Genetic testing for colon cancer: Joint statement of the American College of Medical Genetics and American Society of Human Genetics

 [bib_ref] Genetic testing and counseling for hereditary forms of colorectal cancer, Petersen [/bib_ref] [bib_ref] Alleles of the APC gene: an attenuated form of familial polyposis, Spirio [/bib_ref] [bib_ref] Variable phenotype of familial adenomatous polyposis in pedigrees with 3 mutation in..., Brensinger [/bib_ref] [bib_ref] Genotype-phenotype correlations in attenuated adenomatous polyposis coli, Soravia [/bib_ref] [bib_ref] Novel germline APC variants in patients with multiple adenomas, Pedemonte [/bib_ref] [bib_ref] The APC variants I1307K and E1317Q are associated with colorectal tumors, but..., Frayling [/bib_ref] [bib_ref] Familial colorectal cancer in Ashkenazim due to a hypermutable tract in APC, Laken [/bib_ref] [bib_ref] The APC I1307K allele and cancer risk in a communitybased study of..., Woodage [/bib_ref] 

## Hereditary non-polyposis colon cancer
Germline mutations in five mismatch repair-related genes (hMSH2, hMLH1, hMSH6, hPMS1, and hPMS2) cause
This guideline is designed primarily as an educational resource for medical geneticists and other health care providers to help them provide quality medical genetic services. Adherence to this guideline does not necessarily ensure a successful medical outcome. This guideline should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed toward obtaining the same results. In determining the propriety of any specific procedure or test, the geneticist should apply his or her own professional judgment to the specific clinical circumstances presented by the individual patient or specimen. It may be prudent, however, to document in the patient's record the rationale for any significant deviation from this guideline.
HNPCC, and are associated with specific somatic alterations in the tumor, characterized by high microsatellite instability (MSI-H). [bib_ref] A National Cancer Institute Workshop on Hereditary Colorectal Cancer Syndrome: meeting highlights..., Rodriguez-Bigas [/bib_ref] [bib_ref] A National Cancer Institute workshop on microsatellite instability for cancer detection and..., Boland [/bib_ref] HNPCC is characterized by young onset colorectal cancer (mean age 44 years), proximal colon location, multiple primary cancers, and increased risk of endometrial cancer, transitional cell cancer of the ureters, small bowel cancer, gastric cancer, bile duct cancer, and ovarian cancer. [bib_ref] Overview of natural history, pathology, molecular genetics and management of HNPCC (Lynch..., Lynch [/bib_ref] [bib_ref] Hereditary nonpolyposis colorectal cancer: the syndrome, the genes and historical perspective, Marra [/bib_ref] Microsatellite instability (MSI) in the tumor is associated with HNPCC and tumors with specific pathologic characteristics, suggesting a group of patients on whom such analysis may be useful. [bib_ref] A National Cancer Institute Workshop on Hereditary Colorectal Cancer Syndrome: meeting highlights..., Rodriguez-Bigas [/bib_ref] [bib_ref] A National Cancer Institute workshop on microsatellite instability for cancer detection and..., Boland [/bib_ref] Recently, in a large retrospective study, MSI has been found to be associated with improved survival in young colorectal cancer patients, at all stages, potentially heralding the use of MSI in treatment decisions or risk management. [bib_ref] Tumor microsatellite instability and clinical outcome in young patients with colorectal cancer, Gryfe [/bib_ref] 

## Technology

## Germline genetic testing
Gene tests for colorectal cancer (see [fig_ref] Table 1: Genetic tests for susceptibility to colon cancer [/fig_ref] in the clinical setting primarily consist of the following:
- Protein truncation testing (in vitro synthesized protein assay) for truncating germline mutations of APC, MSH2, and MLH1 in patients where the mutation is not known. This method detects disease-causing mutations (nonsense or frameshift) that result in premature protein termination. In particular, APC mutations in the majority of patients with classic FAP phenotype may be detected this way. This method is less sensitive for HNPCC, be-cause other types of mutations contribute to the disease phenotype. [bib_ref] Various mutation screening techniques in the DNA mismatch repair genes hMSH2 and..., Wahlberg [/bib_ref] If the truncated protein is detected by this assay in the affected patient, then the defect can be similarly assayed in a focused evaluation of at-risk relatives. - CSGE, SSCP, or other screening assay, followed by DNA sequencing for MSH2 and MLH1 in HNPCC families where the mutation is not known. These methods are more sensitive than protein truncation testing, but may still not detect all mutations. [bib_ref] Various mutation screening techniques in the DNA mismatch repair genes hMSH2 and..., Wahlberg [/bib_ref] [bib_ref] Conversion of diploidy to haploidy, Yan [/bib_ref] [bib_ref] Interpretation of genetic test results for hereditary nonpolyposis colorectal cancer: implications for..., Syngal [/bib_ref] Once a disease-associated germline mutation is detected in an affected patient, then ASO methods (described next) can be employed to evaluate at-risk relatives. - Allele specific oligonucleotide (ASO) hybridization is appropriate when the specific disease-causing mutation is known, in cases such as APC I1307K, [bib_ref] Familial colorectal cancer in Ashkenazim due to a hypermutable tract in APC, Laken [/bib_ref] or predictive testing in an FAP or HNPCC kindred where the causal mutation has been identified. [bib_ref] Various mutation screening techniques in the DNA mismatch repair genes hMSH2 and..., Wahlberg [/bib_ref] [bib_ref] Presymptomatic direct detection of APC gene mutations in familial adenomatous polyposis, Petersen [/bib_ref] Tumor assays for genetic predisposition The availability of archival paraffin-preserved colorectal tissue (normal and tumor) enhances our ability to evaluate patients or families suspected of harboring HNPCC mutations. There are two approaches that are moving into clinical application:
- Microsatellite instability (MSI) is considered to be a feasible and informative option in evaluating a family suspected of HNPCC. [bib_ref] A National Cancer Institute Workshop on Hereditary Colorectal Cancer Syndrome: meeting highlights..., Rodriguez-Bigas [/bib_ref] [bib_ref] A National Cancer Institute workshop on microsatellite instability for cancer detection and..., Boland [/bib_ref] [bib_ref] Incidence of hereditary nonpolyposis colorectal cancer and the feasibility of molecular screening..., Aaltonen [/bib_ref] Patients with colorectal cancer The majority of MSI observed in sporadic cases appear to be due to somatic hypermethylation of the MLH1 promoter. [bib_ref] Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability, Cunningham [/bib_ref] [bib_ref] The frequency and mechanism of defective DNA mismatch repair in unselected colorectal..., Cunningham [/bib_ref] It has also been observed that absent MSH2 IHC expression is associated with germline MSH2 mutation, and a minority of absent MLH1 IHC expression is associated with germline MLH1 mutation. [bib_ref] The frequency and mechanism of defective DNA mismatch repair in unselected colorectal..., Cunningham [/bib_ref] The translation of these new methods into routine clinical practice awaits large-scale validation studies.

## Future technology: conversion of diploidy to haploidy
Recently, Vogelstein and coworkers have developed a new technique whereby mutations in APC, MSH2, or MLH1 may be detected by converting diploid cells to haploid cells by selectively fusing with mouse cells and screening the human chromosome of interest. Also termed monoallelic mutation analysis, this method may be more widely available in the near future. [bib_ref] Conversion of diploidy to haploidy, Yan [/bib_ref] [bib_ref] Analysis of masked mutations in familial adenomatous polyposis, Laken [/bib_ref] Unresoved issues
There are a number of issues that need to be considered in the context of colon cancer genetic testing options. The characterization of genetic susceptibility to colon cancer is a very active area of research, and until studies on large populations of patients are completed, precision in risk assessment and screening or preventive interventions will be based on current best evidence. With respect to FAP and HNPCC, genetic tests are part of the spectrum of clinical information gathering, through which management options can be developed. The clinical, technical, and psychosocial frameworks (both the medical as well as the patients' point of view) should be incorporated in testing strategies.

## How many genes are going to turn out to have an effect on colon cancer, especially the hereditary form?
For example, the syndrome HNPCC is due to a class of genes that are involved in the DNA mismatch repair pathway, so that essentially the identical clinical diagnosis can be explained by any of five different genes. While a majority of mutations are found in just two genes (MSH2 and MLH1), if no mutation is found in these two, there are few other clinical testing options.
Only a few research laboratories are studying mutations in the other three genes. Yan et al. [bib_ref] Conversion of diploidy to haploidy, Yan [/bib_ref] suggest that the majority of HNPCC families that manifest MSI tumors will be accounted for by MSH2 and MLH1. In total, however, mutations in these genes only account for approximately one half of the families that fit the Amsterdam criteria (particularly those that do not manifest MSI); thus, additional genes are likely to be involved that may have other underlying mechanisms of action.

## What is the clinical spectrum associated with mutations in "colon cancer" genes?
Molecular diagnostic testing can better characterize syndromes or even split syndromes that were originally considered a single entity on clinical observation grounds. For example, there are more cancers of the renal pelvis, ureter, stomach, and ovaries when the family harbors a mutation in MSH2 (vs. MLH1), while mutations in MLH1 that completely silence the gene product (i.e., no protein produced) have a paucity of extracolonic tumors. An atypical phenotype is seen in families with MSH6 mutations where endometrial/ovarian cancers outnumber colorectal cancers. Further differentiation must await long-term follow-up of multiple families with well-characterized mutations.
In another example, Turcot syndrome was thought to be an autosomal recessive condition characterized by colonic polyposis and brain tumors. Mutation analyses have now shown that Turcot syndrome can be split into at least two dominant polyposis syndromes: (1) the true Turcot syndrome, which is associated with glioblastoma and is due to mutations at MLH1 and PMS2; and (2) the Crail syndrome, which is associated with a different brain tumor and medulloblastoma, and is due to mutations in APC. [bib_ref] The molecular basis of Turcot's syndrome, Hamilton [/bib_ref] As geneticists study the action of these genes and the mutations in different regions, more subtle phenotypes are being associated with each [e.g., [bib_ref] Novel germline APC variants in patients with multiple adenomas, Pedemonte [/bib_ref] [bib_ref] The APC variants I1307K and E1317Q are associated with colorectal tumors, but..., Frayling [/bib_ref]. This finding has the potential to complicate the management of carriers until such time that the most complete spectrum of potential tumors has been identified. Individuals shown to harbor pathologic mutations should remain in an ongoing surveillance program.

## What is the best surveillance for carriers?
The American Cancer Society, American Gastroenterology Association, and National Cancer Center Network have developed recommendations and practice guidelines that consider genetic risk for surveillance of individuals from familial or hereditary colorectal cancer pedigrees. [bib_ref] Colorectal cancer screening: clinical guidelines and rationale, Winawer [/bib_ref] These recommendations are largely based on retrospective clinical observations and expert opinion, as there are no data from large-scale population-based or prospective studies of cancer risk in carriers.
Molecular testing has the potential to improve management of mutation carriers. For example, the attenuated form of FAP (AFAP), which is characterized by fewer adenomas and may be of later onset, should be diagnostically followed by colonoscopy rather than sigmoidoscopy as is standard for classic FAP. [bib_ref] Classification of familial adenomatous polyposis: a diagnostic nightmare, Lynch [/bib_ref] HNPCC also requires colonoscopy to monitor for polyps. [bib_ref] Recommendations for follow-up care of individuals with an inherited predisposition to cancer:..., Burke [/bib_ref] It is the hope, anecdotally observed, that there would be concomitant improvement in adherence to cancer prevention recommendations.

## Informed consent
The American Society of Clinical Oncology 49 has developed a list of elements of informed consent for cancer genetic testing, including (1) information on the specific test being performed, (2) implications of a positive and negative results, [bib_ref] Genetic instability in colorectal cancers, Lengauer [/bib_ref] possibility that the test will not be informative, (4) options for risk estimation without genetic testing, (5) risk of passing a mutation to children, [bib_ref] Genetic testing in hereditary colorectal cancer, Giardiello [/bib_ref] technical accuracy of the test, (7) fees involved in testing and counseling, (8) risks of psychologic distress, (9) risks of insurer or employment discrimination, (10) confidentiality issues, and (11) options and limitations of medical surveillance and screening following testing. It is recommended that all testing be done with a comprehensive informed consent document.

## Summary
Colorectal cancer gene discoveries have led to clinical application in the form of improved cancer genetic risk assessment and genetic testing for hereditary colon cancer, including FAP and HNPCC. However, much more continues to be learned about the biology and clinical aspects of colon cancer susceptibility.
- Germline gene test for FAP utilizes a protein truncation test of APC. - Germline gene test for APC I1307K is an ASO-based assay and may identify persons of Ashkenazi Jewish origin who are at increased risk for colon neoplasia. - HNPCC is genetically heterogeneous but is largely due to mutations in MSH2 and MLH1. Mutations in these mismatch repair genes often result in microsatellite instability (MSI) in the tumor. Tumor assays for MSI may help identify persons who are likely to carry germline MSH2 or MLH1 mutations. - Germline gene tests for MSH2 and MLH1 utilize protein truncation and DNA sequencing strategies. - Genetic counseling is an important and crucial component of the genetic risk assessment process. Informed consent for genetic testing is an integral part of the process, and a clear understanding by the patient can only be arrived at by careful counseling. - In view of the complicated and evolving technology and clinical issues, individuals with a family history of colon cancer would be best served in programs with appropriate laboratory and clinical expertise.

## Approved by the american college of medical genetics and american society of human genetics boards of directors in
[table] Table 1: Genetic tests for susceptibility to colon cancer [/table]