002 Feb 10;97(5):643-8. Genetic testing not compared 

with index test 
D-16

Plaschke J, Kruppa C, Tischler R, Bocker T, Pistorius S, 

Dralle H, et al. Sequence analysis of the mismatch repair 

gene hMSH6 in the germline of patients with familial and 

sporadic colorectal cancer. Int J Cancer 2000 Mar 

1;85(5):606-13. hMLH1 and hMSH2 sequencing not 

performed 

Plaschke J, Ruschoff J, Schackert HK. Genomic 

rearrangements of hMSH6 contribute to the genetic 

predisposition in suspected hereditary non-polyposis 

colorectal cancer syndrome. J Med Genet 2003 

Aug;40(8):597-600. hMLH1 and hMSH2 sequencing 

not performed 

Plevova P, Krepelova A, Papezova M, Sedlakova E, Curik 

R, Foretova L, et al. Immunohistochemical detection of 

the hMLH1 and hMSH2 proteins in hereditary nonpolyposis colon cancer and sporadic colon cancer. 

Neoplasma 2004;51(4):275-84. Not population of 

interest 

Ponz dL, Benatti P, Borghi F, Pedroni M, Scarselli A, Di 

Gregorio C, et al. Aetiology of colorectal cancer and 

relevance of monogenic inheritance. Gut 2004 

Jan;53(1):115-22. Patients reported in Ponz 1999, data 

not extractable 

Ponz dL, Benatti P, Percesepe A, Rossi G, Viel A, 

Santarosa M, et al. Clinical and molecular diagnosis of 

hereditary non-polyposis colorectal cancer: problems and 

pitfalls in an extended pedigree. Ital J Gastroenterol 

Hepatol 1999 Aug;31(6):476-80. Genetic testing not 

compared with index test 

Popat S, Hubner R, Houlston RS. Systematic review of 

microsatellite instability and colorectal cancer prognosis. J 

Clin Oncol 2005;23(3):609-18. Review 

Potocnik U, Glavac D, Golouh R, Ravnik-Glavac M. 

Causes of microsatellite instability in colorectal tumors: 

implications for hereditary non-polyposis colorectal 

cancer screening. Cancer Genet Cytogenet 2001 Apr 

15;126(2):85-96. Data not extractable 

Pucciarelli S, Agostini M, Viel A, Bertorelle R, Russo V, 

Toppan P, et al. Early-age-at-onset colorectal cancer and 

microsatellite instability as markers of hereditary 

nonpolyposis colorectal cancer. Dis Colon Rectum 2003 

Mar;46(3):305-12. Data not extractable 

Quaresima B, Grandinetti C, Baudi F, Tassone P, Barbieri 

V, Conforti S, et al. Hereditary nonpolyposis coloretal 

cancer: identification of novel germline mutations in two 

kindreds not fulfulling the Amsterdam criteria. Mutations 

in brief no. 203. Online. Hum Mutat 1998;12(6):433. 

Meeting abstract 

Raedle J, Brieger A, Trojan J, Hardt T, Herrmann G, 

Zeuzem S. Evaluation of rapid microsatellite analysis of 

paraffin-embedded specimens in screening for hereditary 

nonpolyposis colorectal cancer. Mod Pathol 1999 

May;12(5):485-91. Not relevant 

Raedle J, Schaffner M, Esser N, Sahm S, Trojan J, 

Kriener S, et al. Frequency of the Amsterdam criteria in a 

regional German cohort of patients with colorectal cancer. 

Z Gastroenterol 2002 Aug;40(8):561-8. Patients reported 

in Raedle 2001 

Raevaara TE, Korhonen MK, Lohi H, Hampel H, Lynch 

E, Lonnqvist KE, et al. Functional significance and 

clinical phenotype of nontruncating mismatch repair 

variants of MLH1. Gastroenterology 2005;129(2):537-49. 

Not population of interest, genetic testing not 

compared with an index test 

Rajkumar T, Soumittra N, Pandey D, Nancy KN, Mahajan 

V, Majhi U. Mutation analysis of hMSH2 and hMLH1 in 

colorectal cancer patients in India. Genet Test 

2004;8(2):157-62. Data not extractable 

Ramsey SD, Burke W, Clarke L. An economic viewpoint 

on alternative strategies for identifying persons with 

hereditary nonpolyposis colorectal cancer. Genet Med 

2003 Sep;5(5):353-63. Not relevant 

Ramsey SD, Clarke L, Etzioni R, Higashi M, Berry K, 

Urban N. Cost-effectiveness of microsatellite instability 

screening as a method for detecting hereditary 

nonpolyposis colorectal cancer. Ann Intern Med 

2001;135(8 Pt 1):577-88. Not relevant

Ramsey SD, Clarke L, Etzioni R, Higashi M, Berry K, 

Urban N. Cost-effectiveness of microsatellite instability 

screening as a method for detecting hereditary 

nonpolyposis colorectal cancer. Ann Intern Med 2001 Oct 

16;135(8 Pt 1):577-88. Not relevant - Cost-effectiveness 

study 

Ravnik-Glavac M, Potocnik U, Glavac D. Incidence of 

germline hMLH1 and hMSH2 mutations (HNPCC 

patients) among newly diagnosed colorectal cancers in a 

Slovenian population. Journal of Medical Genetics 2000 

Jul;37(7):533-6. Genetic testing not compared with 

index test 

Renkonen E, Zhang Y, Lohi H, Salovaara R, AbdelRahman WM, Nilbert M, et al. Altered expression of 

MLH1, MSH2, and MSH6 in predisposition to hereditary 

nonpolyposis colorectal cancer. J Clin Oncol 

2003;21(19):3629-37. Not population of interest, 

genetic testing not compared with an index test 
D-17

Reyes CM, Allen BA, Terdiman JP, Wilson LS. 

Comparison of selection strategies for genetic testing of 

patients with hereditary nonpolyposis colorectal 

carcinoma: effectiveness and cost-effectiveness. Cancer 

2002 Nov 1;95(9):1848-56. Comparison of selection 

Ribic CM, Sargent DJ, Moore MJ, Thibodeau SN, French 

AJ, Goldberg RM, et al. Tumor microsatellite-instability 

status as a predictor of benefit from fluorouracil-based 

adjuvant chemotherapy for colon cancer.[see comment]. 

New England Journal of Medicine 2003 Jul 

17;349(3):247-57. Population not well defined 

Riegler G, Savastano A, Selvaggi F, Ciociano R, Martino 

R, Riccio G, et al. Prevalence of HNPCC in a series of 

consecutive patients on the first endoscopic diagnosis of 

colorectal cancer: a multicenter study. The Italian 

Collaborative Group. Endoscopy 1999 Jun;31(5):337-41.

Not relevant 

Rigau V, Sebbagh N, Olschwang S, Paraf F, Mourra N, 

Parc Y, et al. Microsatellite instability in colorectal 

carcinoma. The comparison of immunohistochemistry and 

molecular biology suggests a role for hMSH6 [correction 

of hMLH6] immunostaining. Arch Pathol Lab Med 2003 

Jun;127(6):694-700. hMLH1 and hMSH2 sequencing 

not performed 

Rijcken FE, Mourits MJ, Kleibeuker JH, Hollema H, van 

der Zee AG. Gynecologic screening in hereditary 

nonpolyposis colorectal cancer. Gynecol Oncol 

2003;91(1):74-80. Not relevant

Ripa RS, Katballe N, Wikman FP, Jager AC, Bernstein I, 

Orntoft T, et al. Presymptomatic diagnosis using a 

deletion of a single codon in families with hereditary nonpolyposis colorectal cancer. Mutat Res 2005 Feb 

15;570(1):89-96. Genetic testing not compared with 

index test 

Rodriguez-Bigas MA, Boland CR, Hamilton SR, Henson 

DE, Jass JR, Khan PM, et al. A National Cancer Institute 

Workshop on Hereditary Nonpolyposis Colorectal Cancer 

Syndrome: meeting highlights and Bethesda guidelines. J 

Natl Cancer Inst 1997;89(23):1758-62. Meeting paper 

Rubin SC, Blackwood MA, Bandera C, Behbakht K, 

Benjamin I, Rebbeck TR, et al. BRCA1, BRCA2, and 

hereditary nonpolyposis colorectal cancer gene mutations 

in an unselected ovarian cancer population: relationship to 

family history and implications for genetic testing. Am J 

Obstet Gynecol 1998 Apr;178(4):670-7. Not population 

of interest 

Ruszkiewicz A, Bennett G, Moore J, Manavis J, Rudzki 

B, Shen L, et al. Correlation of mismatch repair genes 

immunohistochemistry and microsatellite instability status 

in HNPCC-associated tumours. Pathology 2002 

Dec;34(6):541-7. Genetic testing not compared with 

index test 

Salahshor S, Kressner U, Fischer H, Lindmark G, 

Glimelius B, Pahlman L, et al. Microsatellite instability in 

sporadic colorectal cancer is not an independent 

prognostic factor. Br J Cancer 1999 Sep;81(2):190-3. Not 

population of interest 

Salvesen HB, MacDonald N, Ryan A, Iversen OE, Jacobs 

IJ, Akslen LA, et al. Methylation of hMLH1 in a 

population-based series of endometrial carcinomas. Clin 

Cancer Res 2000 Sep;6(9):3607-13. Not relevant 

Sarroca C, Valle AD, Fresco R, Renkonen E, Peltomaki P, 

Lynch H. Frequency of hereditary non-polyposis 

colorectal cancer among Uruguayan patients with 

colorectal cancer. Clin Genet 2005 Jul;68(1):80-7. Data 

not extractable 

Sasaki S, Tokino T, Miyatsu T, Muto T, Nakamura Y. 

Mutational analysis of the hMLH1 gene using an 

automated two-dimensional DNA typing system. Hum 

Mutat 1997;9(2):164-71. Not relevant 

Scapoli C, Ponz De Leon M, Sassatelli R, Benatti P, 

Roncucci L, Collins A, et al. Genetic epidemiology of 

hereditary non-polyposis colorectal cancer syndromes in 

Modena, Italy: results of a complex segregation analysis. 

Ann Hum Genet 1994;58 ( Pt 3):275-95. Not relevant 

Scartozzi M, Bianchi F, Rosati S, Galizia E, Antolini A, 

Loretelli C, et al. Mutations of hMLH1 and hMSH2 in 

patients with suspected hereditary nonpolyposis colorectal 

cancer: correlation with microsatellite instability and 

abnormalities of mismatch repair protein expression. J 

Clin Oncol 2002 Mar 1;20(5):1203-8. Not population of 

interest 

Schiemann U, Muller-Koch Y, Gross M, Daum J, Lohse 

P, Baretton G, et al. Extended microsatellite analysis in 

microsatellite stable, MSH2 and MLH1 mutation-negative 

HNPCC patients: genetic reclassification and correlation 

with clinical features. Digestion 2004;69(3):166-76. Data 

not extractable 

Schiemann U, Muller-Koch Y, Gross M, Glas J, Baretton 

G, Muders M, et al. Detection of occult high graded 

microsatellite instabilities in MMR gene mutation 

negative HNPCC tumors by addition of complementary 

marker analysis. Eur J Med Res 2005 Jan 28;10(1):23-8. 

Population not well defined 
D-18

Schlegel J, Bocker T, Zirngibl H, Hofstadter F, Ruschoff 

J. Detection of microsatellite instability in human 

colorectal carcinomas using a non-radioactive PCR-based 

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Not relevant 

Schulmann K, Brasch FE, Kunstmann E, Engel C, 

Pagenstecher C, Vogelsang H, et al. HNPCC-associated 

small bowel cancer: clinical and molecular characteristics. 

Gastroenterology 2005;128(3):590-9. Not outcomes of 

interest

Schweizer P, Moisio AL, Kuismanen SA, Truninger K, 

Vierumaki R, Salovaara R, et al. Lack of MSH2 and 

MSH6 characterizes endometrial but not colon carcinomas 

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2001 Apr 1;61(7):2813-5. Mixed population 

Shia J, Ellis NA, Paty PB, Nash GM, Qin J, Offit K, et al. 

Value of histopathology in predicting microsatellite 

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Nov;27(11):1407-17. Not relevant 

Shin YK, Heo SC, Shin JH, Hong SH, Ku JL, Yoo BC, et 

al. Germline mutations in MLH1, MSH2 and MSH6 in 

Korean hereditary non-polyposis colorectal cancer 

families. Hum Mutat 2004 Oct;24(4):351. Unclear 

whether genetic testing compared with index test 

Singer G, Kallinowski T, Hartmann A, Dietmaier W, Wild 

PJ, Schraml P, et al. Different types of microsatellite 

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20;112(4):643-6. Not population of interest, genetic test 

not compared to index test 

Sinicrope FA, Lemoine M, Xi L, Lynch PM, Cleary KR, 

Shen Y, et al. Reduced expression of cyclooxygenase 2 

proteins in hereditary nonpolyposis colorectal cancers 

relative to sporadic cancers. Gastroenterology 1999 

Aug;117(2):350-8. Not relevant 

Smyth EF, Sharma A, Sivarajasingham N, Hartley J, 

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Dec;47(12):2086-91. Genetic testing not compared with 

index test 

Soliman PT, Broaddus RR, Schmeler KM, Daniels MS, 

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of interest 

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(Williston Park) 2002 Feb;16(2):161-71. Review 

Stanley AJ, Gaff CL, Aittomaki AK, Fabre LC, Macrae 

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report 

Stone JG, Robertson D, Houlston RS. 

Immunohistochemistry for MSH2 and MHL1: a method 

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extractable 

Stone JG, Tomlinson IP, Houlston RS. Optimising 

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Cancer Lett 2000 Feb 28;149(1-2):15-20. Didn't report 

any of 5 options

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D-19

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D-20

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82. hMLH1 and hMSH2 sequencing not performed; 

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Peltomaki P, Lindblom A. Low frequency of hMSH2 

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Journal of Cancer 1997 Feb 20;74(1):134-7. Family level 

data reported, unsure all pts were CRC 

Wang L, Baudhuin LM, Boardman LA, Steenblock KJ, 

Petersen GM, Halling KC, et al. MYH mutations in 

patients with attenuated and classic polyposis and with 

young-onset colorectal cancer without polyps. 

Gastroenterology 2004 Jul;127(1):9-16. Genetic testing 

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sequencing not performed 

Wang Y, Friedl W, Lamberti C, Jungck M, Mathiak M, 

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cancer: frequent occurrence of large genomic deletions in 

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20;103(5):636-41. Not population of interest, data not 

extractable 

Wang Y, Friedl W, Lamberti C, Nothen MM, Kruse R, 

Propping P. A novel missense mutation in the DNA 

mismatch repair gene hMLH1 present among East Asians 

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index test 
D-21

Wang Y, Friedl W, Lamberti C, Ruelfs C, Kruse R, 

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the same somatic mutation in tumour tissue. Hum Genet 

1997 Sep;100(3-4):362-4. Not population of interest; 

genetic testing not compared with index test 

Wang Y, Friedl W, Sengteller M, Jungck M, Filges I, 

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Mutat 2002 Mar;19(3):279-86. Didn't report any of 5 

options 

Ward R, Meldrum C, Williams R, Mokany E, Scott R, 

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2002 Aug;128(8):403-11. Not population of interest 

Ward RL, Turner J, Williams R, Pekarsky B, Packham D, 

Velickovic M, et al. Routine testing for mismatch repair 

deficiency in sporadic colorectal cancer is justified. J 

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compared with an index test; hMLH1 and hMSH2 

sequencing not performed 

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85. Not relevant 

Weber TK, Chin HM, Rodriguez-Bigas M, Keitz B, 

Gilligan R, O'Malley L, et al. Novel hMLH1 and hMSH2 

germline mutations in African Americans with colorectal 

cancer. Jama 1999 Jun 23;281(24):2316-20. Genetic 

testing not compared with index test 

Weber TK, Conlon W, Petrelli NJ, Rodriguez-Bigas M, 

Keitz B, Pazik J, et al. Genomic DNA-based hMSH2 and 

hMLH1 mutation screening in 32 Eastern United States 

hereditary nonpolyposis colorectal cancer pedigrees. 

Cancer Res 1997 Sep 1;57(17):3798-803. Not population 

of interest 

Weber TK, Conroy J, Keitz B, Rodriguez-Bigas M, 

Petrelli NJ, Stoler DL, et al. Genome-wide allelotyping 

indicates increased loss of heterozygosity on 9p and 14q 

in early age of onset colorectal cancer. Cytogenet Cell 

Genet 1999;86(2):142-7. Genetic testing not compared 

with index test 

Wei SC, Shun CT, Tsai-Wu JJ, Wu CH, Sheu JC, Wang 

CY, et al. Microsatellite instability and hMLH1 and 

hMSH2 gene expression in Taiwanese hereditary 

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Wei SC, Yu CY, Tsai-Wu JJ, Su YN, Sheu JC, Wu CH, et 

al. Low mutation rate of hMSH2 and hMLH1 in 

Taiwanese hereditary non-polyposis colorectal cancer. 

Clin Genet 2003 Sep;64(3):243-51. Not population of 

interest 

Whelan AJ, Babb S, Mutch DG, Rader J, Herzog TJ, 

Todd C, et al. MSI in endometrial carcinoma: absence of 

MLH1 promoter methylation is associated with increased 

familial risk for cancers. Int J Cancer 2002 Jun 

10;99(5):697-704. Not population of interest 

Whitehall VL, Wynter CV, Walsh MD, Simms LA, 

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heterogeneity within nonmicrosatellite instability-high 

colorectal cancer. Cancer Res 2002 Nov 1;62(21):6011-4. 

Genetic testing not compared with index test, hMLH1 

and hMSH2 sequencing not performed 

Whiteside D, McLeod R, Graham G, Steckley JL, Booth 

K, Somerville MJ, et al. A homozygous germ-line 

mutation in the human MSH2 gene predisposes to 

hematological malignancy and multiple cafe-au-lait spots. 

Cancer Res 2002 Jan 15;62(2):359-62. Not population of 

interest 

Wijnen J, Khan PM, Vasen H, van der KH, Mulder A, 

Leeuwen-Cornelisse I, et al. Hereditary nonpolyposis 

colorectal cancer families not complying with the 

Amsterdam criteria show extremely low frequency of 

mismatch-repair-gene mutations. Am J Hum Genet 1997 

Aug;61(2):329-35. Not population of interest 

Wijnen JT, Vasen HF, Khan PM, Zwinderman AH, van 

der KH, Mulder A, et al. Clinical findings with 

implications for genetic testing in families with clustering 

of colorectal cancer. N Engl J Med 1998 Aug 

20;339(8):511-8. Family level data reported, no 

primary data 

Wikman FP, Katballe N, Christensen M, Laurberg S, 

Orntoft TF. Efficient mutation detection in mismatch 

repair genes using a combination of single-strand 

conformational polymorphism and heteroduplex analysis 

at a controlled temperature. Genet Test 2000;4(1):15-21. 

Didn't report any of 5 options 
D-22

Winawer S, Fletcher R, Rex D, Bond J, Burt R, Ferrucci J, 

et al. Colorectal cancer screening and surveillance: 

clinical guidelines and rationale-Update based on new 

evidence. Gastroenterology 2003;124(2):544-60. Not 

primary study 

Woerner SM, Gebert J, Yuan YP, Sutter C, Ridder R, 

Bork P, et al. Systematic identification of genes with 

coding microsatellites mutated in DNA mismatch repairdeficient cancer cells. Int J Cancer 2001 Jul 1;93(1):12-9.

Not outcomes of interest 

Woerner SM, Kloor M, Mueller A, Rueschoff J, 

Friedrichs N, Buettner R, et al. Microsatellite instability of 

selective target genes in HNPCC-associated colon 

adenomas. Oncogene 2005 Apr 7;24(15):2525-35. Not 

population of interest 

Wu Y, Berends MJ, Mensink RG, Kempinga C, Sijmons 

RH, Der Zee AG, et al. Association of hereditary 

nonpolyposis colorectal cancer-related tumors displaying 

low microsatellite instability with MSH6 germline 

mutations. Am J Hum Genet 1999 Nov;65(5):1291-8. Not 

population of interest 

Wu Y, Berends MJ, Post JG, Mensink RG, Verlind E, van 

der ST, et al. Germline mutations of EXO1 gene in 

patients with hereditary nonpolyposis colorectal cancer 

(HNPCC) and atypical HNPCC forms. Gastroenterology 

2001 Jun;120(7):1580-7. Not outcomes of interest 

Wu Y, Berends MJ, Sijmons RH, Mensink RG, Verlind E, 

Kooi KA, et al. A role for MLH3 in hereditary 

nonpolyposis colorectal cancer. Nat Genet 2001 

Oct;29(2):137-8. Not population of interest 

Wullenweber HP, Sutter C, Autschbach F, Willeke F, 

Kienle P, Benner A, et al. Evaluation of Bethesda 

guidelines in relation to microsatellite instability. Dis 

Colon Rectum 2001;44(9):1281-9. No data 

Xu DQ, Mattox W. Identification of a splicing enhancer in 

MLH1 using COMPARE, a new assay for determination 

of relative RNA splicing efficiencies. Human Molecular 

Genetics 2006 Jan 15;15(2):329-36. Not relevant 

Yagi OK, Akiyama Y, Nomizu T, Iwama T, Endo M, 

Yuasa Y. Proapoptotic gene BAX is frequently mutated in 

hereditary nonpolyposis colorectal cancers but not in 

adenomas. Gastroenterology 1998 Feb;114(2):268-74. 

Not outcomes of interest 

Yamamoto H, Min Y, Itoh F, Imsumran A, Horiuchi S, 

Yoshida M, et al. Differential involvement of the 

hypermethylator phenotype in hereditary and sporadic 

colorectal cancers with high-frequency microsatellite 

instability. Genes Chromosomes Cancer 2002 

Mar;33(3):322-5. Not outcomes of interest

Yamamoto H, Sawai H, Weber TK, Rodriguez-Bigas MA, 

Perucho M. Somatic frameshift mutations in DNA 

mismatch repair and proapoptosis genes in hereditary 

nonpolyposis colorectal cancer. Cancer Res 1998 Mar 

1;58(5):997-1003. hMLH1 and hMSH2 sequencing not 

performed 

Yamashita K, Arimura Y, Kurokawa S, Itoh F, Endo T, 

Hirata K, et al. Microsatellite instability in patients with 

multiple primary cancers of the gastrointestinal tract. Gut 

2000 Jun;46(6):790-4. Not population of interest 

Young J, Simms LA, Biden KG, Wynter C, Whitehall V, 

Karamatic R, et al. Features of colorectal cancers with 

high-level microsatellite instability occurring in familial 

and sporadic settings: parallel pathways of tumorigenesis. 

Am J Pathol 2001 Dec;159(6):2107-16. Genetic testing 

not compared with an index test 

Yuen ST, Chan TL, Ho JW, Chan AS, Chung LP, Lam 

PW, et al. Germline, somatic and epigenetic events 

underlying mismatch repair deficiency in colorectal and 

HNPCC-related cancers. Oncogene 2002 Oct 

24;21(49):7585-92. No data 

Zhao B, Wang ZJ, Xu YF, Wan YL, Li P, Huang YT. 

Report of 16 kindreds and one kindred with hMLH1 

germline mutation. World J Gastroenterol 2002 

Apr;8(2):263-6. Not relevant 

Zhou XL, Eriksson U, Werelius B, Kressner U, Sun XF, 

Lindblom A. Definition of candidate low risk APC alleles 

in a Swedish population. Int J Cancer 2004 Jul 

1;110(4):550-7. Not population of interest, different 

question 

Zhou XP, Kuismanen S, Nystrom-Lahti M, Peltomaki P, 

Eng C. Distinct PTEN mutational spectra in hereditary 

non-polyposis colon cancer syndrome-related endometrial 

carcinomas compared to sporadic microsatellite unstable 

tumors. Hum Mol Genet 2002 Feb 15;11(4):445-50. Not 

outcomes of interest 

Zhou XP, Loukola A, Salovaara R, Nystrom-Lahti M, 

Peltomaki P, de la CA, et al. PTEN mutational spectra, 

expression levels, and subcellular localization in 

microsatellite stable and unstable colorectal cancers. Am J 

Pathol 2002 Aug;161(2):439-47. Not outcomes of 

interest 
E-1

Appendix E. Peer Reviewers 

We gratefully acknowledge the following individuals who reviewed the initial draft of this 

Report and provided us with constructive feedback. Acknowledgments are made with the 

explicit statement that this does not constitute endorsement of the report by the peer reviewers. 

Linda Bradley, Ph.D.* 

Evaluation of Genomic Applications 

 in Practice and Prevention (EGAPP) 

Office of Genomics and Disease Prevention 

Centers for Disease Control and Prevention 

Atlanta, Georgia 

Celia Kaye, M.D., Ph.D.* 

University of Colorado School of Medicine 

Denver, Colorado 

National Newborn Screening and Genetics 

 Resource Center 

University of Texas Health Science Center 

San Antonio, Texas 

Ralph J. Coates, Ph.D. 

National Center for Chronic Disease 

 Prevention and Health Promotion 

Centers for Disease Control and Prevention 

Atlanta, Georgia 

Michael P. Pignone, M.D., M.P.H. 

Department of Medicine 

University of North Carolina 

Chapel Hill, North Carolina 

Albert de la Chapelle, M.D., Ph.D. 

Human Cancer Genetics Program 

Comprehensive Cancer Center 

Ohio State University 

Philip Prorok, Ph.D. 

National Cancer Institute 

Rockville, Maryland 

Robert H. Fletcher, M.D., M.Sc.* 

University of North Carolina 

Chapel Hill, North Carolina 

Carolyn Sue Richards, Ph.D., F.A.C.M.G.* 

Molecular Diagnostic Center 

Oregon Health & Science University 

Portland, Oregon 

Heather Hampel, M.S., C.G.C. 

Department of Internal Medicine 

James Cancer Hospital & Solove Research 

 Institute 

Columbus, Ohio 

Yvette Seger, Ph.D. 

Office of Biotechnology Activities 

Office of the Director 

National Institutes of Health 

James Haddow, M.D.* 

Women and Infants Hospital 

Providence, Rhode Island 

* Also served on the Technical Expert Panel, which advised the Evidence-based Practice Center. 
F-1

Appendix F. Summary Tables on Clinical Validity 

 Appendix F contains summary Tables (Appendices F-1 to F-29). Appendices F-1 to F-5 

summarize the prevalence on mismatch repair (MMR) gene mutations, microsatellite instability or 

negative immunohistochemistry among patients fulfilling Amsterdam I or II criteria. Appendices F-6 

to F-29 summarize the diagnostic ability of clinical and laboratory predictors to detect MMR gene 

mutations among different populations. Table 14 of the evidence report provides a roadmap to the 

different summary tables. For convenience, the information included in Table 14 of the Evidence 

Report is repeated after the following table of contents. 

 Note that usually only MLH1 and MSH2 were assessed. Whenever additional genes were 

assessed (MSH6 or PMS2), they are clearly noted in the Tables and footnotes. 

Table of contents for Appendix F 

Appendix F-1: Prevalence of mismatch repair gene mutations among colorectal cancer probands 

who fulfill Amsterdam I criteria .................................................................................4 

Appendix F-2: Prevalence of MLH1 and MSH2 mutations among colorectal cancer probands who 

fulfill Amsterdam II criteria........................................................................................7 

Appendix F-3: Prevalence of microsatellite instability among colorectal cancer probands who fulfill 

Amsterdam I criteria ...................................................................................................9 

Appendix F-4: Prevalence of microsatellite instability among colorectal cancer probands who fulfill 

Amsterdam II criteria ................................................................................................11 

Appendix F-5: Prevalence of negative immunostaining for MLH1 or MSH2 among colorectal cancer 

probands who fulfill Amsterdam I criteria................................................................12 

Appendix F-6: Ability of Amsterdam I criteria to identify MLH1 and MSH2 mutation carriers 

among colorectal cancer probands............................................................................13 

Appendix F-7: Ability of Amsterdam I criteria to identify MLH1 and MSH2 mutation carriers 

among unselected colorectal cancer probands ..........................................................18 

Appendix F-8: Ability of Amsterdam I criteria to identify MLH1 and MSH2 mutation carriers 

among colorectal cancer probands fulfilling the Bethesda guidelines......................19 

Appendix F-9: Ability of Amsterdam I criteria to identify MLH1 and MSH2 mutation carriers 

among colorectal cancer probands fulfilling Amsterdam II criteria .........................20 

Appendix F-10: Ability of Amsterdam II criteria to identify MLH1 and MSH2 mutation carriers 

among selected colorectal cancer probands ..............................................................22 

Appendix F-11: Ability of Amsterdam II criteria to identify MLH1 and MSH2 mutation carriers 

among unselected colorectal cancer probands. .........................................................25 

Appendix F-12: Ability of Amsterdam II criteria to identify MLH1 and MSH2 mutation carriers 

among selected colorectal cancer probands fulfilling the Revised Bethesda criteria

...................................................................................................................................26 

Appendix F-13: Ability of Amsterdam II criteria to identify MLH1 and MSH2 mutation carriers 

among selected colorectal cancer probands fulfilling the Bethesda guidelines........27 

Appendix F-14: Ability of modified Amsterdam criteria to identify MLH1 and MSH2 mutation 

carriers among selected colorectal cancer probands .................................................28 

Appendix F-15: Ability of Bethesda guidelines to identify MLH1 and MSH2 mutation carriers......29 
F-2

Appendix F-16: Ability of Bethesda guidelines to identify MLH1 and MSH2 mutation carriers 

among unselected patients with colorectal cancer ....................................................31 

Appendix F-17: Ability of revised Bethesda guidelines to identify MLH1 and MSH2 mutation 

carriers among unselected patients with colorectal cancer .......................................32 

Appendix F-18: Ability of Ability of young age at diagnosis (early disease onset) to identify MLH1 

and MSH2 mutation carriers among colorectal cancer probands .............................33 

Appendix F-19: Ability of Ability of young age at diagnosis (early disease onset) to identify MLH1 

and MSH2 mutation carriers among unselected colorectal cancer probands ...........35 

Appendix F-20: Ability of familial history of malignancy to identify MLH1 and MSH2 mutation 

carriers among colorectal cancer probands...............................................................37 

Appendix F-21: Ability of familial history of malignancy to identify MLH1 and MSH2 mutation 

carriers among unselected colorectal cancer probands .............................................40 

Appendix F-22: Ability of presence of multiple tumors to identify MLH1 and MSH2 mutation 

carriers among unselected colorectal cancer probands .............................................42 

Appendix F-23: Ability of presence of combined family history of colorectal cancer, young age at 

onset or presence of multiple tumors to identify MLH1 and MSH2 mutation carriers 

among unselected colorectal cancer probands ..........................................................43 

Appendix F-24: Ability of microsatellite instability to identify MLH1 and MSH2 mutation carriers 

among unselected colorectal cancer probands ..........................................................45 

Appendix F-25: Ability of microsatellite instability to identify MLH1 and MSH2 mutation carriers 

among unselected colorectal cancer probands ..........................................................51 

Appendix F-26: Ability of microsatellite instability to identify MLH1 and MSH2 mutation carriers 

among colorectal cancer probands fulfilling the revised Bethesda criteria ..............52 

Appendix F-27: Ability of microsatellite instability to identify MLH1 and MSH2 mutation carriers 

among unselected colorectal cancer probands. .........................................................53 

Appendix F-28: Ability of immunohistochemistry to identify MLH1 and MSH2 mutation carriers 

among selected colorectal cancer probands ..............................................................54 

Appendix F-29: Ability of immunohistochemistry to identify MLH1 and MSH2 mutation carriers 

among colorectal cancer probands fulfilling Amsterdam I criteria ..........................57 
F-3

Overview of available evidence on the sensitivity and specificity of various predictors for MMR mutation rates 

Predictor All Specific CRC populations defined by increasingly selective criteria 

studies Nonselected 

CRC 

probands 

Revised 

Bethesda 

guidelines 

Bethesda 

guidelines 

Modified 

Amsterdam 

criteria 

Amsterdam 

II criteria 

Amsterdam I 

criteria 

Amsterdam I criteria F-6 

(n=17) 

F-7 

(n=2) 

ND F-8 

(n=1) 

ND F-9 

(n=4) 

Amsterdam II 

criteria 

F-10 

(n=11) 

F-11 

(n=2) 

F-12 

(n=1) 

F-13 

(n=2) 

ND 

Modified 

Amsterdam criteria 

F-14 

(n=2) 

ND ND ND 

Bethesda guidelines F-15 

(n=4) 

F-16 

(n=1) 

ND 

Revised Bethesda 

guidelines 

F-17 

(n=1) 

F-17 

(n=1) 

 

Young age of onset F-18 

(n=5) 

F-19 

(n=4) 

ND ND ND ND ND 

Family history F-20 

(n=9) 

F-21 

(n=4) 

ND ND ND 

Multiple tumors in 

the same patient 

F-22 

(n=3) 

F-22 

(n=3) 

ND ND ND ND ND 

Age <50 years, 

family history, or 

multiple tumors in 

same patient 

F-23 

(n=3) 

F-23 

(n=3) 

ND ND ND ND ND 

Replication errors 

(MSI-high; MSI-high 

and MSI-low) 

F-24 

(n=16) 

F-25 

(n=2) 

F-26 

(n=1) 

ND ND ND F-27 

(n=3) 

Loss of protein 

expression 

(negative 

immunostaining) 

F-28 

(n=9) 

ND ND ND ND ND F-29 

(n=2) 

Table rows represent different predictors; the table columns describe the populations addressed in the summary tables. 

Each cell in the table refers to a corresponding Summary Table of the appendix. For example, the second cell of the 

second row shows that there were 17 studies that described the ability of Amsterdam I criteria to predict MMR status. The 

studies are described in Appendix F-6. The third cell of the second row shows that of these 17 studies, only two 

performed MMR testing in nonselected patients; these studies are described in Appendix F-7, and so on. 

ND: No data 
F-4

Appendix F-1: Prevalence of mismatch repair gene mutations among colorectal cancer probands who fulfill Am

Study, year (Ref ID); MLH1 and MSH2 Country Single-/multi-center 

A. Comments on sampling 

B. Genetic testing strategy 

C. Definition of deleterious mutations 

NAm1/ 

Ntotal

Positive/ 

Analyzed 

Pro

(

De Abajo 2005; Spain Single-center 

A. Selection among referrals to a specialized center 

B. PCR Æ DGGE Æ sequencing (MSH6 assessed in MLH1/MSH2 

negative cases, included in the counts) 

A. Predicted non-conservative transcription alteration; literature; 

comparison with healthy controls 

56/132 32/56 5

Syngal 2000 (1672) & Wahlberg, 2002 (1158); US Single-center 

C. Selection among referrals to a specialized center 

D. PCR, sequencing 

E. Predicted non-conservative transcription alteration; literature 

28/70 11/28 3

Wang, 1999 (1939); France Single-center 

A. Sampled among referrals to a genetic consultation center 

B. ∅RT-PCR Æ IVSP; 

♦PCR Æ HD; 

in vivo hML1 expression 

C. Predicted transcription alteration; literature 

22/70 14/22 6

Curia, 1999 (1959); Italy Single-center 

A. Sampled from pathology registries, unclear selection criteria B. ♦RT-PCR Æ SSCP Æ Sequencing of abnormal patterns C. Predicted non-conservative transcription alteration; literature; 

comparison with non-cancer controls 

15/30 1/15 

Luce, 1995 (2703); US Single-center 

A. Selection among referrals to a specialized centera

B. ♦PCR Æ IVTT Æ Sequencing of abnormal peptides 

C. Unclear 

12/19a 6/12 5

Katballe, 2002 (1310) & Christensen, 2002 (1038); Denmark Single-center 

A. Selection from a population of 1514 incident CRC 

B. ♦PCR Æ SSCP & HD ÆSequencing of abnormal patterns 

C. Predicted non-conservative transcription alteration; literature 

11/45 5/11 4

Dieumegard, 2000 (1791); France Multi-center 

A. Sample assembled with unclear selection process 

B. ♦PCR Æ SSCP Æ Sequencing of abnormal patterns C. Predicted non-conservative transcription alteration; literature 

10/34 6/10 6

Aaltonen, 1998 (2282); Finland Multi-center 

A. Selection of all incident unrelated CRC from 9 hospitals between 

05/1994 and 04/1996 

B. ♦PCR Æ DGGE (for some samples) Æ Sequencing; 

 PCR Æ Sequencing (for all other samples); ∅ and PCR for founder mutations (all samples) 

C. Literature; and comparison with healthy controls 

4/509 4/4 10
F-5

Study, year (Ref ID); MLH1 and MSH2 Country Single-/multi-center 

A. Comments on sampling 

B. Genetic testing strategy 

C. Definition of deleterious mutations 

NAm1/ 

Ntotal

Positive/ 

Analyzed 

Pro

(

Rossi, 2002 (1146); Brazil Single-center 

A. Selected from consecutive CRC referrals 

B. PCR Æ Sequencing 

C. Unclear 

4/25 1/4 2

Park, 1999 (2007); 7 countries Multi-center (8 centers) 

A. Study sample assembled from the ICG-HNPCC database 

B. ♦PCR Æ SSCP; and 

∅ southern blotting (no details) 

C. Unclearb

154/277 77/154b 5

Lamberti, 1999 (2036); Italy Single-center 

A. Study sample assembled with unclear selection process 

B. ♦PCR Æ SSCP, and RT-PCR Æ PTA 

C. Literature 

57/160 15/57 2

de Leon, 1999 (2012); Italy Single-center 

A. Sample selected from CRC patient registries 

B. ♦PCR Æ SSCP Æ Sequencing of abnormal patterns C. Unclear 

18/36 3/18 1

Liu, 2004 (445); China Single center 

A. Study sample assembled with unclear selection process 

B. ♦PCR Æ DHPLC Æ Sequencing of abnormal patterns C. Unclear 

15/28 5/15 3

Moslein 1996 (2545); US & Germany Multi-center 

A. Sample assembled from various databases; only cases stated to 

have had CRC are analyzedc

B. PCR Æ Sequencing C. Predicted non-conservative transcription alteration; literature 

14/46 6/14c 4

Callistri, 2000 (1797); Italy Multi-center 

A. Study sample assembled with unclear selection process 

B. ♦PCR Æ SSCP C. Unclear 

13/45 7/13 5

Colombino, 2005 (1058); Italy Single-center 

A. Selection from consecutive CRC cases enrolled over 3 yearsa

B. ♦PCR Æ DHPLCÆSequencing of abnormal patterns 

C. Compared mutations to 103 people without cancer 

13/362a 10/13 7

Stormorken, 2001 (721) Norway Single-center 

A. First 56 families in the Norwegian Radium hospital registry 

B. Not described (was already done) (MSH6 was assessed also, 

included in the counts) 

C. Not described 

12/56 3/12 2

Debniak, 2000 (1784); Poland Single-center(?) 

A. Sampled from consecutive CRC, selection process not transparent B. PCR Æ Sequencing 

C. Unclear 

3/168 1/3 3

Yuan, 2004 (303); China 

A. Sampled with unclear selection process; all fulfill Chinese HNPCC 

criteriad

3/14 1/3 3
F-6

Study, year (Ref ID); MLH1 and MSH2 Country Single-/multi-center 

A. Comments on sampling 

B. Genetic testing strategy 

C. Definition of deleterious mutations 

NAm1/ 

Ntotal

Positive/ 

Analyzed 

Pro

(

Single-center B. ♦PCR Æ DHPLC Æ Sequencing of abnormal products 

C. Predicted non-conservative transcription alteration; literature; 

comparison with non-cancer controls 

Studies are ordered by quality and then by decreasing number of patients fulfilling Amsterdam I criteria. Demographic data (on age and gender dis

available for probands fulfilling Amsterdam I criteria. Primary studies did not describe how many of the MMR mutations were MLH1 or MSH2 for th

Amsterdam I criteria. CI: confidence interval; CRC: colorectal cancer; DHPLC: denaturating high performance liquid chromatography; HD: heteroduplex formation; IVSP

assay; IVTT: in vitro transcription-translation; NAm1: number fulfilling Amsterdam I criteria; Ntotal: total number of studied CRC; PCR: polymerase ch

truncation assay; RT-PCR: reverse transcriptase PCR; SSCP: Single-stranded conformation polymorphism Conversion analysis was not used in any study. ♦: Gene screening as been used ∅: Analysis for large deletions has been used 

a Unclear if all probands come from unrelated families. b This study probed for hPSM1 or hPSM2 mutations in some but not all patients; none was found c Although the paper states that 20 patients fulfilled Amsterdam 1 criteria, only 14 were described to have had CRC in the pertinent data table. The d The Chinese HNPCC criteria ≥2 relatives with histologically proven CRC (≥2 must be first degree relatives) and one of the following: multiple co

diagnosed at age younger than 50 years, or development of extracolonic HNPCC-related cancer in family members.
F-7

Appendix F-2: Prevalence of MLH1 and MSH2 mutations among colorectal cancer probands who fulfill Amsterdam 

Study, year (Ref ID); MLH1 and MSH2 mCountry Single-/multi-center 

A. Comments on sampling 

B. Genetic testing strategy C. Definition of deleterious mutations 

NAm2/ 

Ntotal

Positive/A

nalyzed 

Prop

(9

De Abajo 2005; Spain Single-center 

A. Selection among referrals to a specialized center B. PCR Æ DGGE Æ sequencing (MSH6 assessed in MLH1/MSH2 

negative cases) 

C. Predicted non-conservative transcription alteration; literature; 

comparison with healthy controls 

67/132 36/67 54 

Katballe, 2002 (1310) & Christensen, 2002 (1038); Denmark Single-center 

A. Selection from a population of 1514 incident CRC B. ♦PCR Æ SSCP & HD Æ Sequencing of abnormal patterns C. Predicted transcription alteration; literature 

18/45 8/18 44 

Wolf, 2005 (23) Austria Single-center 

A. Retrospective cohort of referral cancer patients fulfilling the modified 

Bethesda criteria 

B. PCR Æ Sequencing 

C. Predicted transcription alteration; literature; comparison with healthy 

controls 

35/81 13/35 37 

Syngal 2000 (1672) & Wahlberg, 2002 (1158); US Single-center 

A. Selection among referrals to specialized center B. PCR Æ Sequencing C. Predicted transcription alteration; literature 

34/70 14/34 41 

Zhu, 2005 (138) China Single-center 

A. Study sample assembled with unclear selection process B. ∅ MLPA Æ Sequencing of detected aberrations C. Unclear 

21/78 4/21 19 

Lee, 2005 (105) Singapore Single-center 

A. Sample selected from referrals to a tertiary center B. PCR Æ Sequencing C. Predicted transcription alteration; literature 

5/46 3/5 60 

Salovaara, 2000 (1740) Finland Multi-center 

A. Selection of all new unrelated CRC from 9 hospitals between 

03/1996 and 06/1998 

B. PCR Æ Sequencing 

∅ PCR for founder mutations in MLH1 gene 

C. Literature; comparison with non-cancer controls 

5/535 4/5 80 

Rossi, 2002 (1146) Brazil Single-center 

A. Selected from consecutive CRC referrals B. PCR, Æ Sequencing C. Unclear 

5/25 2/5 40 

Lamberti, 1999 (2036); Italy Single-center 

A. Study sample assembled with unclear selection process B. ♦PCR Æ SSCP; and RT-PCR, PTA C. Literature 

69/160 17/69 25 

Stormorken, 2001 (721) A. First 56 families in the Norwegian Radium hospital registry 20/56 7/20a 35 
F-8

Study, year (Ref ID); MLH1 and MSH2 mCountry Single-/multi-center 

A. Comments on sampling 

B. Genetic testing strategy C. Definition of deleterious mutations 

NAm2/ 

Ntotal

Positive/A

nalyzed 

Prop

(9

Norway Single-center 

B. Not described (was already done) (MSH6 was assessed also) C. Not described 

Studies are ordered by quality and then by decreasing number of patients fulfilling Amsterdam II criteria. Demographic data (on age and gender distr

for probands fulfilling Amsterdam II criteria. Primary studies did not describe how many of the MMR mutations were MLH1 or MSH2 for the subset of C

criteria. Conversion analysis was not used in any study. ♦: Gene screening as been used ∅: Analysis for large deletions has been used CI: confidence interval; CRC: colorectal cancer; HD: heteroduplex formation; MLPA: Multiple ligation-dependent probe amplification; NAm2: number fNtotal: total number of studied CRC; PCR: polymerase chain reaction; PTA: protein truncation assay; RT-PCR: reverse transcriptase PCR; SSCP: Sinpolymorphism 

a 2/7 mutations shown are MSH6
F-9

Appendix F-3: Prevalence of microsatellite instability among colorectal cancer probands who fulfill Amsterdam I c

Study, year (Ref ID); Tumor microsatellite instability Country Single-/multi-center 

A. Comments on sampling 

B. Definition of MSI-H 

NAm1/ 

Ntotal

MSI-H/ 

Analyzed 

MSI-H [%] 

(95% CI) 

MSI-H&L/ 

Analyzed 

MSI-H&L [%] 

(95% CI) 

Wahlberg, 2002 (1158) & Syngal 2000 (1672); US Multi-center 

A. Selection among referrals to specialized 

center 

B. ≥2 out of 5 dinucleotide repeats (NCI set) 

28/70 15/19 79 (54, 94) 18/19 95 (74, 100) 

Curia, 1999 (1959); Italy Single-center 

A. Sampled from pathology registries, unclear 

selection criteria 

B. Screened with 3 markers and if none was 

unstable up to a total of 7. MSI-H defined as ≥2 markers 

15/30 12/15 80 (52, 96) ND ND 

Katballe, 2002 (1310) & Christensen, 2002 (1038); Denmark Single-center 

A. Selected from a population of 1514 incident 

CRC 

B. ≥2 out of 5 dinucleotide repeats 

11/45 7/10 70 (34, 93) 7/10 70 (34, 93) 

Dieumegard, 2000 (1791); France Multi-center 

A. Sample assembled with unclear selection 

process 

B. ≥10% of up to 23 markers (But all were 

≥30%) 

10/34 9/10 90 (55, 100) 9/10 90 (55, 100) 

Aaltonen, 1998 (2282); Finland Multi-center 

A. Selection of all new unrelated CRC from 9 

hospitals between 05/1994 and 04/1996 

B. ≥30% of 16 markers for tumor analyzed with 

fluorescence methods or ≥2 out of 7 (≈30%) 

markers analyzed with radioactive technique 

4/509 4/4 100 

(40, 100) 

4/4 100 (40, 100) 

Lamberti, 1999 (2036); Italy Single-center 

A. Study sample assembled with unclear 

selection process 

B. ≥40% out of up to 10 mono- di- and 

tetranucleotide repeats 

57/16

0 

35/49 71 (57, 83) 37/49 76 (61, 87) 

de Leon, 1999 (2012); Italy Single-center 

A. Sample selected from CRC patient registries 

B. ≥2 out of ≥5 markers 

18/36 11/18 61 (36, 83) ND ND 

Callistri, 2000 (1797); Italy Multi-center 

A. Study sample assembled with unclear 

selection process 

B. ≥2 out of 13 microsatellite markers 

13/45 11/13 85 (55, 98) 11/13 85 (55, 98) 
F-10

Study, year (Ref ID); Tumor microsatellite instability Country Single-/multi-center 

A. Comments on sampling 

B. Definition of MSI-H 

NAm1/ 

Ntotal

MSI-H/ 

Analyzed 

MSI-H [%] 

(95% CI) 

MSI-H&L/ 

Analyzed 

MSI-H&L [%] 

(95% CI) 

Peel, 1999 (1660); US Multi-center 

A. Referral HNPCC cases, other than the 1134 

CRC probands assessed for other purposes 

B. Unclear; at least 5 markers were used 

11/11 4/9 44 (14, 79) 4/9 44 (14, 79) 

Moslein, 1996 (2545); US & Germany Multi-center 

A. Sample assembled from various databases; 

we present only cases stated to have CRCa

B. ≥30% of 9 to 34 markers analyzed (unclear 

which exactly) 

14/ 

46a

5/9 56 (21, 86) ND ND 

Debniak, 2000 (1784); Poland Single-center(?) 

A. Sampled from consecutive CRC, selection 

not transparent 

B. ≥2 out of 5 dinucleotide repeats (panel I), or 

≥3 out of 10 dinucleotide repeats (panel II, used if only 1 positive in panel I) 

3/168 2/3 67 (9, 99) ND ND 

Studies are ordered by overall quality and then by decreasing number of patients fulfilling the Amsterdam I criteria. Demographic data (on age and ge

available for probands fulfilling Amsterdam I criteria. CI: confidence interval; CRC: colorectal cancer; MSI-H/H&L: microsatellite instability high/combined high and low; ND: Not described; NAm1: number fu

Ntotal: total number of studied CRC; NCI: National cancer institute 

X: Was not used/not stated √: Was used 

a Although the paper states that 20 patients fulfilled Amsterdam I criteria, only 14 were described to have a CRC in the pertinent data table. The other 

14 with CRC are analyzed. 
F-11

Appendix F-4: Prevalence of microsatellite instability among colorectal cancer probands who fulfill Amsterdam II c

Study, year (Ref ID); Tumor microsatellite instability Country Single-/multi-center 

A. Comments on sampling B. Definition of microsatellite 

instability 

NAm2/ 

Ntotal

MSI-H/ 

Analyzed 

MSI-H [%] 

(95% CI) 

MSI-H&L/ 

Analyzed 

MSI-H&L [%] 

(95% CI) 

Wolf, 2005 (23) Austria Single-center 

A. Retrospective cohort of referral cancer 

patients fulfilling the modified Bethesda criteria 

B. ≥30% out of up to 10 markers 

35/81 16/24 67 (45, 84) ND ND 

Katballe, 2002 (1310) & Christensen, 2002 (1038); Denmark Single-center 

A. Selected from a population of 1514 

incident CRC 

B. ≥2 out of 5 dinucleotide repeats 

17/45 10/16 63 (35, 85) 11/16 69 (41, 89) 

Salovaara, 2000 (1740) Finland Multi-center 

A. Selection of all new unrelated CRC 

patients from 9 hospitals between 03/1996 and 06/1998 

B. Single marker (BAT26) analyzed in all 

samples 

5/535 4/5 80 (28, 99) NA NA 

Lamberti, 1999 (2036); Italy Single-center 

A. Study sample assembled with unclear 

selection process 

B. ≥40% out of up to 10 mono- di- and 

tetranucleotide repeats 

69/160 39/57 68 (55, 80) 41/57 72 (58, 83) 

Studies are ordered by quality and then by diminishing number of patients fulfilling the Amsterdam II criteria. Demographic data (on age and gender davailable for probands fulfilling Amsterdam II criteria. CI: confidence interval; CRC: colorectal cancer; MSI-H/H&L: microsatellite instability high/combined high and low; NA: Not applicable; ND: Not describ

Amsterdam II criteria; Ntotal: total number of studied CRC; NCI: National cancer institute 

X: Was not used/not stated √: Was used 
F-12

Appendix F-5: Prevalence of negative immunostaining for MLH1 or MSH2 among colorectal cancer probands who criteria 

Study, year (Ref ID); MLH1 and MSH2 immunostaiCountry Single-/multi-center 

A. Comments on sampling B. Antibodies used for immunostaining 

NAm1/ 

Ntotal

Negative/A

nalyzed 

Proportion [%

(95% CI) 

Curia, 1999 (1959) Italy Single-center 

A. Sampled from pathology registries, but selection process is 

not transparent 

B. Anti-MSH2: FE11 (Oncogene Research Products); antiMLH1: clone 14 (Oncogene Research Products) 

15/30 7/15 47 (21, 73) 

Wahlberg, 2002 (1158) & Syngal 2000 (1672); a

US Single-center 

A. Selection among referrals to specialized center B. Anti-MSH2: FE11 (Oncogene Research Products); antiMLH1: G168-728 (PharMingen) 

28/70 5/14 36 (13, 65) 

Katballe, 2002 (1310) & Christensen, 2002 (1038);b

Denmark Single-center 

A. Selection from a population of 1514 incident CRC 

B. Anti-MSH2: Ab-1, Ab-2 (Oncogene Research Products); 

anti-MLH1: G168-15 (PharMingen) 

11/42 4/11 36 (11, 69) 

Dieumegard, 2000 (1791) France Multi-center 

A. Sample assembled without a transparent selection process B. Anti-MSH2: FE11 (Oncogene Research Products); antiMLH1: Ab-1 (Oncogene Research Products) 

10/34 4/8 50 (16, 84) 

Stormorken, 2001 (721) Norway Single-center 

A. First 56 families in the Norwegian Radium hospital registry B. Anti-MSH2: FE11 (Calbiochem); anti-MLH1: G168-15 

(Pharmingen); anti-MSH6: Clone 44 (transduction laboratories)c

12/56 3/12 25 (5, 57) 

Debniak, 2000 (1784); Poland Single-center(?) 

A. Sampled from consecutive CRC, but selection process is 

not transparent 

B. Not stated 

3/168 2/3 67 (9, 99) 

Studies are ordered by overall quality and then by decreasing number of patients fulfilling Amsterdam I criteria. Demographic data (on age and gende

available for probands fulfilling Amsterdam I criteria. None of these studies assessed any other mismatch repair genes apart from MLH1 and MSH2. CI: confidence interval; CRC: colorectal cancer; NAm1: number fulfilling Amsterdam I criteria; Ntotal: total number of studied CRC 

a Data from the Wahlberg et al. 2002 paper; comments on sampling from the Syngal et al. 2000 paper. b Data from the Christensen et al. 2002 paper; comments on sampling from the Katballe et al. 2002 paper. c 2 MSH6 MMR mutations were also picked up by the anti-MSH2 IHC exam. 
F-13

Appendix F-6: Ability of Amsterdam I criteria to identify MLH1 and MSH2 mutation carriers among colorectal cance

Mutation No 

mutation 

D

(9

Study, year (Ref ID); Country Single-/multi-center 

A. Study sample characteristics B. Verification bias 

C. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

NAm1/ 

Ntotal

Am1 

(+) 

Am1 

(-) 

Am1 

(+) 

Am1 

(-) 

Se

Genetic testing irrespectively of tumor microsatellite instability status Aaltonen, 1998 (2282) Finland Multi-center 

A. Selection of all new unrelated 

CRC from 9 hospitals between 05/1994 and 04/1996 

B. Yes: patients without MSI were 

screened only for MLH1 founder mutations 

C. ND, ND 

A.

∅All CRC: PCR for founder 

mutations 1 & 2 in MLH1 

♦CRC with MSI: (some) PCR 

Æ

DGGE 

Æ Sequencing 

(Remaining) PCR 

Æ Sequencing 

B. 9/1 

C. Literature; comparison with 

healthy controls 

4/509 4 6 0 499 6

De Abajo 2005; Spain Single-center 

A. Selection among referrals to a 

specialized center 

B. No C. ND; ND 

A. PCR 

Æ DGGE 

Æ sequencing 

(MSH6 assessed in MLH1/MSH2 negative cases) 

B. 24/17 (and 3 MSH6) 

C. Predicted non-conservative 

transcription alteration; literature; comparison with healthy controls 

56/ 

132 

33 11 23 65 7

Syngal, 2000 (1672) & Wahlberg, 2002 (1158); US Single-center 

A. Selection among referrals to 

specialized center 

B. No C. ND; ND 

A. PCR

Æsequencing 

B. 18 (MLH1 and MSH2) 

C. Predicted transcription alteration; 

literature 

28/70 11 7 17 35 6

Wang, 1999 (1939); France Single-center 

A. Sampled among referrals to a 

genetic consultation center: Familial CRC with Am1 or up to 2 Am1 criteria missing, or aggregation of HNPCC-related tumors, or sporadic CRC aged <50 y at diagnosis 

B. No C. ND; ND 

A.

∅RT-PCR

Æ IVSP; 

♦ PCR

ÆHD

Æsequencing; 

in vivo MLH1 protein expression 

B. 19/7 

C. Predicted transcription alteration; 

literature 

22/70 14 12 8 41 50 

Katballe, 2002 (1310) & Christensen, 2002 (1038); Denmark Single-center 

A. Familial CRC selected from a 

population of 1514 newly diagnosed CRC: patients fulfilling Am2 (in extended families and relaxing the age criterion to <55y) and familial CRC with early age of onset 

A.

♦PCR

ÆSSCP & HD

Æ

Sequencing of abnormal patterns 

B. 4/6 

C. Predicted transcription alteration; 

literature 

11/45 5 5 6 25 50 
F-14

Mutation No 

mutation 

D

(9

Study, year (Ref ID); Country Single-/multi-center 

A. Study sample characteristics B. Verification bias 

C. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

NAm1/ 

Ntotal

Am1 

(+) 

Am1 

(-) 

Am1 

(+) 

Am1 

(-) 

Se

B. No C. ND; ND 

Curia, 1999 (1959) Italy Single-center 

A. Sampled from pathology 

registries, unclear selection criteria: HNPCC related cancersa

B. No 

C. <50y, ND 

A. ♦RT-PCRÆ SSCPÆ

Sequencing of abnormal products 

B. 2/1 C. Predicted non-conservative 

transcription alteration; literature; comparison with non-cancer controls 

15/30a 1 2 16 11 3

Rossi, 2002 (1146) Brazil Single-center 

A. Selected from consecutive CRC 

referrals: Am2 or familial CRC (including HNPCC-related cancer in family), or sporadic CRC aged <50y or with multiple tumors 

B. No C. 46 (all sample); 60 (all sample) 

A. PCRÆ sequencing B. 4/6 

C. Unclear 

5/25 1 9 3 12 1

Luce, 1995 (2703); US Single-center 

A. Selection among referrals to 

specialized center, familial CRC: In 1st degree family ≥2 CRC or 

endometrial cancer; or ≥1 CRC aged <50y at diagnosis b

B. No 

C. ND; ND 

A. ♦RT-PCRÆ IVTT Æ

sequencing of abnormal peptides; PCR Æ IVTTÆ sequencing of 

abnormal peptides 

B. 4/2 C. Unclear 

12/19b 6 0 6 7 

(

Colombino, 2005 (181); Italy Single-center 

A. Consecutive CRC cases enrolled 

over 3 years in a tertiary center b

B. Yes: Patients without family 

history of cancer were screened only for the mutations identified in the familial cases 

C. 62; 49 

A. ♦PCRÆ DHPLCÆ Sequencing 

of abnormal patterns 

B. 11/10 C. Compared to 103 people without 

cancer 

13 

/362b

10 11 3 338 48 

Park, 1999 (2007); 7 countries Multi-center (8 centers) 

A. Study sample assembled from 

the ICG-HNPCC database of families: Am1; and familial CRC not fulfilling the Am1 criteria 

B. No C. ND; ND 

D. ♦ PCR Æ SSCP; and 

∅ Southern blotting (no details) 

E. 101 (MLH1 and hMLH2) 

F. Unclear 

154/ 

277 

77c 24c 77 99 7

Lamberti, 1999 A. Study sample assembled with A. ♦ PCR Æ SSCP, and 69/ 15 2 42 10 
F-15

Mutation No 

mutation 

D

(9

Study, year (Ref ID); Country Single-/multi-center 

A. Study sample characteristics B. Verification bias 

C. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

NAm1/ 

Ntotal

Am1 

(+) 

Am1 

(-) 

Am1 

(+) 

Am1 

(-) 

Se

(2036); Italy Single-center 

unclear selection process; includes Am1, Am2, other familial CRC and CRC <50y at diagnosis or multiple tumorsd

B. No 

C. 44 for Am1, 37 for Am2; ND 

RT-PCR Æ PTA 

B. 11/6 C. Literature 

160d 

de Leon, 1999 (2012) Italy Single-center 

A. Selected from CRC patient 

registries: Am1 or familial CRC (2 generations with CRC or HNPCC-tumor, ≥1 CRC aged <50y at diagnosis and ≥50% of 

siblings with CRC) 

B. No C. ND, ND 

A. ♦PCRÆSSCPÆ Sequencing of 

aberrant products 

B. 1/2 

C. Unclear 

18/36 3 0 15 18 

(

Liu, 2004 (445); China Single center 

A. Study sample assembled with 

unclear selection process; all fulfill the Japanese HNPCC criteriae

B. No 

C. 46; 55 

A. ♦PCRÆDHPLCÆSequencing 

of abnormal products 

B. 5/7 

C. Unclear 

15/28 5 2 10 11 7

Moslein 1996 (2545) US & Germany Multi-center 

A. Sample assembled from various 

databases; includes familial and sporadic casesf

B. No C. 51, ND 

A. PCRÆsequencing 

B. 7/6 

C. Predicted non-conservative 

transcription alteration; literature 

14/46f 6 6 8 17 50 

Callistri, 2000 (1797); Italy Multi-center 

A. Study sample assembled with 

unclear selection process: Familial CRC fulfilling Am1 criteria or missing up to 2/3 Am1 criteria, or a CRC among 1st

degree family or CRC diagnosis at age <50y, or multiple tumors in the same patient 

B. No C. ND; 56 (all sample) 

A. ♦PCRÆ SSCP 

B. 4/4 C. Unclear 

13/40g 7 1 6 5 

(

Yuan, 2004 (303) China Single-center 

A. Sampled with unclear selection 

process; all fulfill Chinese HNPCC criteriah

A. ♦PCRÆDHPLCÆ Sequencing 

of abnormal products 

∅ Multiplex PCR for large deletions 

3/14 2 1 1 10 6
F-16

Mutation No 

mutation 

D

(9

Study, year (Ref ID); Country Single-/multi-center 

A. Study sample characteristics B. Verification bias 

C. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

NAm1/ 

Ntotal

Am1 

(+) 

Am1 

(-) 

Am1 

(+) 

Am1 

(-) 

Se

B. No C. ND, ND 

B. 2/1 

C. Predicted non-conservative 

transcription alteration; literature; comparison with non-cancer controls 

Genetic testing only among patients with suggestive MSI or ICH. Casey, 2005 Practically among patients with IHC US Multi-center 

A. Referrals to the Colon Cancer 

Family Registry 

B. Yes: 85/89 were pre-selected 

based on suggestive MSI or IHC 

C. Aprox 50 ; ND 

A. CRC with MSI/IHC: 

PCR Æ Sequencing; (also for MSH6) Conversion analysis

B. 42 in total (1 had an MSH6 

mutation) 

C. Predicted non-conservative 

transcription alteration; literature; mRNA expression for missence mutations and additional data for splice site mutations 

63/89 

49 14 15 10 78 

Raedle, 2001 (1363) Among patients with MSI-H tumorsGermany Multi-center 

A. Consecutive referrals to a 

tertiary center. 

B. Yes: patients without MSI were 

not tested for mutation 

C. 52, 55 

A. CRC with MSI:

PCRÆ Sequencing 

B. 8/4 (1 had mutation in both 

genes) 

C. Predicted transcript alteration; 

literature 

12/ 

125 

6 5 0 11 5

Nakahara, 1997 Among patients with MSI-H tumors(2354) Japan Single-center 

A. Sample assembled from hospital 

registriesb

B. Yes: patients without MSI were 

not tested for mutations 

C. 59, 41 

A. CRC with MSI:

♦PCRÆ SSCP Æ Sequencing of 

abnormal patterns 

B. 5/6 (4 CRC had mutations in both 

genes) 

C. Unclear 

≥5/32b

4 3 1 3 5

Studies are ordered by quality and then by decreasing number of patients available for the calculation of sensitivity and specificity (2 by 2 tables). 

Am1/2: Amsterdam I criteria/II; CRC: colorectal cancer; DGGE: Denaturing gradient gel electrophoresis; DHPLC: denaturating high performance liquid 

heteroduplex formation; IVSP: in vitro synthesized protein assay; IVTT: in vitro transcription-translation; MSI(-H): microsatellite instability (high); ND: N

fulfilling Amsterdam I criteria; Ntotal: total number of studied CRC; PCR: polymerase chain reaction; PTA: protein truncation assay; RT-PCR: reverse t

Single-stranded conformation polymorphism ♦: Gene screening method ∅: Detection of large genomic deletions 
F-17

a 4 were not CRC and are excluded from the calculations b Not strictly clear if all CRC probands come from unrelated families. c 0/27 and 0/24 had PMS1 or PMS2 mutations; Not all patients were tested for these other MMR genes. d 69/160 included cases fulfilled Amsterdam II criteria and these are analyzed here. The remaining cases were 45 CRC aged <50 y at diagnosis and 

cancers and 46 CRC aged <50 y at diagnosis or CRC with multiple tumors (no age cutoff). However, these received genetic testing inconsistently and 

analysis 

e The Japanese HNPCC criteria: ≥3 CRC among 1st degree family; or ≥2 CRC among 1st degree family and one of the following: aged <50y at diagno

synchronous or metachronous multiple CRC, or extracolonic malignancy. 

f Interestingly, despite the fact that the study included 46 cancer patients (characterized as CRC), in an analytic description not all were described with 

described with CRC were included in this analysis. 

g Data from 19 (out of the 45 total studied patients) CRC who missed at most 2 out of 3 Amsterdam criteria (and had available information) are analyz

mutations were tested inconsistently among other patient subgroups.

h The Chinese HNPCC criteria: 2 or more relatives with histologically proven CRC (at least 2 first degree relatives) and one of the following: multiple c

diagnosed at age younger than 50y, or development of extracolonic HNPCC-related cancer in family members. 
F-18

Appendix F-7: Ability of Amsterdam I criteria to identify MLH1 and MSH2 mutation carriers among unselected colo

probands

Mutation No 

mutation 

D

(9

Study, year (Ref ID); Country Single-/multi-center 

A. Study sample characteristics B. Verification bias 

C. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

NAm1/ 

Ntotal

Am1 

(+) 

Am1 

(-) 

Am1 

(+) 

Am1 

(-) 

Se

Aaltonen, 1998 (2282) Finland Multi-center 

A. Selection of all new unrelated 

CRC from 9 hospitals between 05/1994 and 04/1996 

B. Yes: patients without MSI were 

screened only for MLH1 founder mutations 

C. ND, ND 

A. ∅All CRC: PCR for founder 

mutations 1 & 2 in MLH1 

♦CRC with MSI: (some) PCR Æ

DGGE Æ Sequencing (Remaining) PCR Æ Sequencing 

B. 9/1 

C. Literature; comparison with 

healthy controls 

4/509 4 6 0 499 6

Colombino, 2005 (1058); Italy Single-center 

A. Consecutive CRC cases enrolled 

over 3 years in a tertiary centera

B. Yes: Patients without family 

history of cancer were screened only for the mutations identified in the familial cases 

C. 62; 49 

A. ♦PCRÆ DHPLCÆ Sequencing of 

abnormal patterns 

B. 11/10 C. Compared to 103 people without 

cancer 

13 

/362a

10 11 3 338 48 

Studies are ordered by quality. None of the studies in this table assessed genes other than MLH1 and hMLH2. None of the studies used conversion a

testing strategies. Am1/2: Amsterdam I criteria/II; CRC: colorectal cancer; DGGE: Denaturing gradient gel electrophoresis; DHPLC: denaturating high performance liquid 

described; NAm1:Number fulfilling Amsterdam I criteria; Ntotal: total number of studied CRC; PCR: polymerase chain reaction 

♦: Gene screening method ∅: Detection of large genomic deletions 

a Not clear if all CRC probands come from unrelated families. 
F-19

Appendix F-8: Ability of Amsterdam I criteria to identify MLH1 and MSH2 mutation carriers among colorectal cance

the Bethesda guidelines

Mutation No 

mutation 

D

(9

Study, year (Ref ID); Country Single-/multi-center 

A. Study sample characteristics B. Verification bias 

C. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

NAm1/ 

Ntotal

Am1 

(+) 

Am1 

(-) 

Am1 

(+) 

Am1 

(-) 

Se

De Abajo 2005; Spain Single-center 

A. Selection among referrals to a 

specialized center 

B. No C. ND; ND 

A. PCR Æ DGGE Æ sequencing 

(MSH6 assessed in MLH1/MSH2 negative cases) 

B. 24/17 (and 3 MSH6) 

C. Predicted non-conservative 

transcription alteration; literature; comparison with healthy controls 

56/ 

132 

33 11 23 37 7

Syngal, 2000 (1672) & Wahlberg, 2002 (1158); US Single-center 

A. Selection among referrals to 

specialized center 

B. No C. ND; ND 

A. PCRÆsequencing B. 18 (MLH1 and MSH2) 

C. Predicted transcription alteration; 

literature 

56/70 11 6 17 22 6

The tabulated study did not assess genes other than MLH1 and MSH2. Genetic testing did not include gene screening, analysis for large genomic de

analysis. Am1: Amsterdam I criteria; ND: Not described; NAm1:Number fulfilling Amsterdam I criteria; Ntotal: total number of studied CRC; PCR: polymerase cha
F-20

Appendix F-9: Ability of Amsterdam I criteria to identify MLH1 and MSH2 mutation carriers among colorectal cance

Amsterdam II criteria

Mutation No 

mutation 

D

(9

Study, year (Ref ID); Country Single-/multi-center 

A. Study sample characteristics B. Verification bias 

C. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

NAm1/ 

Ntotal

Am1 

(+) 

Am1 

(-) 

Am1 

(+) 

Am1 

(-) 

Se

De Abajo 2005; Spain Single-center 

A. Selection among referrals to a 

specialized center 

B. No C. ND; ND 

A. PCR Æ DGGE Æ sequencing 

(MSH6 assessed in MLH1/MSH2 negative cases) 

B. 24/17 (and 3 MSH6) 

C. Predicted non-conservative 

transcription alteration; literature; comparison with healthy controls 

56/ 

132 

33 6 23 5 8

Syngal, 2000 (1672) & Wahlberg, 2002 (1158); US Single-center 

A. Selection among referrals to 

specialized center 

B. No C. ND; ND 

A. PCRÆsequencing B. 18 (MLH1 and MSH2) 

C. Predicted transcription alteration; 

literature 

34/70 11 3 17 3 79 

Katballe, 2002 (1310) & Christensen, 2002 (1038); Denmark Single-center 

A. Familial CRC selected from a 

population of 1514 newly diagnosed CRC: patients fulfilling Am2 (in extended families and relaxing the age criterion to <55y) and familial CRC with early age of onset 

B. No C. ND; ND 

A. ♦PCRÆSSCP & HDÆ

Sequencing of abnormal patterns 

B. 4/6 

C. Predicted transcription alteration; 

literature 

23/45 5 3 6 9 6

Rossi, 2002 (1146) Brazil Single-center 

A. Selected from consecutive CRC 

referrals: Am2 or familial CRC (including HNPCC-related cancer in family), or sporadic CRC aged <50y or with multiple tumors 

B. No C. ND; ND 

A. PCRÆ sequencing B. 4/6 

C. Unclear 

5/25 1 1 3 0 5

Lamberti, 1999 (2036); Italy Single-center 

A. Study sample assembled with 

unclear selection process; includes Am1, Am2, other familial CRC and CRC <50y at diagnosis or multiple tumorsa

B. No 

C. 44 for Am1, 37 for Am2; ND 

A. ♦ PCR Æ SSCP, and 

RT-PCR Æ PTA 

B. 11/6 C. Literature 

69/ 

160a

15 2 42 10 8
F-21

Studies are ordered by quality and then by decreasing number of patients available for the calculation of sensitivity and specificity (2 by 2 tables). Non

deletion analysis to detect large genomic deletions or conversion analysis to detect mismatch repair gene mutations. Most studies did not provide da

gender distribution) for the subgroup of patients fulfilling the Amsterdam II criteria. Am1/2: Amsterdam I criteria/II; CRC: colorectal cancer; HD: heteroduplex formation; ND: Not described; NAm1:Number fulfilling Amsterdam I criteria; N

CRC; PCR: polymerase chain reaction; PTA: protein truncation assay; RT-PCR: reverse transcriptase PCR; SSCP: Single-stranded conformation pol

♦: Gene screening method 

a 69/160 included cases fulfilled Amsterdam II criteria and these are analyzed here. The remaining cases were 45 CRC aged <50 y at diagnosis and 

cancers and 46 CRC aged <50 y at diagnosis or CRC with multiple tumors (no age cutoff). However, these received genetic testing inconsistently and 

analysis 
F-22

Appendix F-10: Ability of Amsterdam II criteria to identify MLH1 and MSH2 mutation carriers among selected color

Mutation No mutation D

(9

Study, year (Ref ID); Country Single-/multi-center 

A. Study sample characteristics 

B. Verification bias 

C. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

NAm2/ 

Ntotal

Am2 

(+) 

Am2 

(-) 

Am2 

(+) 

Am2 

(-) 

Se

Genetic testing irrespectively of MSI or IHC test results Salovaara, 2000 (1740) Finland Multi-center 

A. Selection of all new unrelated 

CRC from 9 hospitals between 03/1996 and 06/1998 

B. Yes: patients without MSI were 

screened only for MLH1 founder mutations 

C. 67, ND 

A. PCR Æ Sequencing 

∅ PCR for founder mutations 

in MLH1 gene 

B. 17/1 

C. Literature; comparison with 

non-cancer controls 

5/535 4 14 1 516 2

De Abajo 2005; Spain Single-center 

A. Selection among referrals to a 

specialized center 

B. No C. ND; ND 

A. PCR Æ DGGE Æ

sequencing (MSH6 assessed in MLH1/MSH2 negative cases) 

B. 24/17 (and 3 MSH6) 

C. Predicted non-conservative 

transcription alteration; literature; comparison with healthy controls 

67/ 

132 

39 5 28 60 89 

Wolf, 2005 (123) Austria Single-center 

A. Retrospective cohort of cancer 

patients fulfilling the modified Bethesda criteria 

B. No C. ND, 48 

A. PCR Æ Sequencing 

B. 12/7 

C. Predicted transcription 

alteration; literature; comparison with healthy controls 

35/81 13 6 22 40 68 

Zhu, 2005 (138) China Single-center 

A. Study sample assembled with 

unclear selection process: Familial CRC and sporadic CRC 

B. No C. ND, ND 

A. ∅ MLPA Æ Sequencing of 

detected aberrations 

B. 4/5 

C. Unclear 

21/78 4 5 17 52 44 

Syngal, 2000 (1672) & Wahlberg, 2002 (1158); US Single-center 

A. Selection among referrals to 

specialized center 

B. No C. ND; ND 

A. PCR Æ Sequencing 

B. 18 (MLH1 and MSH2) 

C. Predicted transcription 

alteration; literature 

34/70 14 4 20 32 78 

Katballe, 2002 (1310) & Christensen, 2002 (1038); Denmark 

A. Familial CRC selected from a 

population of 1514 newly diagnosed CRC: patients fulfilling Amsterdam II criteria (in extended 

A. ♦PCR Æ SSCP & HD Æ

Sequencing of abnormal patterns

18/45 8 2 10 21 80 
F-23

Mutation No mutation D

(9

Study, year (Ref ID); Country Single-/multi-center 

A. Study sample characteristics 

B. Verification bias 

C. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

NAm2/ 

Ntotal

Am2 

(+) 

Am2 

(-) 

Am2 

(+) 

Am2 

(-) 

Se

Single-center families and relaxing the age 

criterion to <55y) and familial CRC with early age of onset 

B. No C. ND; ND 

B. 4/6 

C. Predicted transcription 

alteration; literature 

Rossi, 2002 (1146); Brazil Single-center 

A. Selected from consecutive CRC 

referrals: Am2 or familial CRC (including HNPCC-related cancer in family), or sporadic CRC aged <50y or with multiple tumors 

B. No C. 46; 60 

A. PCR Æ Sequencing 

B. 4/6 

C. Unclear 

5/25 2 8 3 12 2

Lee, 2005 (105); Singapore Single-center 

A. Selection among referrals to 

tertiary center: Amsterdam criteria, familial disease, onset <40y or multiple tumors 

B. No C. 39 (median), 65 

A. PCR Æ Sequencing 

B. 6/1 

C. Predicted transcript 

alteration; literature 

5/46a 3 4 2 37 43 

Genetic testing only in patients selected after MSI and/or ICH 

Assuming no mutations in the absetumors with negative immunostaini

Pinol, 2005 (52) Spain Multi-center 

A. Selection of newly diagnosed 

CRC from 25 centers. 

B. Yes: patients without MSI or with 

negative immunostaining were not sequenced 

C. 70, 60 

A. CRC selected by MSI/IHC:

PCR Æ Sequencing 

∅ also MLPA 

B. 4/7 

C. Predicted transcript 

alteration; literature and databases 

22/ 

1222 

4 7 18 1197 36 

Raedle, 2001 (1363); Among patients with MSI-H tumorsGermany Multi-center 

A. Consecutive referrals to a tertiary 

center. 

B. Yes: patients without MSI were 

not tested for mutations 

C. 52, 55 

A. CRC with MSI:

PCRÆ Sequencing 

B. 8/4 (1 had mutation in both 

genes) 

C. Predicted transcript 

alteration; literature 

20/ 

125 8 3 2 9 73 

Terdiman, 2001 A. Retrospective cohort of patients A. CRC with MSI: 19/ Among patients with MSI-H tumors
F-24

Mutation No mutation D

(9

Study, year (Ref ID); Country Single-/multi-center 

A. Study sample characteristics 

B. Verification bias 

C. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

NAm2/ 

Ntotal

Am2 

(+) 

Am2 

(-) 

Am2 

(+) 

Am2 

(-) 

Se

US Single-center 

who had ≥2 CRC in 1st degree 

family, or age <50 years at CRC diagnosis or multiple tumors in the same patient 

B. Yes: Only patients with MSI-H 

were tested for mutations 

C. ND, ND 

♦PCRÆ DGGE Æ

Sequencing of abnormal products 

B. 11/10 

C. Unclear 

114 15 6 4 7 7

Pistorius, 2000 Among patients with MSI-H tumors(1590); Germany & Czech Republic Multi-center 

A. Selection among referrals to 

tertiary center: Bethesda guidelines met 

B. Yes: patients without MSI were 

not genetically tested 

C. ND, ND 

A. CRC with MSI:

PCR Æ Sequencing 

B. 6/8 (1 MSH6 mutation also 

found) 

C. Predicted transcript 

alteration; literature 

19/72 

12 3 5 18 80 

Only Pistorious et al. assessed genes other than MLH1 and MSH2, namely the MSH6 gene. Am2: Amsterdam II criteria; CRC: colorectal cancer; DGGE: Denaturating gradient gel electrophoresis; HD: heteroduplex formation; MLPA: Multiple liamplification; MSI(-H): microsatellite instability (high); ND: Not described; NAm2:Number fulfilling Amsterdam II criteria; Ntotal: total number of studied C

reaction; SSCP: Single-stranded conformation polymorphism 

Conversion analysis was not used in any study. ♦: Gene screening has been used ∅: Analysis for large deletions has been used 

a Contains two malignant tumors that are not CRC, which could not be separated (>95% are CRC) 
F-25

Appendix F-11: Ability of Amsterdam II criteria to identify MLH1 and MSH2 mutation carriers among unselected coprobands.

Mutation No mutation D

(9

Study, year (Ref ID); Country Single-/multi-center 

A. Study sample characteristics 

B. Verification bias 

C. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

NAm2/ 

Ntotal

Am2 

(+) 

Am2 

(-) 

Am2 

(+) 

Am2 

(-) 

Se

Genetic testing irrespectively of MSI or IHC test results Salovaara, 2000 (1740) Finland Multi-center 

A. Selection of all new unrelated 

CRC from 9 hospitals between 03/1996 and 06/1998 

B. Yes: patients without MSI were 

screened only for MLH1 founder mutations 

C. 67, ND 

A. PCR Æ Sequencing 

∅ PCR for founder mutations 

in MLH1 gene 

B. 17/1 

C. Literature; comparison with 

non-cancer controls 

5/535 4 14 1 516 2

Genetic testing only in patients selected after MSI and/or ICH 

Assuming no mutations in the absetumors with negative immunostaini

Pinol, 2005 (52) Spain Multi-center 

A. Selection of newly diagnosed 

CRC from 25 centers. 

B. Yes: patients without MSI or with 

negative immunostaining were not sequenced 

C. 70, 60 

A. CRC selected by MSI/IHC:

PCR Æ Sequencing 

∅ also MLPA 

B. 4/7 

C. Predicted transcript 

alteration; literature and databases 

22/ 

1222 

4 7 18 1197 36 

As mentioned in the methods, especially for unselected CRC populations we accepted that patients with MSI negative tumors would be negative for m

Am2: Amsterdam II criteria; CRC: colorectal cancer; MLPA: Multiple ligation-dependent probe amplification; MSI(-H): microsatellite instability (high); N

NAm2:Number fulfilling Amsterdam II criteria; Ntotal: total number of studied CRC; PCR: polymerase chain reaction 

Conversion analysis or gene screening were not used in these two studies. ∅: Analysis for large deletions has been used
F-26

Appendix F-12: Ability of Amsterdam II criteria to identify MLH1 and MSH2 mutation carriers among selected color

fulfilling the Revised Bethesda criteria

Mutation No mutation D

(9

Study, year (Ref ID); Country Single-/multi-center 

A. Study sample characteristics 

B. Verification bias 

C. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

NAm2/ 

Ntotal

Am2 

(+) 

Am2 

(-) 

Am2 

(+) 

Am2 

(-) 

Se

Wolf, 2005 (123) Austria Single-center 

A. Retrospective cohort of cancer 

patients fulfilling the modified Bethesda criteria 

B. No C. ND, 48 

A. PCR Æ Sequencing 

B. 12/7 

C. Predicted transcription 

alteration; literature; comparison with healthy controls 

35/81 13 6 22 40 68 

Am2: Amsterdam II criteria; CRC: colorectal cancer; ND: Not described; NAm2:Number fulfilling Amsterdam II criteria; Ntotal: total number of studied CR

reaction Conversion analysis, gene screening or deletion analysis methods were not used.
F-27

Appendix F-13: Ability of Amsterdam II criteria to identify MLH1 and MSH2 mutation carriers among selected color

fulfilling the Bethesda guidelines

Mutation No mutation D

(9

Study, year (Ref ID); Country Single-/multi-center 

A. Study sample characteristics 

B. Verification bias 

C. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

NAm2/ 

Ntotal

Am2 

(+) 

Am2 

(-) 

Am2 

(+) 

Am2 

(-) 

Se

Genetic testing irrespectively of MSI test results De Abajo 2005; Spain Single-center 

A. Selection among referrals to a 

specialized center 

B. No C. ND; ND 

A. PCR Æ DGGE Æ

sequencing (MSH6 assessed in MLH1/MSH2 negative cases) 

B. 24/17 (and 3 MSH6) 

C. Predicted non-conservative 

transcription alteration; literature; comparison with healthy controls 

67/ 

132 

39 5 28 37 89 

Syngal, 2000 (1672) & Wahlberg, 2002 (1158); US Single-center 

A. Selection among referrals to 

specialized center 

B. No C. ND; ND 

A. PCR Æ Sequencing 

B. 18 (MLH1 and MSH2) 

C. Predicted transcription 

alteration; literature 

34/70 14 4 20 32 78 

Genetic testing only in patients selected after MSI Pistorius, 2000 Among patients with MSI-H tumors(1590); Germany & Czech Republic Multi-center 

A. Selection among referrals to 

tertiary center: Bethesda guidelines met 

B. Yes: patients without MSI were 

not genetically tested 

C. ND, ND 

A. CRC with MSI:

PCR Æ Sequencing 

B. 6/8 (1 MSH6 mutation also 

found) 

C. Predicted transcript 

alteration; literature 

19/72 

12 3 5 18 80 

Only Pistorious et al. assessed genes other than MLH1 and MSH2, namely the MSH6 gene. Am2: Amsterdam II criteria; CRC: colorectal cancer; MSI(-H): microsatellite instability (high); ND: Not described; NAm2:Number fulfilling Amsterdam II c

studied CRC; PCR: polymerase chain reaction Conversion analysis, deletion analysis or gene screening were not used in these two studies. 
F-28

Appendix F-14: Ability of modified Amsterdam criteria to identify MLH1 and MSH2 mutation carriers among selecteprobands

Mutation No 

mutation 

(

Study, year (Ref ID); Country Single-/multi-center 

A. Comments on sample 

characteristics 

B. Verification bias C. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of 

deleterious mutations 

NAmMod/ 

Ntotal

Am 

Mod 

(+) 

Am 

Mod 

(-) 

Am 

Mod 

(+) 

Am 

Mod 

(-) 

S

Wolf, 2005 (123) Austria Single-center 

A. Selection from a retrospective 

cohort of cancer patients fulfilling the modified Bethesda criteria a

B. No C. ND, 48 

A. PCR Æ Sequencing B. 12/7 

D. Predicted transcription 

alteration; literature; comparison with healthy controls 

52/81 16 3 36 26 

Syngal, 2000 (1672) & Wahlberg, 2002 (1158); US Single-center 

A. Selection among referrals to 

specialized center 

B. No C. ND; ND 

A. PCR Æ Sequencing 

B. 18 (MLH1 and MSH2) 

C. Predicted transcription 

alteration; literature 

39/70 13 5 26 26 

AmMod: modified Amsterdam criteria; CRC: colorectal cancer; ND: Not described; NAmMod:Number fulfilling Amsterdam criteria; Ntotal: total number of 

Polymerase chain reaction. None of the studies used deletion analysis, conversion analysis or gene screening methods to detect mutations. No other genes apart from MLH1 an

a May include some patients without CRC.
F-29

Appendix F-15: Ability of Bethesda guidelines to identify MLH1 and MSH2 mutation carriers 

Mutation No 

mutation 

Dia

(95% 

Study, year (Ref ID); Country Single-/multicenter 

A. Comments on sampling 

characteristics 

B. Verification bias C. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

NBeth/ 

Ntotal

Beth 

(+) 

Beth 

(-) 

Beth 

(+) 

Beth 

(-) 

Sen

Genetic Testing irrespectively of MSI/IHC results De Abajo 2005; Spain Single-center 

A. Selection among referrals to a 

specialized center 

B. No C. ND; ND 

A. PCR Æ DGGE Æ

sequencing (MSH6 assessed in MLH1/MSH2 negative cases) 

B. 24/17 (and 3 MSH6) 

C. Predicted non-conservative 

transcription alteration; literature; comparison with healthy controls 

67/ 

132 

44 0 60 28 

(92

Wolf, 2005 (123) Austria Single-center 

A. Retrospective cohort of cancer 

patients fulfilling the modified Bethesda criteria 

B. No C. ND, 48 

A. PCR Æ Sequencing 

B. 12/7 

C. Predicted transcription 

alteration; literature; comparison with healthy controls 

72/81 19 0 53 9 

(82

Syngal, 2000 (1672) & Wahlberg, 2002 (1158); US Single-center 

A. Selection among referrals to 

specialized center 

B. No C. ND; ND 

A. PCR Æ Sequencing 

B. 18 (MLH1 and MSH2) 

C. Predicted transcription 

alteration; literature 

56/70 17 1 39 13 

(73

Genetic testing only in patients selected after MSI and/or ICH

Assuming no mutations in the absenctumors with negative immunostaining

Pinol, 2005 (52) Spain Multi-center 

A. Selection of newly diagnosed 

CRC from 25 centers. 

B. Yes: patients without MSI or 

with negative immunostaining were not sequenced 

C. 70, 60 

A. CRC selected by MSI/IHC:

PCR Æ Sequencing 

∅ also MLPA 

B. 4/7 

C. Predicted transcript 

alteration; literature and databases 

224/ 

1222 

8 3 216 995 73 

Raedle, 2001 A. Consecutive referrals to a A. CRC with MSI: 58/ Among patients with MSI-H tumors: 
F-30

(1363) Germany Multi-center 

tertiary center. 

B. Yes: patients without MSI were 

not tested for mutations 

C. 52, 55 

PCRÆ Sequencing 

B. 8/4 (1 had mutation in both 

genes) 

C. Predicted transcript 

alteration; literature 

125 11 0 6 5 

(72

Beth: Bethesda guidelines; CRC: colorectal cancer; IHC: immunohistochemistry; MLPA: Multiple ligation-dependent probe amplification; MSI: microsa

described; Ntotal: total number of studied CRC; PCR: Polymerase chain reaction 

∅: Deletion analysis (detection of large genomic deletions) was used No study used gene screening methods or conversion analysis. All studies assessed only MLH1 and MSH2 genes. 
F-31

Appendix F-16: Ability of Bethesda guidelines to identify MLH1 and MSH2 mutation carriers among unselected patcancer 

Mutation No 

mutation 

Dia

(95% 

Study, year (Ref ID); Country Single-/multicenter 

A. Comments on sampling 

characteristics 

B. Verification bias C. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

NBeth/ 

Ntotal

Beth 

(+) 

Beth 

(-) 

Beth 

(+) 

Beth 

(-) 

Sen

Assuming no mutations in the absenctumors with negative immunostaining

Pinol, 2005 (52) Spain Multi-center 

A. Selection of newly diagnosed 

CRC from 25 centers. 

B. Yes: patients without MSI or 

with negative immunostaining were not sequenced 

C. 70, 60 

A. CRC selected by MSI/IHC:

PCR Æ Sequencing 

∅ also MLPA 

B. 4/7 

C. Predicted transcript 

alteration; literature and databases 

224/ 

1222 

8 3 216 995 73 

Beth: Bethesda guidelines; CRC: colorectal cancer; IHC: immunohistochemistry; MLPA: Multiple ligation-dependent probe amplification; MSI: microsa

described; Ntotal: total number of studied CRC; PCR: Polymerase chain reaction 

∅: Deletion analysis (detection of large genomic deletions) was used Pinol et al. did not use gene screening methods or conversion analysis. They assessed only the MLH1 and MSH2 genes. 
F-32

Appendix F-17: Ability of revised Bethesda guidelines to identify MLH1 and MSH2 mutation carriers among unselecolorectal cancer 

Mutation No 

mutation 

Dia

(95% 

Study, year (Ref ID); Country Single-/multicenter 

A. Comments on sampling 

characteristics 

B. Verification bias C. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

NBeth/ 

Ntotal

Beth 

Rev 

(+) 

Beth 

Rev 

(-) 

Beth 

Rev 

(+) 

Beth 

Rev 

(-) 

Sen

Assuming no mutations in the absenctumors with negative immunostaining

Pinol, 2005 (52) Spain Multi-center 

A. Selection of newly diagnosed 

CRC from 25 centers. 

B. Yes: patients without MSI or 

with negative immunostaining were not sequenced 

C. 70, 60 

A. CRC selected by MSI/IHC:

PCR Æ Sequencing 

∅ also MLPA 

B. 4/7 

C. Predicted transcript 

alteration; literature and databases 

287/ 

1222 

10 1 277 934 

(59

Beth Rev: Revised Bethesda guidelines; CRC: colorectal cancer; IHC: immunohistochemistry; MLPA: Multiple ligation-dependent probe amplification; 

instability; ND: Not described; Ntotal: total number of studied CRC; PCR: Polymerase chain reaction 

∅: Deletion analysis (detection of large genomic deletions) was used Pinol et al. did not use gene screening methods or conversion analysis. They assessed only the MLH1 and MSH2 genes. 
F-33

Appendix F-18: Ability of Ability of young age at diagnosis (early disease onset) to identify MLH1 and MSH2 mutat

colorectal cancer probands 

Mutation No mutation D

(9

Study, year (Ref ID); Country Single-/multi-center 

A. Definition of early onset 

B. Study sample characteristics 

C. Verification bias D. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

Ntotal

Early 

onset 

No early 

onset 

Early 

onset 

No early 

onset 

S

Genetic testing irrespectively of tumor microsatellite instability status Salovaara, 2000 (1740) Finland Multi-center 

A. <50 y 

B. Selection of all new unrelated 

CRC from 9 hospitals between 03/1996 and 06/1998 

C. Yes: patients without MSI were 

screened only for MLH1 founder mutations 

D. 67, ND 

A. PCR 

Æ Sequencing 

∅ PCR for founder mutations in 

MLH1 gene 

B. 17/1 

C. Literature; comparison with noncancer controls 

535 5 13 40 477 2

Aaltonen, 1998 (2282) Finland Multi-center 

A. <50 y 

B. Selection of all new unrelated 

CRC from 9 hospitals between 05/1994 and 04/1996 

C. Yes: patients without MSI were 

screened only for MLH1 founder mutations 

D. ND, ND 

A.

∅All CRC: PCR for founder 

mutations 1 & 2 in MLH1 

♦CRC with MSI: (some) PCR 

Æ

DGGE 

Æ Sequencing 

(Remaining) PCR 

Æ Sequencing 

B. 9/1 

C. Literature; comparison with 

healthy controls 

509 4 6 12 487 6

Colombino, 2005 (1058); Italy Single-center 

A. <45 y 

B. Consecutive CRC cases 

enrolled over 3 years in a tertiary center b

C. Yes: Patients without family 

history of cancer were screened only for the mutations identified in the familial cases 

D. 62; 49 

A.

♦PCR

Æ DHPLC

Æ Sequencing 

of abnormal patterns 

B. 11/10 C. Compared to 103 people without 

cancer 

362a 6 10 5 82 3

Genetic testing only in patients selected after MSI and/or ICH

Assuming no mutations in the absetumors with negative immunostaini

Pinol, 2005 (52) Spain Multi-center 

A. <50 y 

B. Selection of newly diagnosed 

CRC from 25 centers. 

C. Yes: patients without MSI or with 

negative immunostaining were not sequenced 

D. 70, 60 

A. CRC selected by MSI/IHC:

PCR 

Æ Sequencing 

∅ also MLPA 

B. 4/7 

C. Predicted transcript alteration; 

literature and databases 

1222 

3 8 55 1156 2
F-34

Mutation No mutation D

(9

Study, year (Ref ID); Country Single-/multi-center 

A. Definition of early onset 

B. Study sample characteristics 

C. Verification bias D. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

Ntotal

Early 

onset 

No early 

onset 

Early 

onset 

No early 

onset 

S

Samowitz 2001, (34); Assuming no mutations in the abseUS Multicenter 

A. <55y 

B. Selection among incident CRC C. Yes: patients without suggestive 

MSI results were not sequenced 

D. ND, ND 

A. CRC selected by MSI: 

bPCR Æ Sequencing 

∅ Also PCR for founder mutations 

in the MLH1 gene 

B. 5/3c

C. Predicted transcript alteration; 

literature 

1066 

4 3 154 863 5

Studies are ordered by quality and then by decreasing number of patients available for the calculation of sensitivity and specificity (2 by 2 tables). Non

conversion analysis to detect mismatch repair gene mutations. Am1/2: Amsterdam I criteria/II; CRC: colorectal cancer; DGGE: Denaturing gradient gel electrophoresis; DHPLC: denaturating high performance liquid 

Multiple ligation-dependent probe amplification; MSI: microsatellite instability; ND: Not described; Ntotal: total number of studied CRC; PCR: polymeras

♦: Gene screening method ∅: Detection of large genomic deletions 

a Not clear if all CRC probands belong to unrelated families; only data on 103 familial cases are analyzed here, since the pertinent infor

sporadic cases.

b 130 out of 171 of people with tumors with MSI instability could be genetically tested. c One patient had mutations both in the MLH1 and in the MSH2 gene
F-35

Appendix F-19: Ability of Ability of young age at diagnosis (early disease onset) to identify MLH1 and MSH2 mutat

unselected colorectal cancer probands 

Mutation No mutation D

(9

Study, year (Ref ID); Country Single-/multi-center 

A. Definition of early onset B. Study sample characteristics 

C. Verification bias 

D. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

Ntotal

Early 

onset 

No early 

onset 

Early 

onset 

No early 

onset 

S

Genetic testing irrespectively of tumor microsatellite instability status Salovaara, 2000 (1740) Finland Multi-center 

A. <50 y 

B. Selection of all new unrelated 

CRC from 9 hospitals between 03/1996 and 06/1998 

C. Yes: patients without MSI were 

screened only for MLH1 founder mutations 

D. 67, ND 

A. PCR Æ Sequencing 

∅ PCR for founder mutations in 

MLH1 gene 

B. 17/1 

C. Literature; comparison with noncancer controls 

535 5 13 40 477 2

Aaltonen, 1998 (2282) Finland Multi-center 

A. <50 y 

B. Selection of all new unrelated 

CRC from 9 hospitals between 05/1994 and 04/1996 

C. Yes: patients without MSI were 

screened only for MLH1 founder mutations 

D. ND, ND 

A. ∅All CRC: PCR for founder 

mutations 1 & 2 in MLH1 

♦CRC with MSI: (some) PCR Æ

DGGE Æ Sequencing (Remaining) PCR Æ Sequencing 

B. 9/1 

C. Literature; comparison with 

healthy controls 

509 4 6 12 487 6

Genetic testing only in patients selected after MSI and/or ICH

Assuming no mutations in the absetumors with negative immunostaini

Pinol, 2005 (52) Spain Multi-center 

A. <50 y 

B. Selection of newly diagnosed 

CRC from 25 centers. 

C. Yes: patients without MSI or with 

negative immunostaining were not sequenced 

D. 70, 60 

A. CRC selected by MSI/IHC:

PCR Æ Sequencing 

∅ also MLPA 

B. 4/7 

C. Predicted transcript alteration; 

literature and databases 

1222 

3 8 55 1156 2

Samowitz 2001, (34); Assuming no mutations in the abseUS Multicenter 

A. <55y 

B. Selection among incident CRC C. Yes: patients without suggestive 

MSI results were not sequenced 

D. ND, ND 

A. CRC selected by MSI: 

bPCR Æ Sequencing 

∅ Also PCR for founder mutations 

in the MLH1 gene 

B. 5/3c

C. Predicted transcript alteration; 

literature 

1066 

4 3 154 863 5
F-36

Studies are ordered by quality and then by decreasing number of patients available for the calculation of sensitivity and specificity (2 by 2 tables). Non

conversion analysis to detect mismatch repair gene mutations. Am1/2: Amsterdam I criteria/II; CRC: colorectal cancer; DGGE: Denaturing gradient gel electrophoresis; DHPLC: denaturating high performance liquid 

Multiple ligation-dependent probe amplification; MSI: microsatellite instability; ND: Not described; Ntotal: total number of studied CRC; PCR: polymeras

♦: Gene screening method ∅: Detection of large genomic deletions
F-37

Appendix F-20: Ability of familial history of malignancy to identify MLH1 and MSH2 mutation carriers among color

Mutation No mutation 

Study, year (Ref ID); Country Single-/multicenter 

A. Definition of familial history of cancer 

B. Definition of sporadic cancer C. Comments on sample characteristics 

D. Verification bias 

E. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

Ntotal

Familial 

Cancer Hx 

Sporadic 

Cancer

Familial 

Cancer Hx 

Sporadic 

Cancer 

Genetic testing irrespectively of tumor microsatellite instability status Salovaara, 2000 (1740); Finland Multi-center 

A. 1st degree relative with CRC or 

endometrial cancer 

B. All other CRC C. Selection of all new unrelated CRC from 9 

hospitals between 03/1996 and 06/1998 

D. Yes: patients without MSI were screened 

only for MLH1 founder mutations 

E. 67, ND 

A. PCR 

Æ Sequencing 

∅ PCR for founder mutations 

in MLH1 gene 

B. 17/1 

C. Literature; comparison 

with non-cancer controls 

535 15 3 62 455 

Aaltonen, 1998 (2282); Finland Multi-center 

A. 1st degree relative with CRC or 

endometrial cancer 

B. All other CRC C. Selection of all new unrelated CRC from 9 

hospitals between 05/1994 and 04/1996 

D. Yes: patients without MSI were screened 

only for MLH1 founder mutations 

E. ND, ND 

A.

∅ All CRC: PCR for founder 

mutations 1 & 2 in MLH1 

♦CRC with MSI: (some) 

PCR 

Æ DGGE 

Æ

Sequencing (Remaining) PCR 

Æ

Sequencing 

B. 9/1 

C. Literature; comparison 

with healthy controls 

509 9 1 71 428 

Zhu, 2005 (138); China Single center 

A. CRC with cancer in family B. Apparently sporadic CRC consisting of two 

subgroups (<50y or 

≥50y at diagnosis) 

C. Study subgroups assembled without a 

clear selection process 

D. No E. ND, ND 

A.

∅ MLPA 

Æ Sequencing of 

detected aberrations 

B. 4/5 

C. Unclear 

78 7 2 38 31 

Wang, 1999 (1939); France Single-center 

A. CRC from families fulfilling Am1 criteria, or 

from families with 1 or 2 Amsterdam criteria missing, or families with aggregation of HNPCCC-related tumors 

B. CRC with no family Hx aged <50y at 

diagnosis 

C. Sample assembled from referrals to a 

A.

∅ RT-PCR

Æ IVSP; 

♦ PCR

ÆHD

ÆSequencing; 

in vivo MLH1 protein 

expression 

B. 19/7 

C. Predicted transcription 

alteration; literature 

75 26 0 37 12 
F-38

Mutation No mutation 

Study, year (Ref ID); Country Single-/multicenter 

A. Definition of familial history of cancer 

B. Definition of sporadic cancer C. Comments on sample characteristics 

D. Verification bias 

E. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

Ntotal

Familial 

Cancer Hx 

Sporadic 

Cance

r

Familial 

Cancer Hx 

Sporadic 

Cancer 

genetic consultation center 

D. No E. ND, ND 

Yuan 1998 (2229); Korea Single-center 

A. CRC fulfilling the Korean HNPCC criteria B. CRC without family Hx of colorectal cancer 

aged <40y at diagnosis 

C. Sample assembled with unclear selection 

process 

D. No E. Familial: 50, 65; sporadic: 39, 49 

A.♦ PCRÆ SSCP Æ

Sequencing of abnormal products 

B. 2/1 

C. Predicted nonconservative transcription 

alteration; literature; comparison with noncancer controls 

76 7 1 24 44 

Moslein 1996 (2545); US & Germany Multi-center 

A. CRC fulfilling Am1; and CRC with “familial 

Hx of CRC” 

B. CRC without a family Hx of CRC C. Sample assembled with unclear selection 

process 

D. No E. 51, ND 

A. PCRÆsequencing 

B. 7/6 

C. Predicted nonconservative transcription 

alteration; literature 

46a 11 1 18 6 

Lee, 2005 (105) Singapore Single-center 

A. CRC fulfilling Amsterdam II criteria; with 3 

CRC in 1st degree family; and with 2 CRC 

in 1st or 2nd degree family 

B. CRC with no familial Hx and age <40y or 

with multiple HNPCC-related cancers 

C. Sample selected from referrals to a tertiary 

center 

D. No E. 39 (median); 65 

A. PCR Æ Sequencing 

B. 6/1 

C. Predicted transcript 

alteration; literature 

46b 6 1 21 17 
F-39

Mutation No mutation 

Study, year (Ref ID); Country Single-/multicenter 

A. Definition of familial history of cancer 

B. Definition of sporadic cancer C. Comments on sample characteristics 

D. Verification bias 

E. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

Ntotal

Familial 

Cancer Hx 

Sporadic 

Cance

r

Familial 

Cancer Hx 

Sporadic 

Cancer 

Genetic testing only in patients selected after MSI and/or ICH 

Assuming no mutations in the atumors with negative immunost

Pinol, 2005 (52); Spain Multi-center 

A. 1st degree relative with CRC of 

endometrial cancer 

B. All other CRC C. Selection of newly diagnosed CRC from 25 

centers. 

D. Yes: patients without MSI or with negative 

immunostaining were not sequenced 

E. 70, 60 

A. CRC selected by 

MSI/IHC:PCR Æ Sequencing 

∅ also MLPA 

B. 4/7 

C. Predicted transcript 

alteration; literature and databases 

1222 

6 5 151 1060 

Samowitz 2001, Assuming no mutations in the a(34); US Multicenter 

A. Family history of CRC B. All other patients C. Selection among incident CRC 

D. Yes: patients without suggestive MSI 

results were not sequenced 

E. ND, ND 

A. CRC selected by MSI: 

cPCR Æ Sequencing 

∅ Also PCR for founder 

mutations in the MLH1 gene 

B. 5/3d

C. Predicted transcript 

alteration; literature 

1066 

4 3 147 870 

Studies are ordered by quality and then by decreasing number of patients available for the calculation of sensitivity and specificity (2 by 2 tables). Non

conversion analysis to detect mismatch repair gene mutations. Am1: Amsterdam I criteria; CRC: colorectal cancer; DGGE: Denaturing gradient gel electrophoresis; HD: heteroduplex formation; Hx: History; IHC: imin vitro synthesized protein assay; MSI: microsatellite instability; ND: Not described; Ntotal: total number of studied CRC; PCR: polymerase chain react

conformation polymorphism ♦: Gene screening method ∅: Detection of large genomic deletions 

a Interestingly, only 26/46 patients were described to have had CRC in an analytic petioent-level description, and only these are analyzed b Contains 2 HNPCC-related non-CRC tumors that could not be separated (>95% of tumors in these data are CRC) c 130 out of 171 of people with tumors with MSI instability could be genetically tested. d One patient had mutations both in the MLH1 and in the MSH2 gene
F-40

Appendix F-21: Ability of familial history of malignancy to identify MLH1 and MSH2 mutation carriers among unselcancer probands 

Mutation No mutation 

Study, year (Ref ID); Country Single-/multicenter 

A. Definition of familial history of cancer 

B. Definition of sporadic cancer C. Comments on sample characteristics 

D. Verification bias 

E. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

Ntotal

Familial 

Cancer Hx 

Sporadic 

Cance

r

Familial 

Cancer Hx 

Sporadic 

Cancer 

Genetic testing irrespectively of tumor microsatellite instability status Salovaara, 2000 (1740); Finland Multi-center 

A. 1st degree relative with CRC or 

endometrial cancer 

B. All other CRC C. Selection of all new unrelated CRC from 9 

hospitals between 03/1996 and 06/1998 

D. Yes: patients without MSI were screened 

only for MLH1 founder mutations 

E. 67, ND 

A. PCR Æ Sequencing 

∅ PCR for founder mutations 

in MLH1 gene 

B. 17/1 

C. Literature; comparison 

with non-cancer controls 

535 15 3 62 455 

Aaltonen, 1998 (2282); Finland Multi-center 

A. 1st degree relative with CRC or 

endometrial cancer 

B. All other CRC C. Selection of all new unrelated CRC from 9 

hospitals between 05/1994 and 04/1996 

D. Yes: patients without MSI were screened 

only for MLH1 founder mutations 

E. ND, ND 

A. ∅ All CRC: PCR for founder 

mutations 1 & 2 in MLH1 

♦CRC with MSI: (some) 

PCR Æ DGGE Æ

Sequencing (Remaining) PCR ÆSequencing 

B. 9/1 

C. Literature; comparison 

with healthy controls 

509 9 1 71 428 

Genetic testing only in patients selected after MSI and/or ICH 

Assuming no mutations in the atumors with negative immunost

Pinol, 2005 (52); Spain Multi-center 

A. 1st degree relative with CRC of 

endometrial cancer 

B. All other CRC C. Selection of newly diagnosed CRC from 25 

centers. 

D. Yes: patients without MSI or with negative 

immunostaining were not sequenced 

E. 70, 60 

A. CRC selected by 

MSI/IHC:PCR Æ Sequencing 

∅ also MLPA 

B. 4/7 

C. Predicted transcript 

alteration; literature and databases 

1222 

6 5 151 1060 

Samowitz 2001, (34); 

A. Family history of CRC B. All other patients 

A. CRC selected by MSI: 

aPCR Æ Sequencing 

1066 Assuming no mutations in the a
F-41

Mutation No mutation 

Study, year (Ref ID); Country Single-/multicenter 

A. Definition of familial history of cancer 

B. Definition of sporadic cancer C. Comments on sample characteristics 

D. Verification bias 

E. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

Ntotal

Familial 

Cancer Hx 

Sporadic 

Cance

r

Familial 

Cancer Hx 

Sporadic 

Cancer 

US Multicenter 

C. Selection among incident CRC 

D. Yes: patients without suggestive MSI 

results were not sequenced 

E. ND, ND 

∅ Also PCR for founder 

mutations in the MLH1 gene 

B. 5/3b

C. Predicted transcript 

alteration; literature 

4 3 147 870 

Studies are ordered by quality and then by decreasing number of patients available for the calculation of sensitivity and specificity (2 by 2 tables). Non

conversion analysis to detect mismatch repair gene mutations. Am1: Amsterdam I criteria; CRC: colorectal cancer; DGGE: Denaturing gradient gel electrophoresis; Hx: History; IHC: immunohistochemistry; MSI: m

Not described; Ntotal: total number of studied CRC; PCR: polymerase chain reaction 

♦: Gene screening method ∅: Detection of large genomic deletions 

a 130 out of 171 of people with tumors with MSI instability could be genetically tested. b One patient had mutations both in the MLH1 and in the MSH2 gene
F-42

Appendix F-22: Ability of presence of multiple tumors to identify MLH1 and MSH2 mutation carriers among unsele

probands 

Mutation No mutation 

Study, year (Ref ID); Country Single-/multicenter 

A. Definition of multiple tumors 

B. Comments on sample characteristics C. Verification bias 

D. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

Ntotal

Multiple 

tumors 

No multiple 

tumors 

Multiple 

tumors 

No multiple 

tumors 

Genetic testing irrespectively of tumor microsatellite instability status Salovaara, 2000 (1740); Finland Multi-center 

A. Synchronous or metachronous 

endometrial cancer or CRC 

B. Selection of all new unrelated CRC from 9 

hospitals between 03/1996 and 06/1998 

C. Yes: patients without MSI were screened 

only for MLH1 founder mutations 

D. 67, ND 

A. PCR 

Æ Sequencing 

∅ PCR for founder mutations 

in MLH1 gene 

B. 17/1 

C. Literature; comparison 

with non-cancer controls 

535 7 11 5 512 

Aaltonen, 1998 (2282); Finland Multi-center 

A. Synchronous or metachronous 

endometrial cancer or CRC 

B. Selection of all new unrelated CRC from 9 

hospitals between 05/1994 and 04/1996 

C. Yes: patients without MSI were screened 

only for MLH1 founder mutations 

D. ND, ND 

A.

∅ All CRC: PCR for founder 

mutations 1 & 2 in MLH1 

♦CRC with MSI: (some) 

PCR 

Æ DGGE 

Æ

Sequencing (Remaining) PCR 

Æ

Sequencing 

B. 9/1 

C. Literature; comparison 

with healthy controls 

509 4 6 12 487 

Genetic testing only in patients selected after MSI and/or ICH 

Assuming no mutations in the atumors with negative immunost

Pinol, 2005 (52); Spain Multi-center 

A. Synchronous or metachronous 

endometrial cancer or CRC 

B. Selection of newly diagnosed CRC from 25 

centers. 

C. Yes: patients without MSI or with negative 

immunostaining were not sequenced 

D. 70, 60 

A. CRC selected by 

MSI/IHC:PCR 

Æ Sequencing 

∅ also MLPA 

B. 4/7 

C. Predicted transcript 

alteration; literature and databases 

1222 

4 7 90 1121 

Studies are ordered by quality and then by decreasing number of patients available for the calculation of sensitivity and specificity (2 by 2 tables). Non

conversion analysis to detect mismatch repair gene mutations. CRC: colorectal cancer; DGGE: Denaturing gradient gel electrophoresis; Hx: History; IHC: immunohistochemistry; MSI: microsatellite instability; ND: N

number of studied CRC; PCR: polymerase chain reaction ♦: Gene screening method ∅: Detection of large genomic deletions
F-43

Appendix F-23: Ability of presence of combined family history of colorectal cancer, young age at onset or presencto identify MLH1 and MSH2 mutation carriers among unselected colorectal cancer probands 

Mutation No mutation 

Study, year (Ref ID); Country Single-/multicenter 

A. Definition of combined criteria 

B. Comments on sample characteristics C. Verification bias 

D. Mean age (y); Males (%) 

A. Genetic testing B. MLH1/MSH2 C. Definition of deleterious 

mutations 

Ntotal

Combined 

criteria 

Other 

Combined 

criteria 

Other 

Genetic testing irrespectively of tumor microsatellite instability status Salovaara, 2000 (1740); Finland Multi-center 

A. Age <50 or CRC or endometrial cancer in 

1st degree family or personal history of 

CRC or endometrial cancer 

B. Selection of all new unrelated CRC from 9 

hospitals between 03/1996 and 06/1998 

C. Yes: patients without MSI were screened 

only for MLH1 founder mutations 

D. 67, ND 

A. PCR 

Æ Sequencing 

∅ PCR for founder mutations 

in MLH1 gene 

B. 17/1 

C. Literature; comparison 

with non-cancer controls 

535 17 1 100 417 

Aaltonen, 1998 (2282); Finland Multi-center 

A. Age <50 or CRC or endometrial cancer in 

1st degree family or personal history of 

CRC or endometrial cancer 

B. Selection of all new unrelated CRC from 9 

hospitals between 05/1994 and 04/1996 

C. Yes: patients without MSI were screened 

only for MLH1 founder mutations 

D. ND, ND 

A.

∅ All CRC: PCR for founder 

mutations 1 & 2 in MLH1 

♦CRC with MSI: (some) 

PCR 

Æ DGGE 

Æ

Sequencing (Remaining) PCR 

Æ

Sequencing 

B. 9/1 

C. Literature; comparison 

with healthy controls 

509 10 0 112 387 

Genetic testing only in patients selected after MSI and/or ICH 

Assuming no mutations in the atumors with negative immunost

Pinol, 2005 (52); Spain Multi-center 

A. Age <50 or CRC or endometrial cancer in 

1st degree family or personal history of 

CRC or endometrial cancer 

B. Selection of newly diagnosed CRC from 25 

centers. 

C. Yes: patients without MSI or with negative 

immunostaining were not sequenced 

D. 70, 60 

A. CRC selected by 

MSI/IHC:PCR 

Æ Sequencing 

∅ also MLPA 

B. 4/7 

C. Predicted transcript 

alteration; literature and databases 

1222 

8 3 306 905 

Studies are ordered by quality and then by decreasing number of patients available for the calculation of sensitivity and specificity (2 by 2 tables). Non

conversion analysis to detect mismatch repair gene mutations. CRC: colorectal cancer; DGGE: Denaturing gradient gel electrophoresis; Hx: History; IHC: immunohistochemistry; MSI: microsatellite instability; ND: N

number of studied CRC; PCR: polymerase chain reaction ♦: Gene screening method 
F-44

∅: Detection of large genomic deletions 
F-45

Appendix F-24: Ability of microsatellite instability to identify MLH1 and MSH2 mutation carriers among colorectal ca

Mutation No mutation Diagnostic performa

(95% 

confidence interv

Study, year (Ref ID); Country Single-/multi-center 

A. Comments on sample characteristics B. Verification bias 

C. Mean age (y); Males (%) 

D. Definition of MSI 

Ntotal

MSI MSS MSI MSS Sensitivity [%] Specif

A. Selection of all incident CRC aged <55 y at 

diagnosis between 02/1999 and 07/2003 

B. No C. 48; 53 D. ≥2 out of ≥5 markers (MSI-H) 

Barnetson, 2006 20 10 24 298 67 (47, 83) 93 ((NA) Scotland Multi-center 

≥1 out of ≥5 markers (MSI-H&L) 

870a

28 2 50 272 93 (78, 99) 84 (

Salovaara, 2000 (1740) Finland Multi-center 

A. Selection of all new unrelated CRC from 9 

hospitals between 03/1996 and 06/1998 

B. Yes: patients without MSI were screened only 

for MLH1 founder mutations 

C. 67, ND D. MSI based on BAT26 only 

535 18 0 48 469 100 (81, 100) 91 (

Aaltonen, 1998 (2282) Finland Multi-center 

A. Selection of all new unrelated CRC from 9 

hospitals between 05/1994 and 04/1996 

B. Yes: patients without MSI were screened only 

for MLH1 founder mutations 

C. ND, ND D. ≥30% of 16 markers for tumor analyzed with 

fluorescence methods or ≥2 out of 7 (≈30%) markers analyzed with radioactive technique (MSI-H) 

509 10 0 53 446 100 (69, 100) 89 (
F-46

Mutation No mutation Diagnostic performa

(95% 

confidence interv

Study, year (Ref ID); Country Single-/multi-center 

A. Comments on sample characteristics B. Verification bias 

C. Mean age (y); Males (%) 

D. Definition of MSI 

Ntotal

MSI MSS MSI MSS Sensitivity [%] Specif

A. Randomly selected from a prospective cohort 

of all CRC with age of onset <45 y; only patients who received genetic testing are presented b

B. No C. <45y; ND D. >5 out of 10 markers (MSI-H) 

13b 5b Southey, 2005 (66); 4 36 72 (47, 90) 90 (

Australia Single-center 

>1 out of 10 markers (MSIH&L) 

105 

17 b 1b 16 60 94 (73, 100) 79 (

Wolf, 2005 (123); Austria Single-center 

A. Selection from a retrospective cohort of cancer 

patients fulfilling the modified Bethesda criteria 

B. No C. ND, 48 D. ≥30% out of up to 10 markers (MSI-H) 

81 13 0 9 33 100 (75, 100) 79 (

Syngal, 2000 (1672) & Wahlberg, 2002 (1158); US Single-center 

A. Selection among referrals to specialized 

centerc

B. No C. ND; ND D. ≥2 out of ≥5 markers (MSI-H) 

70c 14 0 14 20 100 (77, 100) 59 (

Farrington, 1998 (2205); Scotland Single-center 

A. Retrospective cohort of CRC aged <30 y at 

diagnosis who were still alive (since 1970) 

B. No C. <30y, ND D. ≥2 out of 7 markers (MSI-H) 

50 12 2 7 19 86 (57, 98) 73 (
F-47

Mutation No mutation Diagnostic performa

(95% 

confidence interv

Study, year (Ref ID); Country Single-/multi-center 

A. Comments on sample characteristics B. Verification bias 

C. Mean age (y); Males (%) 

D. Definition of MSI 

Ntotal

MSI MSS MSI MSS Sensitivity [%] Specif

A. Familial CRC selected from a population of 

1514 newly diagnosed CRC: patients fulfilling Amsterdam II criteria (in extended families and relaxing the age criterion to <55y) and familial CRC with early age of onset 

B. No C. ND; ND D. ≥2 out of ≥5 markers (MSI-H) 

Katballe, 2002 (1310) 10 0 3 20 100 (69, 100) 87 (& Christensen, 2002 (1038); Denmark Single-center 

≥1 out of ≥5 markers (MSI-H&L) 

45 

10 0 5 20 100 (69, 100) 80 (

A. Sample assembled with unclear selection 

process: we present analyses only among familial CRC who were at most 1 criterion short of fulfilling Am1 (n=17); study also included 17 apparently sporadic CRC aged <50y at diagnosis. 

B. Yes: not all sporadic CRC underwent MMR 

mutation testing (these are not included in this analysis) 

C. Oldest at diagnosis was 56, ND D. ≥10% of up to 23 markers (But all were ≥30%) 

(MSI-H) 

Dieumegard, 2000 9 0 6 9 100 (66, 100) 60 ((1791); France Multi-center 

≥10% of up to 23 markers (MSI-H&L) 

34d

9 0 6 9 100 (66, 100) 60 (

Curia, 1999 (1959); Italy Single-center 

A. Sampled from pathology registries, unclear 

selection criteria: HNPCC related cancerse

B. No 

C. <50y, ND D. ≥2 markers out of 3 markers or up to a total of 

7 markers (MSI-H) 

30e 1 0 15 3 100 (0, 100) 17 
F-48

Mutation No mutation Diagnostic performa

(95% 

confidence interv

Study, year (Ref ID); Country Single-/multi-center 

A. Comments on sample characteristics B. Verification bias 

C. Mean age (y); Males (%) 

D. Definition of MSI 

Ntotal

MSI MSS MSI MSS Sensitivity [%] Specif

Debniak,, 2000 (1784); Poland Single-center(?) 

A. Sampled from consecutive CRC, selection 

process not transparent. All patient with available data are included. 

B. Yes: Only 43/143 apparently sporadic CRC 

were tested, but it is unclear how they were selected 

C. ND, ND D. ≥2 out of ≥5 markers or ≥3 out of ≥10 

markers (MSI-H) 

168 5 1 8 54 83 (36, 100) 87 (

A. Selection among referrals to tertiary center: 

Amsterdam criteria, familial disease, onset <40y or multiple tumors 

B. No C. 39 (median); 65 D. ≥2 out of ≥5 markers (MSI-H) 

Lee, 2005 (105); 4 1 7 21 80 (28, 99) 75 (Singapore Single-center 

≥1 out of ≥5 markers (MSI-H&L) 

46f

4 1 9 21 80 (28, 99) 70 (

Moslein 1996 (2545) US & Germany Multi-center 

A. Sample assembled from various databases; 

we present analyses only among familial cases (39/46), and only those described to have CRC. Study also included 7 sporadic CRC.g

B. No 

C. 51, ND D. ≥30% of 9 to 34 markers analyzed (unclear 

which exactly) (MSI-H) 

46g 5 4 4 8 56 (21, 86) 67 (
F-49

Mutation No mutation Diagnostic performa

(95% 

confidence interv

Study, year (Ref ID); Country Single-/multi-center 

A. Comments on sample characteristics B. Verification bias 

C. Mean age (y); Males (%) 

D. Definition of MSI 

Ntotal

MSI MSS MSI MSS Sensitivity [%] Specif

Callistri, 2000 (1797); Italy Multi-center 

A. Study sample assembled with unclear 

selection process: Familial CRC fulfilling Am1 criteria or missing up to 2/3 Am1 criteria, or a CRC among 1st degree family or CRC 

diagnosis at age <50y, or multiple tumors in the same patient 

B. No C. ND; 56 (all sample) D. ≥2 out of ≥5 markers (MSI-H) 

40h 7 0 5 4 100 (59, 100) 44 (

Durno, 2005 (195); Canada Multi-center 

A. Retrospective cohort of CRC aged <24 y at 

diagnosis who were still alive (since 1960) 

B. No C. <24y, 38 D. ≥2 out of ≥5 markers, or (MSI-H) ≥40% out of 

up to 10 markers (MSI-H) 

16 5 0 3 1 100 (48, 100) 25 

Peel, 1999 (1660); US Multi-center 

A. Referral HNPCC cases, other than the 1134 

CRC probands who were also included but were not assessed with laboratory tests 

B. No C. ND; ND D. Unclear; at least 5 markers were used (MSIH?) 

11 3 0 1 5 100 (29, 100) 83 (3

Note that studies that assessed genetic mutations only in patients with MSI cannot be used to construct 2 by 2 tables for the ability of MSI testing to detesuch studies are not included in this table. Not included in the table are data from Lamberti 1999, where only the contrast of MSI-H versus combined MSextractable. CRC: colorectal cancer; MSI(-H/-L): microsatellite instability (-high/-low); MSS: microsatellite-stable (no instability); NA: Not applicable; ND: Not describe

studied CRC 

a Overall, 359 patients had available tumor tissue of good quality. Study assessed for mutations in the MSH6 gene also. b This study assessed MSH6 and hPSM2 also. The mutation counts include pathogenic mutations in these genes also. c Data from the Wahlberg et al. publication d Only 7/17 apparently sporadic CRC were genetically tested (unclear how they were selected). e Four were not CRC and were excluded from the calculations; numbers among patients with available tumors; only pathogenic mutations are shown.
F-50

f Included are 2 malignant tumors other than CRC that could not be separated. g Interestingly, despite the fact that all were characterized as CRC, in an analytic description not all were described with CRC. Only familial cases that w

included.

h Data out of 20 CRC (of 45 total studied patients) who missed at most 2 out of 3 Amsterdam I criteria are analyzed here, because MMR gene mutations 

among the remaining patients. 
F-51

Appendix F-25: Ability of microsatellite instability to identify MLH1 and MSH2 mutation carriers among unselected cprobands 

Mutation No mutation Diagnostic performa

(95% 

confidence interv

Study, year (Ref ID); Country Single-/multi-center 

A. Comments on sample characteristics B. Verification bias 

C. Mean age (y); Males (%) 

D. Definition of MSI 

Ntotal

MSIH MSS MSI- H MSS Sensitivity [%] Specif

Salovaara, 2000 (1740) Finland Multi-center 

A. Selection of all new unrelated CRC from 9 

hospitals between 0.3/1996 and 06/1998 

B. Yes: patients without MSI were screened only 

for MLH1 founder mutations 

C. 67, ND D. MSI based on BAT26 only 

535 18 0 48 469 100 (81, 100) 91 (

Aaltonen, 1998 (2282) Finland Multi-center 

A. Selection of all new unrelated CRC from 9 

hospitals between 05/1994 and 04/1996 

B. Yes: patients without MSI were screened only 

for MLH1 founder mutations 

C. ND, ND D. ≥30% of 16 markers for tumor analyzed with 

fluorescence methods or ≥2 out of 7 (≈30%) markers analyzed with radioactive technique (MSI-H) 

509 10 0 53 446 100 (69, 100) 89 (

Note that studies that assessed genetic mutations only in patients with MSI cannot be used to construct 2 by 2 tables for the ability of MSI testing to detesuch studies are not included in this table. CRC: colorectal cancer; MSI(-H): microsatellite instability (-high); MSS: microsatellite-stable (no instability); ND: Not described; Ntotal: total number of stu
F-52

Appendix F-26: Ability of microsatellite instability to identify MLH1 and MSH2 mutation carriers among colorectal ca

fulfilling the revised Bethesda criteria 

Mutation No mutation Diagnostic performa

(95% 

confidence interv

Study, year (Ref ID); Country Single-/multi-center 

A. Comments on sample characteristics B. Verification bias 

C. Mean age (y); Males (%) 

D. Definition of MSI 

Ntotal

MSI MSS MSI MSS Sensitivity [%] Specif

Wolf, 2005 (123); Austria Single-center 

A. Selection from a retrospective cohort of cancer 

patients fulfilling the modified Bethesda criteria 

B. No C. ND, 48 D. ≥30% out of up to 10 markers (MSI-H) 

81 13 0 9 33 100 (75, 100) 79 (

Note that studies that assessed genetic mutations only in patients with MSI cannot be used to construct 2 by 2 tables for the ability of MSI testing to detesuch studies are not included in this table. CRC: colorectal cancer; MSI(-H/-L): microsatellite instability (-high/-low); MSS: microsatellite-stable (no instability); NA: Not applicable; ND: Not describe

studied CRC 
F-53

Appendix F-27: Ability of microsatellite instability to identify MLH1 and MSH2 mutation carriers among colorectal ca

fulfilling the Amsterdam I criteria. 

Mutation No mutation Diagnostic performa

(95% 

confidence interv

Study, year (Ref ID); Country Single-/multi-center 

A. Comments on sample characteristics B. Verification bias 

C. Definition of MSI 

NAm1/

Ntotal

MSI MSS MSI MSS Sensitivity [%] Specif

Moslein 1996 (2545) US & Germany Multi-center 

A. Sample assembled from various databases; 

we present analyses only among familial cases (39/46), and only those described to have CRC. Study also included 7 sporadic CRC. 

B. No C. ≥30% of 9 to 34 markers analyzed (unclear 

which exactly) (MSI-H) 

14/46 5 0 0 4 100 (48, 100) 100 (

A. Sample assembled with unclear selection 

process: we present analyses only among familial CRC who were at most 1 criterion short of fulfilling Am1 (n=17); study also included 17 apparently sporadic CRC aged <50y at diagnosis. 

B. Yes: not all sporadic CRC underwent MMR 

mutation testing (these are not included in this analysis) 

C. ≥10% of up to 23 markers (But all were ≥30%) 

(MSI-H) 

Dieumegard, 2000 6 0 3 1 100 (54, 100) 25 (1791); France Multi-center 

≥10% of up to 23 markers (MSI-H&L) 

10/34 

6 0 3 1 100 (54, 100) 25 

Peel, 1999 (1660); US Multi-center 

A. Referral HNPCC cases, other than the 1134 

CRC probands who were also included but were not assessed with laboratory tests 

B. No C. Unclear; at least 5 markers were used (MSIH?) 

11/11 3 0 1 5 100 (29, 100) 83 (3

Demographics (data on age and gender distributions) were not available among people with fulfilling the Amsterdam I criteria. Note that studies that ass

in patients with MSI cannot be used to construct 2 by 2 tables for the ability of MSI testing to detect MMR mutations. Thus such studies are not included CRC: colorectal cancer; MSI(-H/-L): microsatellite instability (-high/-low); MSS: microasatellite-stable (no instability); NA: Not applicable; ND: Not describ

people fulfilling Amsterdam I criteria in the study population; Ntotal: total number of studied CRC
F-54

Appendix F-28: Ability of immunohistochemistry to identify MLH1 and MSH2 mutation carriers among selected coloprobands 

Mutation No mutation Diagnostic perform

(95% 

confidence inter

Study, year (Ref ID); Country Single-/multi-center 

A. Comments on sampling B. Verification bias C. Mean age (y); Males (%) 

D. Antibodies used 

Ntotal

No staining 

Staining 

No staining 

Staining Sensitivity [%] Specif

Genetic testing irrespectively of tumor microsatellite instability statusBarnetson, 2006 (NA) Scotland Multicenter 

A. Selection of all incident CRC aged 

<55 y ate diagnosis between 02/1999 and 07/2003 

B. No C. 48; 53 D. Anti-MSH2: (Oncogene Research 

Products); anti-MLH1: (PharMingen); anti-MSH6: (Transduction laboratories); 

870a 24 3 39 285 89 (71, 98) 88 (8

Southey, 2005 (66) Australia Single-center 

A. Randomly selected from a 

prospective cohort of all CRC with age of onset <45y; only patients who received genetic testing are presented b

B. No C. <45y; ND D. Anti-MSH2: FE-11 (Oncogene 

Research Products); anti-MLH1: G168-728 (PharMingen); anti-MSH6: clone 44 (BD transduction laboratories); anti-PMS2: clone A16-4 (PharMingen) 

105b 18b 0b 8b 33b 100 

(81, 100) 

80 (6

Syngal, 2000 (1672) & Wahlberg, 2002 (1158); US Single-center 

A. Selection among referrals to 

specialized center c

B. No C. ND; ND D. Anti-MSH2: FE11 (Oncogene 

Research Products); anti-MLH1: G168-728 (PharMingen) 

70c 6 5 3 22 55 (23, 83) 88 (6
F-55

Mutation No mutation Diagnostic perform

(95% 

confidence inter

Study, year (Ref ID); Country Single-/multi-center 

A. Comments on sampling B. Verification bias C. Mean age (y); Males (%) 

D. Antibodies used 

Ntotal

No staining 

Staining 

No staining 

Staining Sensitivity [%] Specif

Katballe, 2002 (1310) & Christensen, 2002 (1038); Denmark Single-center 

A. Selected from a population of 1514 

incident CRC 

B. No C. ND; ND D. Anti-MSH2: Ab-1, Ab-2 (Oncogene 

Research Products); anti-MLH1: G168-15 (PharMingen) 

42d 9 4 3 15 69 (39, 91) 83 (5

Curia, 1999 (1959) Italy Single-center 

A. Sampled from pathology registries, 

unclear selection criteria: HNPCC related cancerse

B. No C. <50y, ND D. Anti-MSH2: FE11 (Oncogene 

Research Products); anti-MLH1: clone 14 (Oncogene Research Products) 

30e 1 0 13 10 100 

(0, 100) 

57 (

Dieumegard, 2000 (1791) France Multicenter 

A. Sample assembled with unclear 

selection process: familial CRC missing at most 1 Am1 criterion and sporadic CRC aged <50y at diagnosis 

B. Yes: only 7 sporadic CRC underwent 

genetic testing 

C. Oldest age at diagnosis 56, ND D. Anti-MSH2: FE-11 (Oncogene 

Research Products); anti-MLH1: Ab-1 (Oncogene Research Products) 

34 4 3 5 9 57 (18, 90) 64 (3
F-56

Mutation No mutation Diagnostic perform

(95% 

confidence inter

Study, year (Ref ID); Country Single-/multi-center 

A. Comments on sampling B. Verification bias C. Mean age (y); Males (%) 

D. Antibodies used 

Ntotal

No staining 

Staining 

No staining 

Staining Sensitivity [%] Specif

Debniak,, 2000 (1784); Poland Single-center(?) 

E. Sampled from consecutive CRC, 

selection process not transparent. All patient with available data are included. 

F. Yes: Only 43/143 apparently sporadic 

CRC were tested, but it is unclear how they were selected 

G. ND, ND H. Unclear 

168 2 9 0 56 18 (2, 51) 100 (9

Durno, 2005 (195) Canada Multi-center 

E. Retrospective cohort of CRC 

aged<24 y at diagnosis who were still alive (since 1970) 

F. No G. <24y; 38 H. Anti-MSH2: FE-11 (Oncogene 

Research Products); anti-MLH1: G168-728 (PharMingen 

16 3 1 1 3 75 (19, 99) 75 (

Genetic testing among patients with MSI-H tumors Terdiman, 2001 (1572) US Single-center 

A. Retrospective cohort of CRC 

probands with ≥2 CRC in first degree family, age <50y at diagnosis or multiple tumors in the same patient 

B. Yes: only patients with MSI-H were 

assessed 

C. ND, ND 

D. Unclear 

114 16 1 29 4 94 

(71, 100) 

13 (

No (nuclear immuno-) staining is the abnormal response. The number of patients in the 2 by 2 tables is the number of patients with available data and is total number of patients included in the study. CRC: colorectal cancer; ND: Not described; Ntotal: total number of studied CRC a Overall 359 patients had available tumor tissue of good quality. b This study assessed PMS2 and MSH6 also. The mutation and IHC testing include pathogenic mutations in these genes also. c Data from the publication by Wahlberg et al. d Data from the publication by Christensen et al. e 4 were not CRC and are excluded from the calculations; Only 24 specimens had available IHC data.
F-57

Appendix F-29: Ability of immunohistochemistry to identify MLH1 and MSH2 mutation carriers among colorectal canfulfilling Amsterdam I criteria 

Mutation No mutation Diagnostic perform

(95% 

confidence inter

Study, year (Ref ID); Country Single-/multi-center 

A. Comments on sampling 

B. Verification bias C. Mean age (y); Males (%) D. Antibodies used 

NAm1/ 

Ntotal

No staining 

Staining 

No staining 

Staining Sensitivity [%] Specif

Katballe, 2002 (1310) & Christensen, 2002 (1038); Denmark Single-center 

A. Selected from a population of 1514 

incident CRC 

B. No C. ND; ND 

D. Anti-MSH2: Ab-1, Ab-2 (Oncogene 

Research Products); anti-MLH1: G168-15 (PharMingen) 

11/42a 2 2 0 5 50 (7, 93) 100 (4

Dieumegard, 2000 (1791) France Multicenter 

A. Sample assembled with unclear 

selection process: familial CRC missing at most 1 Am1 criterion and sporadic CRC aged <50y at diagnosis 

B. Yes: only 7 sporadic CRC underwent 

genetic testing 

C. Oldest age at diagnosis 56, ND 

D. Anti-MSH2: FE-11 (Oncogene 

Research Products); anti-MLH1: Ab-1 (Oncogene Research Products) 

10/34 2 2 2 2 50 (7, 93) 50 (

No (nuclear immuno-) staining is the abnormal response. The number of patients in the 2 by 2 tables is the number of patients with available data and is total number of patients included in the study. CRC: colorectal cancer; ; NAm1: number fulfilling Amsterdam I criteria; ND: Not described; Ntotal: total number of studied CRC a Data from the publication by Christensen et al. 
G-1

Appendix G: One-Way Sensitivity Analyses for the 

Decision Trees 

 The three variables with the most important influence for each strategy are depicted in the 

corresponding figures. 

Number of tests 

Figure G-1 shows the one-way sensitivity analyses on the expected number of MMR genetic 

tests with each treatment strategy. Among the three variables that exerted the most influence on 

the expected number of MMR genetic tests were the diagnostic characteristics (mainly the 

specificities) of the clinical criteria, IHC or MSI, and the prevalence of MMR mutations. For 

strategies using serial screening with clinical criteria and MSI or IHC, the diagnostic 

characteristics of both tests (mainly their specificities) were among the three more influential 

variables. 

 

Figure G-1. One-way sensitivity analysis for the number of MMR genetic tests expected from each strategy 

among a hypothetical population of 100,000 incident CRC. Shown are the effects of one-way sensitivity analyses 

on the number of MMR tests performed in each strategy. The horizontal bars illustrate the different values that the 

outcome takes when a variable is varied from its lower to its higher plausible value in the sensitivity analyses. Only 

the three variables that had the greatest influence on each strategy are depicted (variable names are explained in 

Table 15 and in the text). Since all other variables had an even smaller influence, they are not depicted at all. The first 

strategy (MMR-All) is not shown. The first strategy assumes that all patients receive MMR testing; thus the number of 

expected MMR tests is always 100% of the population. 
G-2

Figure G-2 shows the variation in the expected number of MSI and IHC tests for the 

strategies that are affected by the sensitivity analyses. The remaining strategies either do not use 

MSI or IHC at all (MMR-All, BethR-All, 3Clinical-All) or use one test in all patients and the 

other test in none (MSI-All and IHC-All). 

C_BethR

Prev

S_BethR

C_3Clinical

Prev

S_3Clinical

C_BethR

Prev

S_BethR

C_3Clinical

Prev

S_3Clinical

BethR-MSI

3Clinical-MSI

BethR-IHC

3Clinical-IHC

15,000 20,000 25,000

Number of MSI or IHC tests

for a hypothetical cohort of 100,000 incident CRC

Figure G-2. One-way sensitivity analysis for the number of MSI or IHC genetic tests expected from each 

strategy among a hypothetical population of 100,000 incident cases of CRC. The bars for the first two strategies 

(BethR-MSI and 3Clinical-MSI) refer to the expected number of MSI tests. The expected number of IHC tests is zero 

since IHC is not used in these strategies. Similarly, the bars for the last two strategies (BethR-IHC and 3Clinical-IHC) 

refer to the expected number of IHC tests. The expected number of MSI tests is zero since MSI is not used in the 

latter strategies. Layout similar to Figure 27. 

Number of people with positive test 

 The most important influence on the number of people with positive or true positive MMR 

was prevalence of MMR mutation carriers in the population. Diagnostic characteristics of the 

various tests also have a role. Figure G-3 and Figure G-4 illustrate the pertinent one-way 

sensitivity analyses. 
G-3

Figure G-3. One-way sensitivity analysis for the number of positive MMR tests expected from each strategy 

among a hypothetical population of 100,000 incident cases of CRC. The layout is similar to Figure 27. 
G-4

Figure G-4. One-way sensitivity analysis for the number of true positive MMR tests expected from each 

strategy among a hypothetical population of 100,000 incident cases of CRC. The first strategy (MMR-All) is 

affected only by two variables in the sensitivity analyses. The layout is similar to Figure 27.