Exhibit 10.22

 

AMENDMENT #1 TO

 

CLINICAL STUDY AGREEMENT

 

The following is Amendment #1 to the Clinical Study Agreement dated July 17,
2017 between Volition America, Inc. (“Laboratory”) and the Regents of the
University of Michigan (the “University”) for Dean Brenner, ORSP Reference #
AWD005981, and incorporates all of the terms therein. This Amendment is
effective as of September 1, 2019.

 

The purpose of this Amendment is to acknowledge that the requirements of the
GLNE010 study have been satisfied. This Amendment serves to end the completed
sections of the Study Protocol in Exhibit A (GLNE010) add Study Protocol Exhibit
A-1 {GLNE007}, Exhibit B and Exhibit C. This Amendment shall also replace
Section 3.1 in its entirety:

 

3.1In consideration of its participation in the Clinical Study on the terms and
conditions of this Agreement, Laboratory shall provide direct and indirect
funding in the amount of up to One Million, Five Hundred Thousand United State
Dollars (US$1,500,000). Direct and indirect payments by the Laboratory for the
Clinical Study has concluded, are paid in full, and no additional future funding
will be provided. 

 

All terms of the Clinical Study Agreement shall remain unchanged.

 

 

ACCEPTED AND AGREED TO:

 

 

Volition America, Inc.

 

/s/  Jason Terrell                                                   02/17/2020 

Signature of Responsible Officer for LaboratoryDate 

 

Jason Terrell, MD, Chief Executive Officer

Typed Name and Title

 

 

Regents of the University of Michigan:

 

/s/  Julie Olivero
                                                   02/11/2020 

Signature of Responsible Officer for LaboratoryDate 

 

Julie Olivero, Project Representative

Typed Name and Title

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

Evaluation of Stool Based Markers for the Early Detection of Colorectal Cancers
and Adenomas

Great Lakes New England (GLNE) Clinical Validation Center

NCI Early Detection Research Network

 

STUDY INVESTIGATORS:

Clinical Validation Center Investigators (GLNE)

 

Henry Appelman, M.D.1

John A. Baron, M.D.2

Dean E. Brenner, M.D.1

Robert Bresalier, M.D.3

Tim Church Ph.D.4

Seth Crockett, M.D., MPH2

Marcia Cruz-Correa, M.D., Ph.D.9

William M. Grady, M.D.8

Mack Ruffin, M.D., M.P.H5

Ananda Sen, Ph.D.1

Elena Stoffel, M.D., M.P.H.1

Sapna Syngal, M.D. 6

Christopher Teshima M.D.7

 

 

1University of Michigan Medical Center, Ann Arbor, MI

2University of North Carolina, Chapel Hill, NC

3MD Anderson Cancer Center, Houston, TX

4University of Minnesota, Minneapolis, MN

5Hershey Medical Center-PSU, Hershey, PA6

6Dana-Farber Harvard Cancer Center, Boston, MA

7Saint Michael’s Hospital, University of Toronto, Toronto, Ontario

8University of Washington Medical Center, Seattle, WA

9University of Puerto Rico Comprehensive Cancer Center, San Juan, Puerto Rico

 

Contact information for Great Lakes New England Clinical Validation Center

 

2150 Cancer Center and Geriatrics Center

University of Michigan Medical Center Ann Arbor, MI 48109-0930

Telephone: (734) 647-1417

Fax: (734) 647-9817

Email:dbrenner@umich.edu (PI)  

kherman@umich.edu (Lead CRA)

 

EDRN Data Management and Coordinating Center (DMCC)

Fred Hutchinson Cancer Research Center

1100 Fairview Ave N Seattle, WA 98109-1024

206-667-6038

Ziding Feng, Ph.D. (PI)

spage@fredhutch.org (DMCC GLNE 007 Project Manager)

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

GLNE 007

EVALUATION OF STOOL BASED MARKERS FOR THE EARLY DETECTION OF

COLORECTAL CANCERS AND ADENOMAS

 

1.0

Summary of Study

1

2.0

Schema

2

3.0

Objectives

3

4.0

Background and Significance

3

4.1

Biomarkers

3

4.2

The Early Detection Research Network (EDRN)

3

4.3

Current State of the Art: Recommended Early Detection

4

4.4

Current State of the Art: Serum Based Biomarkers for Colorectal Neoplasia

4

4.5

Rationale and Current State of the Art: Stool Based Biomarkers for Detection of
Colorectal Neoplasia

6

4.6

Key Issues Driving Research Questions in CRC Early Detection Biomarkers

8

5.0

Study Design

9

5.1

Summary of Study Plan

9

5.2

Rationale for tissue collection

9

6.0

Inclusion and Exclusion Criteria

9

6.1

Inclusion Criteria

9

6.2

Exclusion Criteria

11

7.0

Study Procedures

11

7.1

Subject Recruitment

11

7.2

Clinical Procedures

11

7.3

Biological Sample and Data Collection

13

7.4

Circulating methylated genes BCAT1/IKZF1 (Clinical Genomics)

14

7.5

Hypomethylated LINE1 from circulating cell free DNA (VolitionRx)

15

7.6

Disclosure of results to subjects

15

7.7

Evaluable subjects

15

7.8

Completion of Study

15

7.9

Subject Compensation

15

8.0

Study Calendar

16

9.0

Statistical Considerations

16

9.1

Study Population

16

9.2

Data Analysis Plan

17

9.3

Justification of Design and Sample Size

18

10.0

Data Safety and Monitoring

19

10.1

Data Safety and Monitoring

19

11.0

Adverse Event Reporting

20

12.0

Data Management

20

12.1

Registration

20

12.2

Timeliness

20

12.3

Completeness and Accuracy

20

12.4

Accuracy--Revisions and Corrections

20

12.5

On Site Data Audits

21

12.6

Sample Tracking

21

12.7

Confidentiality

21

12.8

Security

21

13.0

Ethical and Regulatory Considerations

22

13.1

Institutional Review

22

14.0

References

23

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

 

1.0SUMMARY OF STUDY 

 

As part of the National Cancer Institute-funded Early Detection Research Network
(EDRN), the Great Lakes-New England Clinical Epidemiological Center (GLNE CEC)
proposes a research study that validates potential molecular markers
(“biomarkers”) for the detection of precancerous and cancerous conditions and
cancer risk assessment. Although examples of such biomarkers are currently in
clinical use (i.e. CEA, CA-125), there are limitations to all of them. Our
consortium focuses on gastrointestinal neoplasia.

 

The goals of this phase of the proposed research are:

 

1.Assessment of the utility of individual stool-based, and serum-based
biomarkers for discriminating between patients with adenocarcinomas, patients
with adenomas with high grade dysplasia, patients with advanced adenomas defined
as adenoma histology of any combination including sessile serrated adenoma,
tubulovillous adenoma, villous adenoma, sessile serrated polyp/adenoma,
traditional serrated adenoma OR any adenoma ≥1 cm OR three or more adenomas,
patients with adenomas that are not advanced, and normal colonoscopy subjects
both at normal and high risk for developing colon cancer. 

 

2.Construction of a panel of markers from those considered in Objective 1 to
discriminate, under a number of assumptions concerning prevalence and cost of
misclassification, between: 

 

a.(Primary) Subjects with normal colons or non-advanced adenomas versus patients
with cancers 

 

b.(Secondary) Subjects with normal colons versus patients with cancers. 

 

3.Comparison of the characteristics of individual markers and panels as
discriminators to those of the established current standard, fecal
immunochemical test (FIT). 

 

4.Development of a bank of stool samples linked to serum, tissue, and clinical
data from patients with colorectal cancer, adenomas and normal controls for
validation of stool-based markers that may be developed in the future. 

 

To build our collection, we propose to collect stool, FIT, serum, plasma, and
tissue samples from 1,000 new subjects. EDRN Common Data Elements (CDEs) will be
completed by the recruiting sites and provided for the laboratories developing
the assay. Each biomarker will be analyzed individually and considered as a
potential panel marker to be used for future large-scale screening longitudinal
trials.

 

This protocol had previously recruited subjects from January 2006 to June 2010.
The samples from this recruitment period are 9-13 years old as of the
development of this protocol in April, 2019. Prior recruited subjects:

 

262 adenomas (54 of those advanced)

191 cancers

65 high risk, colonoscopic normal

164 colonoscopic normal

 

From each subject, we collected 30 serum, 30 plasma, 5 stool, 20 5- ml urine
aliquots

Current Status of GLNE 007 repository:

 

Total circulating space samples collected: 16,900 serum, 15,700 plasma, 3,000
stool, and 7,000 urine aliquots (42,600 total)

Total circulating space samples disbursed: 12,600 aliquots of various types.

Remaining in the collection: (total 30,000)

10,200 Serum aliquots

11,500 Plasma aliquots

2,100 stool aliquots (representing 585 unique subjects with at least 1 aliquot
left)

6,200 urine aliquots

Total tissue samples: 2,100 tissue pieces snap frozen in liquid nitrogen

Total tissue samples disbursed: 1,050

Total tissue samples remaining: 1,050

 

This amended protocol (version 7) proposes to restart GLNE 007 to recruit 1,000
new subjects, (400 colorectal cancers, 200 adenomas, 200 higher risk but
endoscopically normals and 200 endoscopically normal colons for controls). Thus,
bringing our total from 682 to 1,682 total subjects.

--------------------------------------------------------------------------------

1

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

2.0SCHEMA 

 

pg 2.jpg [f10k123119ex10z221.jpg] 

 

NOTES:

 

Nursing women who otherwise meet the eligibility criteria may participate. 

Subjects who had CRC that was successfully treated at least three years ago are
eligible. 

Recent screening colonoscopy (within 3 weeks of enrollment), poor preparation
found at colonoscopy and returning for repeat colonoscopy are eligible. 

--------------------------------------------------------------------------------

2

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

 

 

3.0OBJECTIVES 

 

1.Assessment of the utility of individual stool-based, and serum-based
biomarkers for discriminating between patients with adenocarcinomas, patients
with adenomas with high grade dysplasia, patients with advanced adenomas defined
as adenoma histology of any combination including sessile serrated adenoma,
tubulovillous adenoma, villous adenoma, sessile serrated polyp/adenoma,
traditional serrated adenoma OR any adenoma ≥1 cm OR three or more adenomas,
patients with adenomas that are not advanced, and normal colonoscopy subjects
both at normal and high risk for developing colon cancer. 

 

2.Construction of a panel of markers from those considered in Objective 1 to
discriminate, under a number of assumptions concerning prevalence and cost of
misclassification, between: 

 

a.(Primary) Subjects with normal colons or non-advanced adenomas versus patients
with cancers; 

 

b.(Secondary) Subjects with normal colons versus patients with cancers. 

 

3.Comparison of the characteristics of individual markers and panels as
discriminators to those of the established current standard, fecal
immunochemical test (FIT). 

 

4.Continued support of a renewal of a bank of stool samples linked to serum,
tissue, and clinical data from patients with colorectal cancer, adenomas and
normal controls for validation of stool- based markers that may be developed in
the future. 

 

4.0BACKGROUND AND SIGNIFICANCE 

 

4.1Biomarkers 

 

Definitions, underlying assumptions, and rationale.

 

A biomarker is defined as a characteristic that is measured and evaluated as an
indicator of normal biologic processes, pathogenic processes, or pharmacologic
responses to therapeutic interventions (3). An NCI Working Group further
characterized a biomarker as a Clinical Endpoint--a characteristic or variable
that measures how a patient feels, functions or survives; as a Surrogate
Endpoint--a biomarker intended to substitute for a clinical endpoint in a
clinical trial-- and as a Global Assessment: an evaluation of risk and benefit
balance for a patient or group of patients. However, the working group did not
address biomarkers specific to the carcinogenesis process or for cancer
detection.

 

The underlying assumption of a surrogate endpoint for cancer prevention is that
a measured biological event will predict a cancer outcome, either immediately or
at a later time (4) and, in the same circumstances, be affected by the
intervention.  The primary motivations for development of such surrogate
endpoints concerns the ability to diagnose cancer at an early stage, to identify
individuals at high risk for development of cancer and to enable reduction of
sample size and trial duration for an interventional trial such that a rare or
distal endpoint can be replaced by a more frequent and more proximate endpoint
(5).

 

Specifications for a useful biomarker.

 

An “ideal” biomarker will have the following characteristics (6):

 

(i)Variability of expression between phases of the carcinogenesis process (i.e.
normal, pre- malignant, malignant). 

(ii)Detectable early in the carcinogenesis process. 

(iii)Associated with the risk of developing cancer or the occurrence of
pre-cancer. 

(iv)Detected in body fluids (e.g. blood, urine, sputum) or tissues obtained via
biopsy. 

(v)Capability for development of adequate quality control procedures. 

(vi)Potential for modification by a chemo preventive agent. 

 

4.2The Early Detection Research Network (EDRN) 

 

The mission of the Early Detection Research Network.

 

The Early Detection Research Network (EDRN) is a comprehensive effort supported
by the NCI to develop highly sensitive, specific, and clinically reliable early
detection tools. The Network is harnessing scientific expertise from national
and international institutions to identify and validate molecular markers for
the detection of precancerous and cancerous cells and to assess risk for
developing cancer.

--------------------------------------------------------------------------------

3

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

Great Lakes New England Clinical Validation Center (GLNE CVC)

 

The GLNE CVC is a funded EDRN consortium dedicated to the characterization and
validation of biomarkers for the early detection and risk assessment of
colorectal adenocarcinoma. The consortium provides the EDRN with expertise in
population epidemiology, biostatistics, pharmacology and medical oncology.

 

4.3Current State of the Art: Recommended Early Detection 

 

Randomized controlled trials have shown that annual or biennial fecal occult
blood tests (FOBT) reduce colorectal cancer (CRC) mortality by 15% to 33% (7-9).
The reduction is durable over 3 decades (10). Population based cohort studies of
colonoscopic screening demonstrate reduced CRC mortality, primarily in distal
but not in the proximal colon (11-13). This discrepancy has been attributed to
endoscopic quality issues, the technical difficulties in detecting lesions in
the right colon, and the more frequent occurrence of flat and depressed
dysplastic lesions in the right colon (14-17). In tandem colonoscopy studies, a
subset of large polyps may be missed by a single examiner. Shorter withdrawal is
time-linked to a lower adenoma detection rate (18, 19). Flat and depressed
lesions are more challenging to detect and have been described with a relatively
high prevalence in a US colonoscopy cohort (20).  While colonoscopic removal of
adenomatous polyps reduces CRC mortality (21), prospective, randomized
controlled trials of screening colonoscopy have been initiated by the VA and in
Europe (21-23). Over-diagnosis (i.e. early detection of indolent invasive
neoplasms that do not cause mortality) or lead-time bias in early detection of
colorectal neoplasms do not degrade the efficacy of screening and early
detection for colorectal cancers (24).

 

Current screening guidelines for average risk individuals vary world-wide. In
the United States the American Gastroenterology Association recommends testing
for early detection of adenomas and cancer (structural examination) or of cancer
(non-invasive stool tests) beginning at age 50 (25). The United States
Preventive Services Task Force (USPSTF) recommends fecal occult blood testing
(FOBT) every two years with optional endoscopic screening with either flexible
sigmoidoscopy or colonoscopy (26). The majority of developed countries recommend
fecal occult blood testing every two years but do not support endoscopic
screening (27); albeit with some exceptions (e.g. Germany (27, 28)). In 2012,
65.1% of the United States adults adhered to USPSTF colorectal screening
guidelines with colonoscopy the commonly used screening method (61.7%) followed
by FOBT (10.4%) (29) whereas colonoscopic screening adherence in Germany is 16%
(28). Over 20 years of SEER data (1991 to 2011), United States CRC incidence
(all races, males, females) has fallen from 59.5 cases in 1991 to 39.3 cases per
100,000 in 2011 (35% reduction) with a corresponding mortality reduction over
the same time period from 24.0 to 15.1 deaths per 100,000 (37% reduction) (30).
Widespread adherence to screening guidelines in the United States may be driven
by the profound changes in the organization of medical care including enhanced
access via the Affordable Care Act, rigid guideline enforcement by payers with
physician performance incentives and disincentives, and the rapid adaptation of
electronic medical record systems enabling ease referrals for screening,
compliance reminders, and management tracking of compliance to care guidelines
(31).

 

4.4Current State of the Art: Serum Based Biomarkers for Colorectal Neoplasia 

 

Rational for non-adherence with stool based or colonoscopic based CRC screening
include the volume of bowel preparation, inadequate analgesia, no recommendation
from primary physician, embarrassment (32) or cultural taboos surrounding
collection or manipulation of stool provide rationale for discovery and
validation of circulating biomarkers for early detection of colorectal
neoplasia. Circulating signatures may be detected from neoplasm generated
genetic products, antigens, antibodies, glycans, circulating tumor cells.

--------------------------------------------------------------------------------

4

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

Genetic Products

 

In a recent study of 24 CRC patients, mutant DNA fragments (circulating tumor
DNA, ctDNA) are found in relatively high concentrations in the circulation of
most patients with metastatic cancer and at were detected in ~70% of patients
with localized cancers (33). The direct detection of aberrant genes or genetic
material specific to colorectal neoplasms (e.g. APC, b-catenin, K-ras, DCC, and
p53) has been limited by the technical challenge of DNA recovery, the large
number of potential underlying genetic mutations, and by the limited sensitivity
of any single genetic alteration due to the extremely low abundance gene
mutations in circulating plasma or serum (33- 38). DNA hypermethylation, in
contrast, affects residues in regulatory portions of genes and provides major
advantages in designing biomarker assays (37, 39-41). Digital based quantitative
technologies improving upon bisulfite conversion while minimizing bisulfite
associated DNA fragmentation and single molecule detection technologies (42)
permit cost effective development of DNA hypermethylated gene biomarkers. Such
technology detected circulating methylated vimentin with 59% sensitivity (42).
Septin9, a methylated gene discovered in tissues with array technology (43, 44),
detects CRC with 50% sensitivity and 92% specificity in a large (7941
participants) prospective colonoscopy verified screening trial (45). For early
stage CRC, Septin9 sensitivity decreased to 35%. While circulating methylated
CpG DNA promoter sites appear to have higher CRC detection performance than
other genetic detection strategies, they substantially lag behind stool based
detection of blood DNA markers or endoscopy. Nevertheless, for individuals
refusing to use stool based screening, detection sensitivity of circulating
methylated DNA markers appears equivalent to guaiac based stool screening and
has the potential advantage of capturing the 40% of the population refusing
stool screening. miRNAs are stable and detectable in serum and plasma. As in
stool, numerous up and down regulated miRNA stool signatures discovered using
unsupervised array technology may be useful as CRC detection biomarkers. A
recent review identifies 19 miRNAs as individual or groups in panels as
candidates for detection markers; but, insufficient clinical validation renders
the data generated to date using small convenience sets confusing and not
mechanism driven (46).

 

Proteins

 

Antigens: Approximately 50% of all proteins are estimated to be glycosylated
(47). Glycan abundance and their micro- and macro-heterogeneity can be changed
in a disease-specific manner (48). Glycoprotein screening studies, many EDRN
supported, have relied on immunoprecipitation or lectin affinity capture of
whole glycoproteins and mass spectrometry identification of the de- glycosylated
protein portion or probed with lectins in an array format containing up to a few
hundred antibodies (49-53). Sialylated Lewis A and Lewis X moieties carrying
proteins identify panels of potential markers. The Lampe EDRN laboratory has
found seven such proteins (B3GNT5, CD44, HSPG2, IL6, INHBC, NOTCH4 and VWF)
which when combined in discovery set plasma samples ROC AUC of 0.83 (54). GLNE
discovered glycan ligand, galectin- 3 ligand is a circulating glycan biomarker
in large population based prospective validation (55).

 

Antibodies: Serum antibodies recognizing multiple colon cancer antigens can be
detected in colorectal adenocarcinoma patients’ markers (56-58). Preliminary
validation of single or small autoantibody panels have been disappointing (59).
For example, antibodies to the Fas receptor have 17% sensitivity when 100%
specific for CRC detection (60). Experience with p53, Hsp60, and nucleobindin 1
(Calnuc) autoantibodies has been better (~50% sensitivity/70 to 90% specific);
but, they are not specific to CRC (59, 61, 62) and cannot be used as a colon
specific screening tool. Discovery sets that include a miniarray of
autoantibodies with other markers have reported improved detection accuracy
(sensitivity 83%/specificity 90%) (63) but require clinical validation.

 

Cytokines/growth factors: High serum concentrations of insulin-like growth
factors (IGF) and low levels of their binding proteins have been shown to
correlate with CRC risk in large cohort studies (64-67) but have low
sensitivities with high specificities for CRC detection. Other cytokines or
angiogenesis factors such as TGF-b1 (68-74), VEGF (75, 76), angiogenin (77),
endostatin (78), and endothelins (79, 80) also have low sensitivity in small
convenience sets and have not proceeded to clinical validation.

 

Other proteins: Of the matrix metalloproteinases (81-83), plasma TIMP1 is
elevated in CRC but has not had sufficient sensitivity in larger validation
trials to merit development as a detection biomarker (84). Cell adhesion
molecules (85) have low sensitivities for detection of early stage CRC.

--------------------------------------------------------------------------------

5

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

Circulating Tumor Cells

 

Circulating tumor cells (CTCs) entering the vascular space from primary
neoplasms have been considered to be initiators of metastases (86-88) and can be
detected in early stage invasive neoplasms (89, 90). CTC isolation from
epithelial cancers initially used antibody capture technology dependent upon
epithelial adhesion (EpCAM) and cytokeratins (86). This technology limits CTC
detection of early stage neoplasms because CTCs are thought to undergo
epithelial to mesenchymal transition (EMT), epithelial traits are lost and
epithelial marker such as EpCAM and cytokines are downregulated. CTCs present in
as few as 1 cell in 5 x 109 red cells, and up to 5–10 x 106 white blood cells,
are rare events (88). Newer microfluidic or centrifugation devices appear to
more efficiently capture CTCs (89, 91). The inclusion of
mesenchymal/EMT-specific antibodies, for example, vimentin, PLS3 may improve CTC
capture and/or expansion (88). With the emergence of ex-vivo expansion protocols
of CTCs and the increased ability to detect stem like or stem progenitor cells,
CTCs are of future interest as an early cancer detection diagnostic (89, 91),
but remain in the technology development phase.

 

Special consideration—EDRN discovered and preliminarily validated circulating
biomarker: Galectin-3 Ligand ELISA as a Serum Biomarker for the Detection of
Colorectal Neoplasia

 

The galectins are widely distributed and evolutionarily conserved carbohydrate
binding proteins characterized by their binding affinity for β-galactosides and
by conserved sequence elements in the carbohydrate-binding region (92).
Galectin-3 is the galectin that is of most interest in regard to colon cancer
because of its demonstrated role in cancer progression, metastases, and
interaction with mucins(93-97). Galectin-3 ligands include laminin, LAMP-1 and
2, LPS and colon cancer mucin. The major galectin-3 ligand detected in serum is
a 40 kDa band distinct from MUC2 and other mucins CEA, and Mac-2-BP. We reported
a true positive rate for the detection of CRC of 91% and false positive rate of
18% using preliminary data using quantitative Western blot technology on a
convenience set of GLNE serum (55).

 

We developed a sensitive, reproducible ELISA assay for galectin-3 using a new
antibody we created. This was used to assay the GLNE colorectal reference set
(50 colorectal adenocarcinomas/50 adenomas/50 endoscopically normal controls).
The ROC analyses for galectin-3 ligand combined with FOBT (fecal occult blood
test-guaiac based) for detection of colorectal adenocarcinoma versus controls
who had normal colonoscopy shows an area under the ROC curve of 0.91, while
galectin-3 ligand detection of colorectal adenocarcinoma alone versus controls
who had normal colonoscopy shows an area under the curve of 0.84. The true
positive rate of galectin-3 ligand with FOBT for detection of CRC is 64% with a
false positive rate of 5%. Without FOBT, true positive rate of galectin-2 ligand
was 72% with a false positive rate of 20%.

 

4.5Rationale and Current State of the Art: Stool Based Biomarkers for Detection
of Colorectal Neoplasia 

 

Occult blood tests

 

Stool testing as a screening approach offers the potential advantages of
noninvasiveness, low  cost, avoidance of cathartic preparation, and minimal
impact on work time or daily activities. Guaiac based FOBT is not specific for
human blood, and consequently, it has a high false positive rate for colorectal
neoplasia. The fecal immunochemical test (FIT) detects human hemoglobin, thus
eliminating the false positives caused by non-human hemoglobin in the diet (98,
99). FIT tests are more sensitive at detecting CRCs (sensitivity range 61% to
91%) and adenomas (sensitivity range 16% to 31%) than classical unrehydrated
guaiac FOBT (Hemoccult II) (sensitivity range 25% to 38% for CRC; 16% to 31% for
advanced adenomas) (100, 101). A recent meta- analysis that analyzed data from
19 prospective randomized trials or cohorts using 8 different commercially
available FIT tests with colonoscopy or 2-year observation endpoints reported an
overall sensitivity for detection of CRC of 79% (95% CI = 0.69-0.86),
specificity of 94% (95% CI = 0.92-0.95) and overall accuracy (defined as
hierarchical summary receiver operating characteristic (ROC) curve) of 95% (95%
CI = 93% - 97%) (Figure 1). Differences in performance characteristics among FIT
brands were small, particularly between the two major brands used OC-Light
(Eiken Chemical) and OC-Micro/Sensor (Polymedco + Eiken Chemical). The Polymedco
product is widely used in the USA. Quantitative FIT (Eiken OC-SENSOR) >177 µg/gm
stool combined with age and sex predicts 11.46 fold risk of a large adenoma over
lower risk groups (102).

--------------------------------------------------------------------------------

6

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

Doc 1_Page_2.jpg [f10k123119ex10z222.jpg] 

Fig 1 from Lee et al (1): Hierarchical ROC curve of the sensitivity versus
specificity of FIT. The diamond = summary point of the curve to which the pooled
sensitivity and specificity correspond. Dashed line = 95% CI for summary point;
dotted line = 95% confidence area of FIT diagnostic accuracy. AUC = area under
the curve; SENS = sensitivity;

 

Stool DNA tests

 

Since the neoplastic transformation process of the colonic epithelium results in
cells shedding into the stool, collection of fecal material is likely to yield
detectable molecular and biochemical events associated with cellular
transformation (103, 104). First generation multi-marker stool DNA tests
detected 52-73% of CRCs, 41-49% of CRCs plus adenomas with high grade dysplasia,
and 15-46% of adenomas ≥1 cm, with specificities of 84- 95% (105, 106). Stool
DNA test performance in both studies was compromised by failure to use
stabilization buffer with stool collection, inefficient marker recovery from
stool, and relatively insensitive analytical methods. Exact Sciences modified
their previously published stool DNA panel (106) and now uses a panel consisting
of methylated BMP3 and NDRG4 promoter regions, mutant K- ras (7 point mutations,
Exon 2, codons 12,13), and a proprietary FIT test. In a recently published cross
sectional validation study of 9,989 patients undergoing screening colonoscopy,
the panel performed with a sensitivity of 92% for CRC; 84% for CRC + high grade
dysplasia; and 42% for advanced adenomas (Figure 2) (2). The specificity was 87%
for CRC, the ROC AUC for the Exact Sciences DNA stool panel for the detection of
colorectal cancer is 0.94. FIT alone (Polymedco FIT) performed with sensitivity
of 73.8% and specificity of 94.9% for detection of CRC and sensitivity of 23.8%
for screen relevant neoplasia. Stool DNA component of the panels adds ~20%
sensitivity to FIT. The USPSTF is currently assessing the role and contribution
of fecal DNA panels such as the Exact Sciences panel to CRC screening (107).

 

Doc 1_Page_3.jpg [f10k123119ex10z223.jpg] 

Fig 2 from Imperiale et al (2) sensitivity for detection of CRC by Exact
Sciences stool DNA panel + FIT (light blue) vs Fit alone (dark blue) by stage.

--------------------------------------------------------------------------------

7

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

Vimentin Methylation as a Stool DNA Test

 

Aberrant methylation of vimentin exon 1 was initially described as a highly
frequent biomarker of colorectal cancers and adenomas by Markowitz and
co-workers (108). In reproducible studies, aberrant methylation of vimentin has
been detected in 72%-83% of colon cancers and 70%-84% of colon adenomas (108,
109). The current assay for detection of vimentin exon 1 methylation is based on
using methylation specific PCR (MSP).  Adaptation of the vimentin MSP to testing
fecal DNA is accomplished by recovery of vimentin DNA sequences from human stool
using hybrid capture to vimentin specific oligonuclotides (108). Initial study
showed that MSP assay of vimentin purified from feces (fecal vimentin DNA)
detected methylated fecal vimentin DNA in 46% of cancer patients (N=94) at a
specificity of 90% (N=198)(108). This initial study involved collaboration
between the Markowitz laboratory who had discovered the methylated vimentin DNA
marker, and Exact Sciences, who implemented detection of this marker in fecal
DNA. This initial study was limited by use of samples that had suffered problems
of DNA degradation  during sample collection and shipping (106).  A recently
published two stage follow-up study lead by Itzkowitz  et al in collaboration
with Exact Sciences and the Markowitz laboratory showed markedly improved
results with the use of a DNA stabilizing buffer added to stools at the time of
collection (110). Detection of methylated fecal vimentin DNA was found in 77% of
cancers (N=82) at 83% specificity (N=363). Six of 7 adenomas with high-grade
dysplasia were also detected. This assay has successfully detected 55% (N=22) of
adenomas that were greater or equal to 1cm in size (110). This is a published
assay of capture of fecal vimentin DNA and then MSP detection of methylated
vimentin exon 1 sequences (108, 110, 111).

 

Other Stool Based Biomarkers Under Investigation

 

Considerable interest in fecal microbiome populations has triggered EDRN
supported investigators into identifying unique bacterial species that are
associated with colonic carcinogenesis and suggests that a microbiome signature
may be a useful stool biomarker for CRC risk (112, 113). Metabolome signatures
promise to identify amino acid or fatty acid profiles associated with colorectal
cancer or high risk (114) have been preliminarily developed in EDRN supported
research. Micro-RNAs (miRNA) have both oncogenic and suppressor properties, can
be detected in stool, and have been explored as stool based early detection
biomarkers (115, 116). Studies published to date have used small convenience
samples and array technologies that have identified diverse and non-reproducible
miRNAs as classifiers for colonic neoplasms.

 

4.6Key Issues Driving Research Questions in CRC Early Detection Biomarkers 

 

Until therapeutic agents with much greater potency and minimal side effects are
developed, the current best strategy for reducing cancer morbidity and mortality
is early detection of neoplastic disease (117). Key opportunities in the current
state of colorectal screening and early detection include:

 

1.Enhancing adherence to current screening guidelines: Screening and early
detection reduce mortality from colorectal cancer; yet 35% of the population in
the USA remain non-adherent. 

 

Adherence is much lower in other countries (28). The barriers to these
recommendations (cost, discomfort, cultural taboos) may be overcome with
circulating biomarkers that provide individuals with persuasive evidence that
undergoing invasive screening procedures, i.e. colonoscopy, will have important
life-saving benefit that reduces mortality from CRC (11-13, 21). Developing,
validating and bringing circulating biomarkers to population screening use
remains a high priority that will likely increase adherence to endoscopic
screening.

 

2.Tailoring colonoscopic screening to individual risk: Recently published data
from the Clinical Outcomes Research Initiative found the prevalence of large
polyps higher in blacks than whites among both men and women (118). Tailoring
endoscopic screening to those at risk while limiting screening for those with
minimal or no risk (119, 120) will enhance screening adherence and eliminate
excess cost. Recommendations for tailoring were primarily population demographic
based (119, 120); yet, the translation of carcinogenesis biology and genetics
into biomarker panels with extremely high sensitivity (99%), i.e. no false
negative tests, promises precise tailored endoscopic screening. The current
state of art stool using based biomarker tools is coming close—92% sensitivity
(2) but insufficient to permit tailored or individualized risk. 

 

3.Persistently positive stool DNA tests with negative colonoscopic screening:
The stool methylated DNA panel’s report 5% false positives (2, 111). A positive
stool DNA test with a negative screening colonoscopy could potentially arise
from neoplasia in the upper gastrointestinal tract or from occult and missed
lesions in the colorectum. The latter is a particular concern in the right
colon, where flat lesions and/or sessile serrated adenomas are more prevalent.
Preliminary data from the Case Western EDRN BDL found near 100% vimentin
methylation in gastric dysplasia while no methylation in adjacent gastric mucosa
(S. Markowitz, Personal Communication). In Barrett’s esophagus (BE), 7 of 7 high
grade dysplasias (HGD), and 15 of 18 esophageal adenocarcinomas (EAC) and even
in some squamous cancers (SCC) had methylated vimentin, whereas it was absent in
all 9 normal squamous mucosa (121). A “false positive” stool DNA test may detect
dysplasia or invasive neoplasms in the upper GI tract. 

--------------------------------------------------------------------------------

8

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

5.0STUDY DESIGN 

 

5.1Summary of Study Plan 

 

We propose a multi-center, prospective, cross-sectional cohort validation study
of 1,682 subjects. We propose to increment the original GLNE 007 cohort with 400
subjects with diagnosed colorectal cancer, 200 subjects with colorectal
adenomas, 200 subjects a prior history of adenomas, colorectal adenocarcinoma
(>3 years previous), returning for surveillance or positive stool test (DNA or
blood) but have a normal colonoscopy (higher risk normal), and 200 subjects who
have a normal colon with NO prior history of adenomas, colorectal adenocarcinoma
(not returning for surveillance) and who do not have a current (within 12
months) positive stool test (DNA or blood) (normal risk) and have a normal
colonoscopy . Subjects will be recruited as described in Appendix B. The
baseline visit should be done prior to a scheduled colonoscopy. If a subject is
suspected of having a colon adenocarcinoma or an adenoma, the baseline samples
should be collected before any procedure to remove the cancer or adenoma so the
lesions are present when sample collection is done. Patients with cancer must
have their baseline visit and all sample collection completed prior to
endoscopic or surgical resection of CRC and chemotherapy and/or radiation
therapy. Informed consent, demographic information and medical history via
questionnaires, blood, and collection will be done at baseline. Stool
collection, to sample for FIT (x2) and for adenocarcinoma four native stool
specimen vials and 1 slurry will be done as described in the study calendar and
Appendix D. All samples will be collected, handled, transported, processed, and
stored according to detailed standard operating procedures and will be
de-identified by random Specimen ID linked to the Participant ID in VSIMS.
Selected subjects, based upon estimated future biomarker requirements, will have
normal colonic epithelium collected during the colonoscopy procedure for future
biomarker research. For those subjects with a large adenoma found on endoscopy,
a frozen biopsy will be requested. Cancers, for the most part will be identified
following endoscopic diagnosis.

 

5.2Rationale for tissue collection 

 

A primary goal of GLNE 007 is to provide biosamples for training and testing of
biomarkers that the EDRN believes have potential for future validation for
regulatory review. A secondary goal of GLNE 007 meets the EDRN’s discovery and
early phase characterization of biomarkers. The EDRN is a vertically integrated
organization that includes laboratories doing discovery research and early
detection performance characterization research. The GLNE supports all of the
EDRN’s missions—discovery, characterization, training and testing in addition to
large scale regulatory validation.

 

On occasion, investigators need fresh tissue to develop and test new biomarker
technologies. The GLNE maintains a repository of frozen normal and adenomatous
biopsy samples for this purpose. As with other biosamples proposed for GLNE 007,
the frozen tissue samples need revitalization and updating.

 

GLNE collects fresh biopsies from adenomas as made available by local
pathologists. If available, GLNE will also collect fresh biopsies from invasive
cancers at endoscopy.

 

Biopsy tissue samples are not required from every subject entered into GLNE 007.
The GLNE collects biopsies from normal colonic mucosa from subjects undergoing
colonoscopy who are found to have a normal exam on an as needed basis
(approximately 10% or 100 subjects will be asked to undergo biopsy of normal
colonic mucosa) with a small repository, to be made available to EDRN
investigators for discovery and early phase characterization. Because of the
extra risk, time for participants and extra costs to the GLNE involved with
performing these biopsies endoscopically, we do not require all patients with
normal colonoscopies to undergo biopsy and tissue collection of normal colonic
mucosa. The GLNE pays centers extra beyond the usual costs to procure frozen
biopsy samples. Normal biopsies may be used as controls for EDRN laboratory
biomarker discovery research controls for comparison with adenoma tissue and
invasive neoplasm tissue. GLNE 007 has provided this resource to the EDRN over
the last 15 years and continues to do so.

 

6.0INCLUSION AND EXCLUSION CRITERIA 

 

6.1Inclusion Criteria 

 

Willing to sign informed consent

 

Able to physically tolerate removal of up to 60 ml of blood

 

Adults at least 18 years old

 

Willing to collect 2 stool samples to prepare FIT test (x2) and for
adenocarcinoma 4 native specimen vials and 1 slurry

 

Nursing women who otherwise meet the eligibility criteria may participate

 

Subjects undergoing colonoscopy for screening or surveillance (known prior
neoplasms resected).

--------------------------------------------------------------------------------

9

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

Screening Colonoscopy

 

No known colonic neoplastic disease. Undergoing colonoscopic screening based
upon current colon cancer screening guidelines.

 

Subjects whose screening colonoscopy shows any of these types of polyps may be
included in the normal or the higher risk normal bin if they meet the other
criteria noted above.

 

Hyperplastic polyps 

Benign mucosal polyps 

Polypoid granulation tissue 

Prolapsed mucosal polyps 

Inflammatory polyp 

Transitional mucosal polyp 

Lipoma 

Gangleoneuroma 

Neuroma 

Hamartomatous polyp 

 

Subjects who had colorectal adenocarcinoma that was successfully treated at
least three years prior are eligible.

 

Recent screening colonoscopy (within 3 weeks of enrollment), poor preparation
found at colonoscopy and returning for repeat colonoscopy.

 

Recent diagnostic colonoscopy (within 3 weeks of enrollment) with detection of
adenocarcinoma or adenoma.

 

Known colorectal adenocarcinoma or adenoma remains in place after a diagnostic
colonoscopy— adenocarcinoma or adenoma in colon at time of blood and stool
collection.

 

Enrolled participants will be grouped into Bins according to one of the
following:

 

Colorectal Cancer-pathologically confirmed colorectal cancer either present at
time of stool collection or discovered during colonoscopy (Cancer Bin) 

 

Adenoma-pathologically confirmed adenoma (Adenoma Bin) 

 

Higher Risk Normal (normal colonoscopy) 

 

Negative study colonoscopy and:

 

oSubjects with a personal history of adenomas (confirmed by pathology) with none
present on qualifying colonoscopy 

oSubjects with a personal history of CRC (longer than 3 years ago because of
exclusion criteria of cancer within last 3 years) with none present at time of
qualifying colonoscopy 

oAny family history of CRC (1st degree relative) 

oCurrent positive screening stool test for blood, for DNA or for both within 12
months. 

 

Normal Control (normal colonoscopy) 

 

Negative colonoscopy and:

 

oNo prior history of adenomas 

oNo prior history of CRC 

oNo family history of CRC 

oNegative screening test (if performed) for blood, for DNA or for both within 12
months. 

--------------------------------------------------------------------------------

10

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

 

 

6.2Exclusion Criteria 

 

Cancer patients who have had any surgery, radiation, or chemotherapy for their
current colorectal cancer prior to collecting the baseline samples

 

Other active malignancy within 3 years of enrollment except any of the
following:

 

a.Squamous cell carcinoma of the skin 

b.Basal cell carcinoma of the skin 

c.Carcinoma in situ of the cervix, Stages Ia or Ib invasive squamous cell
carcinoma of the cervix treated by surgery only. (Excluded if had pelvic
radiation) 

d.Stage Ia Grade 1 adenocarcinoma of the endometrium treated with surgery  

 

Patient is on active chemotherapy or radiation treatment

 

Patients with a history of or clinically active Inflammatory Bowel Disease

 

Patients with known HNPCC or FAP

 

Subjects with known HIV or chronic viral hepatitis

 

Inability to provide informed consent

 

Women who are pregnant

 

7.0STUDY PROCEDURES 

 

7.1Subject Recruitment 

 

Patients diagnosed with colorectal cancer and adenomas and scheduled for
surgical or endoscopic resection or subjects scheduled for a colonoscopy will be
recruited from collaborating consortium centers.

 

The clinical research associate or study nurse (CRA) at each clinical site will
identify subjects with appointments for colonoscopy, surgery, endoscopic polyp
or cancer removal, or oncology. The study team will obtain permission to review
the schedules from the physicians and the Institutional Review Boards. If the
physician agrees that their patient can be contacted regarding participation,
the research coordinator will meet with the patient in person or send a letter
to the patient describing the study. Advertisements (e.g., newspapers, clinics)
may also be used to recruit subjects from the surrounding communities.

 

The letter to the subject will include an opt-in response card. If we receive
permission from the subject to contact them, the CRA will discuss the overall
study with the potential subject, and arrange for a baseline visit to get
consent, baseline samples, and provide stool kit for FIT and specimen vials.

 

7.2Clinical Procedures 

 

Enrollment and Registration Procedure

 

Eligible subjects will be enrolled into the study after providing informed
consent to analyze stool samples and FIT, and blood samples for biomarkers,
medical records review, and for completion of questionnaires. The subject will
be assigned a Participant ID by the recruiting site and documented in VSIMS.

 

Timing of Sample Collection

 

7.2.1.1Sample collected prior to colonoscopy procedure 

 

Baseline samples, including stool, blood, and FIT must be collected prior to any
colonoscopic preparation procedure.

--------------------------------------------------------------------------------

11

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

7.2.1.2Samples collected with known diagnosis of unresected, untreated colon
adenocarcinomas or adenomas. 

 

If any subjects are eligible to begin the study after their colonoscopy (e.g., a
lesion remains in the colon), at least 7 days must elapse from the diagnostic
colonoscopy, but no more than 3 weeks. Eligibility for the respective bins will
be determined from the pathology and colonoscopy reports. Cancer patients must
have a diagnosis of colon or rectal adenocarcinoma that has been previously
untreated. Any stage is allowed. The baseline stool, FIT, and blood samples must
be collected before any surgical resection or chemotherapy or radiation therapy
is performed.

 

Baseline Visit

 

Informed consent will be obtained prior to any data or sample collection.
Samples will be collected either prior to colonoscopic preparative procedure or
7 days or more after a diagnostic colonoscopy as outlined in Section 7.2.2.2.
Detailed instructions will be provided to the subjects on the collection of the
stool for the FIT tests and for adenocarcinoma four native stool specimen vials
and 1 slurry. Samples will be collected as described below. Subjects will
prepare two FIT tests and for adenocarcinoma four native specimen vials and 1
slurry from the stool sample for shipping to the University of Michigan.

 

Data Collection

 

The subject will be asked to complete EDRN demographic and medical history
questionnaires (Appendix A) at baseline. Clarification or additional information
may be obtained from the medical records. These data forms have gone through
multiple stages of development and testing and are standardized across EDRN
studies. Case report forms (CRFs) will also be used to collect information on
concomitant medications, colonoscopy outcomes, resection information, cancer
treatment, and diagnostics. The Follow up forms and medical record review will
be completed at the follow up visit for the subjects in the adenoma and CRC bins
if seen in clinic, otherwise done over the phone or e-mail. Long term data
collection (medical records review and follow up CDE for all bins) will be done
by a phone call or email once at one year post their last contact.

 

Sample Collection: Blood

 

Blood samples, up to 60 mLs, will be obtained according to standard operating
procedures (Appendix C).

 

Sample Collection: Stool for FIT Testing

 

Adenocarcinoma subjects will be provided with a standard collection kit
including detailed instructions on how to complete the FIT sampling. All other
subjects will only obtain two FIT tests (Appendix D). The first FIT tube will be
shipped inside the same shipping container with the stool sample. The second FIT
tube will be mailed (pre-paid) to the University of Michigan at room temperature
in the manufacturer’s United States Department of Transportation-compliant
envelope. The test will be analyzed at the Central Laboratory at the University
of Michigan using analytic equipment provided by Eiken Chemical Company.
(OC-SENSOR Diana).

 

Sample Collection: Stool for Biomarker Testing (ADENOCARCINOMA SUBJECTS ONLY)

 

Subjects with a known diagnosis of colorectal adenocarcinoma will be asked to
collect their stool in the collection bucket (hat) provided. Subjects will be
given detailed instructions and complete kits to collect the stool samples at
home. They will prepare a FIT tube (FIT #1) from the stool sample. Subjects will
also collect scoops of stool into a container with an EDTA-based buffer
(“buffered stool”) and additional scoops of stool into tubes provided to be sent
on ice packs (“native stool”).

 

The subjects will then package both the stool and the FIT for shipping per
provided instructions. The US and Canadian subjects will ship the stool sample
to the Central Laboratory at the University of Michigan using pre-paid DOT
(Department of Transportation)-compliant packaging. Buffered stool samples will
be homogenized and frozen in four 5 ml aliquots at –70° C or colder for batch
shipment to the analytical labs. The native stool will be placed at –70° C or
colder upon receipt.

 

Sample Labeling

 

All samples will be labeled or have an embedded barcode with a unique bar code
and linked to the participant ID through VSIMS.

--------------------------------------------------------------------------------

12

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

 

 

7.3Biological Sample and Data Collection 

 

Blood Collection and Storage

 

Subjects will provide up to 60 ml of blood in six 10 ml collection vials (2 red,
3 purple tops and 1 ACD-A, for serum and plasma respectively). Purple tops tubes
must be filled to manufacturer’s level to maintain blood: EDTA ratio. Additional
blood draws, prior to prepping for the colonoscopy may be done to get to the
necessary blood volume.

 

The serum samples (red top tubes) will sit at room temperature for a minimum of
30 minutes (maximum of 60 minutes) to allow the clot to form, and if not
processed immediately, they can be held at 4° C for a maximum of 4 hours after
collection. Plasma samples (purple {aka lavender}top tubes) and ACD-A (yellow
top) will be held at 4° C for a maximum of 4 hours after collection. The red top
collection tubes will be centrifuged at >1,300 x g at 4° C for 20 minutes
(centrifuge brake off for first 10 minutes, then on for last 10 minutes). The
serum will be removed, transferred to pre-labeled tubes, and frozen at –70° C or
colder. The purple top and ACD-A collection tubes will be centrifuged at >1,300
x g at 4° C for 10 minutes without the centrifuge brake off for first 10 minutes
and on for the last 10 minutes. The plasma will be transferred to a 15 ml
conical tube for a second centrifugation step (>1,300 x g at 4° C for 10
minutes) prior to aliquoting in pre-labeled tubes, and frozen at –70° C or
colder. All frozen samples will be stored at –70° C or colder at the collection
site and shipped on dry ice monthly to the Central Laboratory at the University
of Michigan and stored at–70° C or colder until assayed. Detailed Standard
Operating Procedures including shipping and sample handling instructions are
provided in Appendix C.

 

Stool Sample Collection and Handling (ADENOCARCINOMA ONLY)

 

Subjects with a known adenocarcinoma will be asked to collect a stool sample at
baseline prior to any therapy or resection (when applicable). Subjects will be
given a standard stool collection basin (hat) with detailed instructions,
shipping container, pre-paid shipping labels, four native specimen collection
vials, 1 stool slurry and cold packs, FIT vials, and all necessary supplies.

 

Subjects will be asked to collect a whole stool sample in the container
provided, ensuring that no other materials (e.g. paper or urine) are collected
in the hat. Subjects will collect scoops of stool into a container with an
EDTA-based buffer (“buffered stool”), additional scoops of stool into tubes
provided to be sent on ice packs (“native stool”), and a sample in a FIT vial.
The subjects will then package both the stool and the FIT for shipping per
provided instructions. Subjects will be asked to prepare four native specimen
vials, 1 slurry and FIT tests (x2) (see appendix D) using the materials and
instructions provided. The specimen vials will be shipped on the cold packs and
frozen at - 80° C at the Brenner laboratory at the University of Michigan.

 

Fecal immunochemical Test (FIT) (All enrolled subjects)

 

Subjects will be asked to prepare two FIT tests (see appendix D) using the
materials and instructions provided. The OC-Sensor®, Eiken Chemical Company
product, will be used according to manufacturer’s instructions. The threshold
for a positive test is 100 ng/ml. The Central Laboratory will process the
samples using equipment provided by Eiken Inc. Technicians will undergo tutorial
and quality assessment with Eiken support technicians prior to study launch. A
quantitative result will be generated and recorded in the database.

 

Sample Collection: Tissue Samples

 

NOTE: Tissue sample collection not required for protocol completion. A limited
number of tissues per bin will be collected. Collection will be performed at
specified designated institutions for incremental payment per accrual

 

7.3.1.1Collection of Frozen Normal and Adenoma or Cancer Tissue 

 

For individuals with large adenomas who are undergoing endoscopic resection, the
fresh surgical sample will be obtained by the endoscopist. Once the adenomas are
located, a digital endoscopic picture will be obtained. Once the adenoma (s) is
(are) removed, two biopsies will be done or two cuts will be made. The biopsies
will then be frozen in liquid nitrogen after being placed in a pre-designated,
labeled container. Normal sigmoid tissue will be collected as described below.

Bar coded vials will be sent to University of Michigan sample storage facility.
The adenoma will then be sent to the institution’s clinical pathology department
according to standard clinical procedures.

--------------------------------------------------------------------------------

13

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

For cancer or adenoma patients who are undergoing surgical resection, the Site
CRA will notify the Pathology Service or the Institutional Tissue Procurement
Service of a surgical sample needed for study purposes. Once the specimens are
removed, two to four biopsies will be done or two- four cuts will be made. At
least one of the biopsies should be from normal colon. The biopsies will be
frozen in liquid nitrogen after being placed in a pre-designated, labeled
container. Bar coded vials will be sent to University of Michigan sample storage
facility. The specimen will be sent to the institution’s clinical pathology
department according to standard clinical procedures.

 

7.3.1.2Collection of Fixed and Frozen Normal Sigmoid Colon Biopsies on
Qualifying Colonoscopy 

 

For all subjects who agree to the biopsy portion of the study and are undergoing
colonoscopy, the endoscopist will take up to 6 biopsies from the normal sigmoid
colon. Of those, at least 2 (and up to 4) will be snap frozen and at least 1
(and up to 2) will be fixed in 10% formalin and sent to the University of
Michigan for paraffin embedding by the Histology Core. The fixed and frozen
samples will be stored at the University of Michigan GLNE Core Laboratory.

 

Sample management procedures including storage, tracking, and shipping
instructions are provided in Appendix E.

 

Tissue samples from pathology specimens may be requested for future biomarker
studies from samples collected during routine clinical management of patients
with adenomas and CRC. Medical records may be re-reviewed to extract data
including, but not limited to size and location of tumor, histopathological
features, patient treatment, and response to therapy. Patient permission will be
obtained via the informed consent document. The University of Michigan core
laboratory may request tissue blocks either to cut slides or to keep for future
biomarker studies.

 

Medical Records Documentation

 

Medical records will be reviewed to collect information regarding the results of
the procedures, pathology analysis, surgery, treatment, history, or outcomes and
documented in the CRFs/CDEs. The medical records will serve as the source
documents and will be maintained at the site enrolling the subject. Since these
records necessarily contain subject identifiers, they will not be sent to the
Data Coordinating Center at Dartmouth or to the University of Michigan. Medical
records may be reviewed at the site during audits or monitoring visits.

 

Sample labels

 

All samples will be labeled with bar-coded labels or have embedded bar-codes.
The labels will be provided by the DMCC and will link to the subject
identification number in VSIMS. Labels will be placed on all tubes in the blood
drawing kit, FIT, and frozen stool sample vials.

 

Sample tracking

 

All samples will be tracked by a bar code through a computerized program called
VSIMS. Upon receipt of the specimen in the University of Michigan Laboratory
Core, the bar code will be read and the date and time of arrival, documented.
The Data Management Center will be notified at completion of each individual
assay performed on a sample.

 

Long-term Follow up

 

The CRA will contact the subject via phone or email one year after their last
sample collected for additional follow up data. Data will be collected on
medical record review and follow up CDE (Appendix A), and include information
related to their GI tract history or cancer history and related treatments,
procedures, and outcomes. The consent form describes the long-term data
collection.

 

7.4Circulating methylated genes BCAT1/IKZF1 (Clinical Genomics) 

 

A Good Laboratory Practice validated bisulfite PCR assay developed by Clinical
Genomics will be used for this assay. Clinical Genomics will perform this assay
on blinded samples at their laboratory facility in Rutherford, NJ. Clinical
Genomics is not responsible for analysis of any other biomarkers other than
their BCAT1/IKZF1 product. Sample distribution schedule is outlined in the
Clinical Study Agreement.

--------------------------------------------------------------------------------

14

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

7.5Hypomethylated LINE1 from circulating cell free DNA (VolitionRx) 

 

A Good Laboratory Practice validated assay developed by VolitionRx will be used
for this assay. VolitionRx will perform this assay on blinded samples at their
laboratory facility in Namur, Belgium. Volition is not responsible for analysis
of any other biomarkers other than their hypomethylated LINE1 assay. Sample
distribution schedule is outlined in the Clinical Study Agreement.

 

7.6Disclosure of results to subjects 

 

Subjects will be informed as part of the consent process that neither they nor
their health care providers will receive any results from participation in this
study.

 

7.7Evaluable subjects 

 

A subject is considered evaluable and on-study if all samples are collected per
protocol. Subjects without a full set of samples or data may need to be replaced
on the study to get 400 evaluable cancers and 200 evaluable subjects the other
three bins.

 

A subject will be asked to provide a replacement sample if:

 

a.The stool specimens are received outside the time window required (i.e.
greater than 36 hours after collection time and/or not kept cold)
(Adenocarcinoma subjects only) 

b.No FIT test 

c.Blood cannot be obtained (must be obtained while target lesion is still
present for adenomas and cancers) 

d.Blood is subject to some kind of handling error (no EDTA, too long at room
temperature, etc.) and subject is still eligible to provide the blood again 

 

Protocol deviations

 

Subjects who do not provide one of the samples or all of the data, but are
otherwise eligible to remain on study, will not be reported as deviations.

 

7.8Completion of Study 

 

A subject has completed the study when the CRF data, blood samples, stool
samples (adenocarcinoma only) and FIT have been obtained, properly processed and
delivered to University of Michigan, and the one year follow up phone call has
been done. A subject may be asked to provide a replacement sample if there is a
problem with one collected, including an additional stool sample. The subject
may decline, if they choose.

 

7.9Subject Compensation 

 

To compensate for the inconvenience and cost of driving and parking, $25 will be
provided to each subject once blood samples, and stool samples are completed or
$50 for adenocarcinoma subjects who have provided all required samples.
Recruiting sites will receive gift cards to distribute to subjects that complete
the requirements to receive payment. Sites are required to account for
distribution of gift cards to subjects. Sites outside the US will receive
reimbursement by invoice, instead of gift cards.

--------------------------------------------------------------------------------

15

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

8.0STUDY CALENDAR 

 

Procedures

 

Baseline1

Baseline stool collection2

Colonoscopy/

Resection

Long-Term

Follow up

Eligibility Checklist

X

 

 

 

Consent Documentation

X

 

 

 

General Information

X

 

 

 

Medical History

X

 

 

 

Concomitant Medications

X

 

 

 

Colonoscopy

 

 

X

 

Surgery

 

 

X

 

Colon Cancer Treatment

 

 

 

X

Blood Collection

X

 

 

 

FIT TESTS

 

X2

 

 

Stool Sample

 

A2,4

 

 

Frozen/Fixed Tissue collection

 

 

S3

 

Follow up CDEs

 

 

 

X

 

1Baseline clinic visit—Prior to treatment of any colon lesion, or prior to a
colonoscopy (specifically, prep), OR at least 7 days post colonoscopy but no
later than 3 weeks post colonoscopy.

2Stool collection any time after baseline visit and subject returns home with
kits

3Frozen tissue is collected at the time of surgical or endoscopic resection of
cancer or colonoscopy findings (fixed and frozen).

4Stool samples in EDTA buffer and 4 vials collected in adenocarcinoma subjects
per stool collection and handling SOPs.

S = Special circumstance. Not a required component of protocol completion unless
institution is registered as Special circumstance. Additional remuneration
provided for a specific number of patients for frozen tissue collection in each
of specified bins.

A = Stool collection required for subjects who have adenocarcinoma of the colon
or rectum. The patient incentive is increased from $25 to $50 because of their
diagnosis and additional effort compared to subjects who do not have cancer.

 

9.0STATISTICAL CONSIDERATIONS 

 

9.1Study Population 

 

This study is stratified: normal subjects (Stratum 1); subjects at high risk or
previously with adenomas who currently are without adenomas (Stratum 2);
subjects with adenomas (screen relevant neoplasia (SRN) and non-screen relevant
neoplasia (Stratum 3) subjects with colorectal adenocarcinoma (Stratum 4).  200
subjects are to be accrued to each stratum except 400 subjects in Strata 4.
Subjects in both Strata 2 and 3 are expected to be more likely to be positive
for upstream markers of carcinogenesis than the normal subjects in Stratum 1
(who are both not at high risk and have never had adenomas), while subjects in
Strata 3 and 4 are expected to be more likely to be positive for downstream
markers indicating the presence of adenomas or adenocarcinomas than those in
Strata 1 and 2. Stratum 2 may, therefore, be pooled with Stratum 1 or Stratum 3,
depending on the context. From the screening perspective, Stratum 3 will be
further divided into SRN or non-SRN. Non-SRN in stratum 3 could be combined with
Strata 1&2 to form a non-SRN group and compared to Stratum 4, or compared to SRN
in Stratum 3. Oversampling of subjects with adenocarcinoma of the colon or
rectum is necessary to provide sufficient dedicated samples and data for
validation trials aimed at regulatory approval. Samples and data from subjects
recruited in this trial may be used to update and enhance reference sets used by
the EDRN to further train, test and/or validate new biomarkers for future
inclusion in validation trials aimed at regulatory approval. Strata 1-3 are
necessary to ensure these comparison groups collected under the same protocol to
Stratum 4 are available.

 

Training and validation: The prospective GLNE010 study has recruited many
subjects in each stratum except adenocarcinoma. The samples from this protocol
will need to be combined with GLNE010 samples to allow both panel building
(training) and panel validation for each of above comparisons.

--------------------------------------------------------------------------------

16

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

9.2Data Analysis Plan 

 

Assessment of the utility of individual biomarkers for discriminating between
patients with adenocarcinomas, patients with adenomas, patients without adenomas
and normal subjects.

 

For markers measured on a continuous scale, the within-class distributions of
the marker values will be assessed by graphical means (e.g., q-q plots). Maximum
likelihood estimates of distribution parameters will be calculated. For markers
measured on a dichotomous scale, the proportions of positive tests in each class
will be determined. For each marker, non-parametric (via SAS PROC LOGISTIC) and
fully parametric ROC curves will be constructed for: Stratum 4 versus all others
except SRN-adenomas (define as adenoma ≥1 cm or adenoma with high grade
dysplasia or sessile serrated polyp ≥1 cm) (primary comparison); Stratum 4
versus Strata 1 and 2 (secondary comparison); and other exploratory comparisons:
Stratum 4 versus Stratum 1; Stratum 4 and SRN-adenomas in Strata 3 versus Strata
1 and 2 and non-SRN adenoma; Strata 2, 3 and 4 versus Stratum 1. While the
non-parametric ROCs are generally preferred, decision rules for population
screens may require very high specificity, which will require accurate
estimation in the distribution tails; parametric ROC curves may be better for
this application. The area under each ROC curve (AUC) for each comparison will
be determined.

 

Construction or testing of a panel of markers from those considered in Objective
1

 

Construction of a panel of markers from those considered in Objectives 1 and 2
to discriminate, under specific of assumptions concerning prevalence and cost of
misclassification, for the primary, secondary, and exploratory comparisons
described above. Candidate markers will be chosen according to both statistical
(e.g., high patient or tissue sensitivity) and practical (less expensive assays,
all markers assessed on blood) criteria. Forward stepwise logistic regression
will be used to construct a panel to discriminate between the two classes of
patients. The ROC curve will be constructed and AUC will be determined. Other
panel building approach will also be used when appropriate, e.g. an “OR” rule
will be used, that a test is positive if either one test is positive, if each of
the biomarker is very specific but only for a subset of cancers.

 

Validation of a panel of biomarkers: If the cutoff has not been locked-down but
the combination rule has been pre-determined, the optimal cutoff will depend on
the intended clinical use. For example, for a blood based biomarker as a first
step screening for those who do not want to do stool FIT tests, we might the
cutoff that is corresponding to sensitivity of FIT test for colorectal cancers,
then evaluate if the specificity of this cutoff is adequate. False positive is
of less concern because it will lead to colonoscopy, a recommended screening in
US. For a stool- based test (only for adenocarcinomas in this study when
combined with the previous GLNE007 or GLNE010 set where stool samples were
collected for all participants), if a test with much lower cost than that of the
Exact Sciences multi-marker panel that includes a fecal immunochemical test and
methylated DNA gene markers is brought for testing, the threshold sensitivity
required to enter a large validation trial might be lower than that of the Exact
Sciences panel (in the range of 85% to 92% for detection of adenocarcinoma) but
better than FIT alone.

 

We would then compare the specificity to that of Exact Sciences multi-marker
panel. If the cutoff has been pre-determined, then the evaluation will be a
simple joint 1-sided 95% confidence region for sensitivity and specificity.

 

Comparison of the characteristics of individual markers and panels as
discriminators to those of the established current standard, Fecal
Immunohistochemistry test (FIT).

 

For biomarker validation we assume at least the panel combination rule has been
locked-down. The biomarkers or panels could be from outside of the consortium or
from the one built in 9.2.2 using previous GLNE007 specimens collected between
2006-2010 and GLNE 010 samples collected between 2011 and 2019. As in Objective
3, the following analysis will be performed for each of the primary, secondary,
and exploratory comparisons. For blood-based biomarkers, we will test whether it
has a similar sensitivity as that of FIT and has a reasonable specificity (e.g.
> 70%) if the cutoff is pre-determined, or whether at a cutoff corresponding to
the same sensitivity of FIT the specificity is better than 50% (target
specificity > 70%) if a cutoff is not pre- determined. This performance
criterion is also used for training set panel building, i.e., a panel will need
to have this performance before it is locked-down for validation. For
stool-based biomarkers, we will test whether the sensitivity is better than that
of FIT, with compatible specificity. With non-screening colonoscopies, we will
collect information whether the colonoscopy was triggered by a positive FIT test
or triggered by symptom. The performance of biomarker will be evaluated with
each of these two groups separately and compared, to gauge the potential bias
caused by FIT positive results triggering colonoscopy that will lead to over-
estimate of sensitivity for FIT.

--------------------------------------------------------------------------------

17

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

Development of a bank of stool samples linked to serum, tissue, and clinical
data from patients with colorectal cancer, adenomas and normal controls for
validation of stool-based markers that may be developed in the future.

 

Markers available in the future will be developed in a similar fashion to
Objectives 1-4. Every effort will be made to ensure that samples from and data
concerning subjects in all four strata are collected, processed and stored
according to the same procedures (Section 8.2 and Appendices), so that data and
sample banking do not introduce bias into future studies.

 

9.3Justification of Design and Sample Size 

 

The primary goals of this protocol are to enhance the already available EDRN
reference set and provide biosamples and data as required to fill in validation
sets for the purposes of regulatory approval. The reference sets and other GLNE
007 samples will be used to assess the ability of different markers to
discriminate between patients with adenocarcinoma, patients with adenomas and
normal subjects (Objective 1) and to strategically use this information to
construct panels of markers to discriminate cases (adenocarcinomas and/or screen
relevant-adenomas) from controls (Objective 2). An additional reference set
might be set aside for the purposes of regulatory validation. Such samples may
not be used for training or testing of a given marker that might be validated
with samples from the GLNE 007 reference set or other validation sets previously
collected by EDRN.

 

We justify the sample size for the primary comparison for training and
validation separately:

 

Panel training and testing: Cancer (n=200) versus 560 normal (normal
colonoscopies (200 average risk subjects, 200 high risk subjects) or non-screen
relevant adenomas (estimated to be 80% of 200 adenomas, i.e. n=160)) for a
blood-based test as the first line test for people who do not do any colorectal
cancer screening. We assume the cutoff has not been locked-down (statistical
power would be much larger if the cutoff is locked-down) so we will use cutoff
corresponding to sensitivity of FIT (75%). We argue that with this sensitivity a
test with at least 70% specificity would have great clinical utility. With the
study sample size, we will have >90% power to reject a null hypothesis
specificity of 58% if the true specificity is at least 70%.

 

For stratified analysis if there is evidence that specificities for normal
colonoscopy high risk subjects and subjects with non-screen relevant adenoma are
significantly lower than that for normal colonoscopy low risk group, suggesting
they may have higher risk for screen relevant neoplasms in the future and should
be analyzed separately. With 200 cancers and 200 normal colonoscopies in low
risk group, we have at least 82% power to reject a null hypothesis specificity
of 58% if the true specificity is at least 70%. With 200 cancers and 360
subjects in high risk or non-SRN adenoma groups, we have at least 89% power to
reject a null hypothesis specificity of 58% if the true specificity is at least
70%.

 

For validation of stool-based test, we use the scenario that using a cutoff
corresponding to 92% sensitivity (that of the Exact Sciences multi-marker panel)
and test the adequacy of specificity, with 200 cancers and 747 normal or
non-screen relevant neoplasms from GLNE010 training set, we have at least 86%
power to reject a null hypothesis of specificity 76% if the true specificity is
at least 85%. Such a test if it is substantially cheaper than that of the Exact
Sciences multi-marker panel will have clinical value to increase the sensitivity
of FIT.

 

Panel validation: Cancer (n=200) versus 560 normal (normal colonoscopies (200
average risk subjects, 200 high risk subjects) or non-screen relevant adenomas
(estimated to be 80% of 200 adenomas, i.e. n=160)) for a blood-based test as the
first line test for people who do not do any colorectal cancer screening. We
assume the cutoff has not been locked-down (statistical power would be much
larger if the cutoff is locked-down) so we will use cutoff corresponding to
sensitivity of FIT (75%). We argue that with this sensitivity a test with at
least 70% specificity would have great clinical utility. With the study sample
size, we will have >90% power to reject a null hypothesis specificity of 58% if
the true specificity is at least 70%. If the validation is done using all 3070
normal and non-screen relevant neoplasms from GLNE010 (the number as of April
2019), then we have >90% power for to reject a null hypothesis specificity of
60%.

 

For validation of stool-based test, we use the scenario that using a cutoff
corresponding to 92% sensitivity (that of the Exact Sciences multi-marker panel)
and test the adequacy of specificity, with 200 cancers from this protocol and
3070 normal and non-screen relevant neoplasms from GLNE010 as of April 2019, we
have at least 90% power to reject a null hypothesis of specificity 76% if the
true specificity is at least 85%. Such a test if it is substantially cheaper
than that of the Exact Sciences multi-marker panel will have clinical value to
increase the sensitivity of FIT.

--------------------------------------------------------------------------------

18

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

10.0DATA SAFETY AND MONITORING 

 

10.1Data Safety and Monitoring 

 

Authority

 

The DSMC reviews, makes recommendations, and acts on the following:

 

a.All protocols being run through the GLNE EDRN will be monitored by the DSMC. 

b.Progress towards completion of the trial—recruitment and retention of study
participants. 

c.Insufficient accrual to warrant continuation of the trial. 

d.Evaluation of interim data analyses. 

e.Evaluation of interim new information. 

f.Evaluation of toxicity events including reporting of adverse events. 

g.Timeliness of data. 

h.Quality of data. 

i.Ethical conduct of research. 

 

The DSMC is empowered with the authority to recommend a trial be suspended or
terminated based upon concerns in any of the above areas of review. The DSMC
reviews all serious adverse events and ensures that these events have been
correctly reported to all institutional review boards, and that adverse events
have been correctly classified as serious or not serious. The Board assesses the
impact of these events upon the conduct of the clinical trial. The Board is
empowered with the authority to suspend or terminate any trials for which there
are concerns of toxicity that endanger human participants. Monitoring also
considers factors external to the study, such as scientific or therapeutic
developments that may have an impact on the safety of the participants or the
ethics of the study. Recommendations that emanate from monitoring activities are
reviewed by the principal investigator and addressed.

 

Composition

 

The principal investigator is present in an open session portion of the meeting
and absent in a closed session. All DSMC official subjects in the review of
confidential data and discussions regarding continuance or stoppage of a study
have no conflict of interest and no financial stake in the research outcome. The
current UM Prevention research base Data and Safety Monitoring Committee is
Chaired by the Research Base Biostatistician and comprised of Faculty members
from Gastroenterology, Family Medicine, Hematology/Oncology. At least 3 faculty
members, not including the study PI, must be present along with the
biostatistician as chair to have quorum. If the DSMC cannot meet face-to-face, a
conference call is acceptable.

 

Meeting Frequency

 

The UM Prevention Research Base DSMC meets monthly by means of regularly
scheduled meetings. Prior to each meeting, the UM Prevention Research Base
clinical research associate distributes a standard summary report detailing
accrual, biomarker modulations data, new publications or presentations relevant
to the ongoing project, quality control audit information, any ethical concerns,
patient-subject complaints and adverse events or serious adverse events of all
prevention protocols.

 

Recommendations and Reporting

 

Recommendations for action are sent to the Principal Investigator. The Principal
Investigator is responsible for implementing DSMC recommendations. In addition
to the Principal Investigator, minutes from the monthly meetings are forwarded
to the following as needed:

 

a.DSMB members and the principal investigators at other sites 

b.The University of Michigan Comprehensive Cancer Center Prevention and Control
Protocol Review Committee Chair, per PRC policies; 

c.IRBMED (University of Michigan Medical School IRB); 

d.NCI/DCP Program Staff; 

e.Any other trial sponsor. 

 

Serious adverse events and adverse events are reported to the institutional
review boards of all clinical sites, University of Michigan IRBMED per standard
SAE reporting guidelines, and the sponsor as required by Federal regulations. A
yearly summary report of trial activities is made to all trial investigators,
supervisory committees and the sponsor. The UM prevention data management office
and the DMCC have the responsibility of informing other trial investigators
concerning the data and safety monitoring policy, procedures, and decisions.

--------------------------------------------------------------------------------

19

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

11.0ADVERSE EVENT REPORTING 

 

Definition

 

An adverse event (AE) is any condition, which appears or worsens after the
participant is enrolled in an investigational study.

 

AE Information

 

No adverse events are expected, as there is no intervention for this trial. Any
adverse events related to the subject’s participation in this study will be
forwarded to the data coordinating center and reported to the UM IRBMed per
Standard Adverse Event Guidelines.

 

Serious Adverse Events

 

One-third of the participants will have colon cancer by study design, and deaths
due to disease progression or serious adverse events due to cancer treatment are
expected. The only procedures that are part of this study are blood, and stool
collection, so it is unlikely that any deaths or hospitalizations will be
related to the sample collection in this study. Only Serious Adverse Events that
are deemed to be directly related to a study procedure (sample collection) by
the DSMB will be reported to any regulatory body.

 

A serious adverse event is defined (by ICH Guideline E2A and Fed. Reg. 62, Oct.
7, 1997) as an event, occurring at any dose, which meets any of the following
criteria:

 

Results in death 

Is immediately life threatening 

Requires inpatient hospitalization or prolongation of existing hospitalization 

Results in persistent or significant disability/incapacity 

Is a congenital anomaly/birth defect 

 

In addition, events that may not meet these criteria, but which the investigator
finds very unusual and/or potentially serious, will be reported in the same
manner.

 

12.0DATA MANAGEMENT 

 

12.1Registration 

 

Institutional collaborators will enter IRB information into the secure VSIMS
database, including IRB approval date, expiration date, and document versions.
Subject registrations will not be allowed without IRB approval. The DMCC will
provide recruiting sites with Participant ID numbers to be assigned in VSIMS. No
exceptions to eligibility requirements will be permitted without prior
permission of the protocol PI.

 

12.2Timeliness 

 

Timeliness is monitored by the DMCC and UM through various reporting mechanisms
within VSIMS.

 

12.3Completeness and Accuracy 

 

The DMCC will assure the completeness of the data by writing data entry programs
that will not allow for empty fields whenever possible. The accuracy of the data
will be checked by identifying appropriate parameters allowed to be entered in a
given data field. Periodic reviews of the paper CDEs and the database data will
be conducted by the lead CRA and DMCC site monitor.

 

12.4Accuracy--Revisions and Corrections 

 

All corrections to paper study documents will be initialed and dated. If
computer-readable data is corrected by replacement of a data set, the replaced
version of the data set will be retained in an archive. The collection of these
auxiliary data sets represents an audit trail of corrections to the database.

--------------------------------------------------------------------------------

20

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

 

 

12.5On Site Data Audits 

 

All consortium sites are subject to periodic on-site audits. The objective of
the on-site audit is to conduct a general review of a random sample of
registered subjects from the selected protocol to assess overall protocol
adherence with respect to subject eligibility, appropriate procedure for
informed consent, registration process, general protocol adherence, sample
shipment process, follow-up and off-study process.

 

An On-Site Audit checklist will be developed which will contain all of the
essential elements of an On- Site audit. Each of the essential elements are
reviewed and discussed with the clinical site. The Checklist is signed by the
auditors and retained at the DMCC.

 

In preparation for a site audit, the study statistician will select the subjects
for review using a randomized selection procedure. Other cases may also be
selected at the discretion of the audit team. A minimum of 10% of the subjects
accrued since the last audit will be reviewed. The on-site audit team will audit
two to three unannounced cases. The consortium site investigator and research
coordinator will be notified of the impending audit not more than 3 months in
advance. Two to four weeks prior to the site visit, the list of selected
subjects will be sent to the consortium clinical site. All data and material
pertinent to the subject will be reviewed including eligibility criteria,
informed consent, and sample shipment logs.

 

Subject data will be extracted at the DMCC prior to the visit. At the audit, the
data from the DMCC will be compared to the original data (source documents
and/or CDEs). On-site audit staff will review the documentation of IRB
approvals, for each audited protocol, any amendments or adverse events, and
consent forms.

 

Based on the findings of the audit, a follow-up schedule will be defined. A
report of the audit is written and faxed to the DMCC and the NCI within 5
working days of the audit. A copy of the report is emailed or faxed to the
consortium site investigator. The site PI has 30 days from receipt of the report
to respond in writing to the DMCC directly. After the 30-day response period,
the report is finalized and sent to NCI and the consortium site investigator.

 

The DMCC will maintain a file containing the latest version of the On-Site Audit
guidelines, a listing of all consortium institutions reviewed to date, a copy of
the On-Site Audit results and all correspondence for each audit conducted. These
results will be reviewed by the Center’s Executive Committee at a monthly
telephone conference and will be made available to the NCI.

 

12.6Sample Tracking 

 

Sites receiving shipments of samples are notified via e-mail, so if samples are
delayed or lost, tracking may be initiated by the sending site. Sample shipment
forms are included with shipments. These data forms describe the date of sample
receipt, and availability of sample, along with tracking information. The
receiving site will evaluate the sample condition on arrival, scan the bar-coded
samples in the VSIMS database, verify samples shipped match samples sent, and
store at appropriate conditions until shipment to analytical labs.

 

12.7Confidentiality 

 

Subjects will be identified in the database by their unique EDRN subject
identification numbers only. Information that could identify subjects, such as
name, address, or social security number will be kept only by the enrolling site
and will not be supplied to the DMCC at Fred Hutch. The Coordinating Center at
UM will have a separate payment form with name, address, and social security
number for payment purposes only as previously described. During an on-site
audit or NCI site visit, staff may review medical records and other information
that contains PHI, but this information will not be removed from the enrolling
site. The Coordinating center at UM will not keep copies of signed informed
consent documents. No information, including copies of the informed consent
unless required by the institution, obtained during the study will be placed in
a subject’s medical record.

 

12.8Security 

 

All subject files will be stored under lock and key at all times. All computer
systems will be password- protected against intrusion; all network-based
communications between sites of confidential information are encrypted.

 

An on-going computer-virus-protection program is available and used, maintained,
and audited on all computers and pathways into the system, including good
practice policies, screening of data files, executable software, diskettes, text
macros, downloads, and other concerns as they arise. The DMCC will assist in
maintaining appropriate levels of network security.

--------------------------------------------------------------------------------

21

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

 

 

13.0ETHICAL AND REGULATORY CONSIDERATIONS 

 

13.1Institutional Review 

 

This study must be approved by an appropriate institutional review committee as
defined by Federal Regulatory Guidelines (Ref. Federal Register Vol. 46, No. 17,
January 27, 1981, part 56). The protocol and informed consent form for this
study must be approved in writing by the appropriate Institutional Review Board
(IRB). The IRB must be from an institution that has a valid Federal Wide
Assurance, Multiple Project Assurance, Single Project Assurance or Cooperative
Oncology Group Assurance on file with the Office for Human Research Protections,
Department of Health and Human Services. The institution must comply with
regulations of the Food and Drug Administration and the Department of Health and
Human Services. Changes to the protocol, consent, as well as a changes to the
investigator list at each site, must also be approved by the IRB and
documentation of this approval provided to the Coordinating center. Records of
the Institutional Review Board review and approval of all documents pertaining
to this study must be kept on file by the investigator and are subject to OHRP
or NCI inspection at any time during the study. Periodic status reports must be
submitted to the Institutional Review Board at least yearly, as well as
notification of completion of the study and a final report within 3 months of
study completion or termination. The investigator must maintain an accurate and
complete record of all submissions made to the Institutional Review Board,
including a list of all reports and documents submitted.

 

Inclusion of New Biomarkers Discovered by EDRN Investigators over the Next Two
Years

 

The design of this project including the collection of serum, DNA and tissue
samples permit the inclusion of new EDRN discovered biomarkers into this panel.
Should EDRN investigators provide sufficient preliminary data to justify
inclusion in this panel; new biomarkers will be included in the validation
program using the procedures described above.

 

--------------------------------------------------------------------------------

22

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

14.0REFERENCES 

 

1Lee JK, Liles EG, Bent S, Levin TR, Corley DA. Accuracy of fecal immunochemical
tests for colorectal cancer: systematic review and meta-analysis. Ann Intern
Med. 2014;160(3):171. 

2.Imperiale TF, Ransohoff DF, Itzkowitz SH, Levin TR, Lavin P, Lidgard GP, et
al. Multitarget Stool DNA Testing for Colorectal-Cancer Screening. N Engl J Med.
2014. 

3.Definitions Working Group, editor Biomarkers and Surrogate Endpoints.
Biomarkers and Surrogate Endpoints; 1999; Bethesda, MD: National Institutes of
Health, Food and Drug Administration. 

4.Shatzkin A, Freedman L, Schiffman M, Sawsey S. Validation of intermediate end
points in cancer research. J Natl Cancer Inst. 1990;82:1746-52. 

5.Prentice RL. Surrogate endpoints in clinical trials: definition and
operational criteria. Stat Med. 1989;8(4):431-40. 

6.Lippman S, Lec J, Lotan R, Hittelman W, Wargovich M, Hong W. Biomarkers as
intermediate endpoints in chemoprevention trials. JNCI. 1990;82:555-60. 

7.Mandel JS, Bond JH, Church TR, Snover DC, Bradley GM, Schuman LM, et al.
Reducing mortality from colorectal cancer by screening for fecal occult blood.
Minnesota Colon Cancer Control Study [published erratum appears in N Engl J Med
1993 Aug 26;329(9):672] [see comments]. N Engl J Med. 1993;328(19):1365-71. 

8.Kronborg O, Fenger C, Olsen J, Jorgensen O, Sondergaard O. Randomised study of
screening for colorectal cancer with faecal-occult-blood test. Lancet.
1996;348:1467-71. 

9.Hardcastle JD, Chamberlain JO, Robinson MH, Moss SM, Amar SS, Balfour TW, et
al. Randomised controlled trial of faecal-occult-blood screening for colorectal
cancer. Lancet. 1996;348(9040):1472-7. 

10.Shaukat A, Mongin SJ, Geisser MS, Lederle FA, Bond JH, Mandel JS, et al.
Long-term mortality after screening for colorectal cancer. N Engl J Med.
2013;369(12):1106-14. 

11.Baxter NN, Goldwasser MA, Paszat LF, Saskin R, Urbach DR, Rabeneck L.
Association of colonoscopy and death from colorectal cancer. Ann Intern Med.
2009;150(1):1-8. 

12.Singh H, Nugent Z, Demers AA, Kliewer EV, Mahmud SM, Bernstein CN. The
reduction in colorectal cancer mortality after colonoscopy varies by site of the
cancer. Gastroenterology. 2010;139(4):1128-37. 

13.Brenner H, Chang-Claude J, Seiler CM, Rickert A, Hoffmeister M. Protection
from colorectal cancer after colonoscopy: a population-based, case-control
study. Ann Intern Med. 2011;154(1):22-30. 

14.Muto T, Kamiya J, Sawada T, Konishi F, Sugihara K, Kubota Y, et al. Small
flat adenoma of the large bowel with special reference to its clinicopathologic
features. Dis Colon Rectum. 1985;28(11):847-51. 

15.Rex DK, Cutler CS, Lemmel GT, Rahmani EY, Clark DW, Helper DJ, et al.
Colonoscopic miss rates of adenomas determined by back-to-back colonoscopies.
Gastroenterology. 1997;112(1):24- 8. 

16.Saitoh Y, Waxman I, West AB, Popnikolov NK, Gatalica Z, Watari J, et al.
Prevalence and distinctive biologic features of flat colorectal adenomas in a
North American population. Gastroenterology. 2001;120(7):1657-65. 

17.Samadder NJ, Curtin K, Tuohy TM, Pappas L, Boucher K, Provenzale D, et al.
Characteristics of missed or interval colorectal cancer and patient survival: a
population-based study. Gastroenterology. 2014;146(4):950-60. 

18.Barclay RL, Vicari JJ, Greenlaw RL. Effect of a time-dependent colonoscopic
withdrawal protocol on adenoma detection during screening colonoscopy. Clin
Gastroenterol Hepatol. 2008;6(10):1091-8. 

19.Barclay RL, Vicari JJ, Doughty AS, Johanson JF, Greenlaw RL. Colonoscopic
withdrawal times and adenoma detection during screening colonoscopy. N Engl J
Med. 2006;355(24):2533-41. 

20.Soetikno RM, Kaltenbach T, Rouse RV, Park W, Maheshwari A, Sato T, et al.
Prevalence of nonpolypoid (flat and depressed) colorectal neoplasms in
asymptomatic and symptomatic adults. JAMA. 2008;299(9):1027-35. 

21.Zauber AG, Winawer SJ, O’Brien MJ, Lansdorp-Vogelaar I, van Ballegooijen M,
Hankey BF, et al. Colonoscopic polypectomy and long-term prevention of
colorectal-cancer deaths. N Engl J Med. 2012;366(8):687-96. 

22.Salas D, Vanaclocha M, Ibanez J, Molina-Barcelo A, Hernandez V, Cubiella J,
et al. Participation and detection rates by age and sex for colonoscopy versus
fecal immunochemical testing in colorectal cancer screening. Cancer Causes
Control. 2014;25(8):985-97. 

23.Quintero E, Castells A, Bujanda L, Cubiella J, Salas D, Lanas A, et al.
Colonoscopy versus fecal immunochemical testing in colorectal-cancer screening.
N Engl J Med. 2012;366(8):697-706. 

24.Welch HG, Black WC. Overdiagnosis in cancer. J Natl Cancer Inst.
2010;102(9):605-13. 

25.Levin B, Lieberman DA, McFarland B, Andrews KS, Brooks D, Bond J, et al.
Screening and surveillance for the early detection of colorectal cancer and
adenomatous polyps, 2008: a joint guideline from the American Cancer Society,
the US Multi-Society Task Force on Colorectal Cancer, and the American College
of Radiology. Gastroenterology. 2008;134(5):1570-95. 

26.Force USPST. Screening for colorectal cancer: U.S. Preventive Services Task
Force recommendation statement.[see comment][summary for patients in Ann Intern
Med. 2008 Nov 4;149(9):I-44; PMID: 18838719]. Annals of Internal Medicine.
2008;149(9):627-37. 

27.Institute NC. International Cancer Screening Network Bethesda, MD2014
[Available from:
http://appliedresearch.cancer.gov/icsn/colorectal/screening.html. 

--------------------------------------------------------------------------------

23

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

28.Pox CP, Altenhofen L, Brenner H, Theilmeier A, Von Stillfried D, Schmiegel W.
Efficacy of a nationwide screening colonoscopy program for colorectal cancer.
Gastroenterology. 2012;142(7):1460-7 e2. 

29.Centers for Disease C, Prevention. Vital signs: colorectal cancer screening
test use--United States, 2012. MMWR Morb Mortal Wkly Rep. 2013;62(44):881-8. 

30.Surveillance E, and End Results Program. SEER Stat Fact Sheets: Colon and
Rectum Cancer: Centers for Disease Control; 2014 [Available from:
http://seer.cancer.gov/statfacts/html/colorect.html. 

31.Green BB, Wang CY, Anderson ML, Chubak J, Meenan RT, Vernon SW, et al. An
automated intervention with stepped increases in support to increase uptake of
colorectal cancer screening: a randomized trial. Ann Intern Med. 2013;158(5 Pt
1):301-11. 

32.Harewood GC, Wiersema MJ, Melton LJ, 3rd. A prospective, controlled
assessment of factors influencing acceptance of screening colonoscopy. Am J
Gastroenterol. 2002;97(12):3186-94. 

33.Bettegowda C, Sausen M, Leary RJ, Kinde I, Wang Y, Agrawal N, et al.
Detection of circulating tumor DNA in early- and late-stage human malignancies.
Sci Transl Med. 2014;6(224):224ra24. 

34.Hibi K, Robinson CR, Booker S, Wu L, Hamilton SR, Sidransky D, et al.
Molecular detection of genetic alterations in the serum of colorectal cancer
patients. Cancer Res. 1998;58(7):1405-7. 

35.Vlems FA, Diepstra JH, Cornelissen IM, Ligtenberg MJ, Wobbes T, Punt CJ, et
al. Investigations for a multi-marker RT-PCR to improve sensitivity of
disseminated tumor cell detection. Anticancer Res. 2003;23(1A):179-86. 

36.Yamaguchi K, Takagi Y, Aoki S, Futamura M, Saji S. Significant detection of
circulating cancer cells in the blood by reverse transcriptase-polymerase chain
reaction during colorectal cancer resection. Ann Surg. 2000;232(1):58-65. 

37.Zou H, Yu B, Wang Z, Sun J, Cang H, Gao F, et al. Detection of aberrant p16
methylation in the serum of colorectal cancer patients. Clin Cancer Res.
2002;8:188-91. 

38.Noh YH, Im G, Ku JH, Lee YS, Ahn MJ. Detection of tumor cell contamination in
peripheral blood by RT-PCR in gastrointestinal cancer patients. J Korean Med
Sci. 1999;14(6):623-8. 

39.Grady WM, Rajput A, Lutterbaugh JD, Markowitz SD. Detection of aberrantly
methylated hMLH1 promoter DNA in the serum of patients with microsatellite
unstable colon cancer. Cancer Res. 2001;61(3):900-2. 

40.Nakayama H, Hibi K, Taguchi M, Takase T, Yamazaki T, Kasai Y, et al.
Molecular detection of p16 promoter methylation in the serum of colorectal
cancer patients. Cancer Lett. 2002;188(1- 2):115-9. 

41.Verma M, Srivastava S. Epigenetics in cancer: implications for early
detection and prevention. Lancet Oncol. 2002;3(12):755-63. 

42.Li M, Chen WD, Papadopoulos N, Goodman SN, Bjerregaard NC, Laurberg S, et al.
Sensitive digital quantification of DNA methylation in clinical samples. Nat
Biotechnol. 2009;27(9):858- 63. 

43.Grutzmann R, Molnar B, Pilarsky C, Habermann JK, Schlag PM, Saeger HD, et al.
Sensitive detection of colorectal cancer in peripheral blood by septin 9 DNA
methylation assay. PLoS One. 2008;3(11):e3759. 

44.Lofton-Day C, Model F, Devos T, Tetzner R, Distler J, Schuster M, et al. DNA
methylation biomarkers for blood-based colorectal cancer screening. Clin Chem.
2008;54(2):414-23. 

45.Church TR, Wandell M, Lofton-Day C, Mongin SJ, Burger M, Payne SR, et al.
Prospective evaluation of methylated SEPT9 in plasma for detection of
asymptomatic colorectal cancer. Gut. 2014;63(2):317-25. 

46.Hofsli E, Sjursen W, Prestvik WS, Johansen J, Rye M, Trano G, et al.
Identification of serum microRNA profiles in colon cancer. Br J Cancer.
2013;108(8):1712-9. 

47.Apweiler R, Hermjakob H, Sharon N. On the frequency of protein glycosylation,
as deduced from analysis of the SWISS-PROT database. Biochim Biophys Acta.
1999;1473(1):4-8. 

48.Adamczyk B, Tharmalingam T, Rudd PM. Glycans as cancer biomarkers. Biochim
Biophys Acta. 2012;1820(9):1347-53. 

49.Patwa TH, Zhao J, Anderson MA, Simeone DM, Lubman DM. Screening of
glycosylation patterns in serum using natural glycoprotein microarrays and
multi-lectin fluorescence detection. Anal Chem. 2006;78(18):6411-21. 

50.Yue T, Maupin KA, Fallon B, Li L, Partyka K, Anderson MA, et al. Enhanced
discrimination of malignant from benign pancreatic disease by measuring the CA
19-9 antigen on specific protein carriers. PLoS One. 2011;6(12):e29180. 

51.Zhao J, Patwa TH, Qiu W, Shedden K, Hinderer R, Misek DE, et al. Glycoprotein
microarrays with multi-lectin detection: unique lectin binding patterns as a
tool for classifying normal, chronic pancreatitis and pancreatic cancer sera. J
Proteome Res. 2007;6(5):1864-74. 

52.Zhao J, Qiu W, Simeone DM, Lubman DM. N-linked glycosylation profiling of
pancreatic cancer serum using capillary liquid phase separation coupled with
mass spectrometric analysis. J Proteome Res. 2007;6(3):1126-38. 

53.Qiu Y, Patwa TH, Xu L, Shedden K, Misek DE, Tuck M, et al. Plasma
glycoprotein profiling for colorectal cancer biomarker identification by lectin
glycoarray and lectin blot. J Proteome Res. 2008;7(4):1693-703. 

54.Rho JH, Mead JR, Wright WS, Brenner DE, Stave JW, Gildersleeve JC, et al.
Discovery of sialyl Lewis A and Lewis X modified protein cancer biomarkers using
high density antibody arrays. J Proteomics. 2014;96:291-9. 

55.Bresalier RS, Byrd JC, Tessler D, Lebel J, Koomen J, Hawke D, et al. A
circulating ligand for galectin-3 is a haptoglobin-related glycoprotein elevated
in individuals with colon cancer. Gastroenterology. 2004;127(3):741-8. 

--------------------------------------------------------------------------------

24

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

56.Scanlan MJ, Welt S, Gordon CM, Chen YT, Gure AO, Stockert E, et al.
Cancer-related serological recognition of human colon cancer: identification of
potential diagnostic and immunotherapeutic targets. Cancer Res.
2002;62(14):4041-7. 

57.Lu H, Goodell V, Disis ML. Targeting serum antibody for cancer diagnosis: a
focus on colorectal cancer. Expert Opin Ther Targets. 2007;11(2):235-44. 

58.Nam MJ, Madoz-Gurpide J, Wang H, Lescure P, Schmalbach CE, Zhao R, et al.
Molecular profiling of the immune response in colon cancer using protein
microarrays: occurrence of autoantibodies to ubiquitin C-terminal hydrolase L3.
Proteomics. 2003;3(11):2108-15. 

59.Zaenker P, Ziman MR. Serologic autoantibodies as diagnostic cancer
biomarkers--a review. Cancer Epidemiol Biomarkers Prev. 2013;22(12):2161-81. 

60.Reipert BM, Tanneberger S, Pannetta A, Bedosti M, Poell M, Zimmermann K, et
al. Increase in autoantibodies against Fas (CD95) during carcinogenesis in the
human colon: a hope for the immunoprevention of cancer? Cancer Immunol
Immunother. 2005;54(10):1038-42. 

61.He Y, Wu Y, Mou Z, Li W, Zou L, Fu T, et al. Proteomics-based identification
of HSP60 as a tumor-associated antigen in colorectal cancer. Proteomics Clin
Appl. 2007;1(3):336-42. 

62.Chen Y, Lin P, Qiu S, Peng XX, Looi K, Farquhar MG, et al. Autoantibodies to
Ca2+ binding protein Calnuc is a potential marker in colon cancer detection. Int
J Oncol. 2007;30(5):1137-44. 

63.Liu W, Wang P, Li Z, Xu W, Dai L, Wang K, et al. Evaluation of
tumour-associated antigen (TAA) miniarray in immunodiagnosis of colon cancer.
Scand J Immunol. 2009;69(1):57-63. 

64.Giovannucci E. Insulin-like growth factor-1 and binding protein-3 and risk of
cancer. Horm Res. 1999;51(Suppl 3):34-41. 

65.Giovannucci E, Pollak MN, Platz EA, Willet WC, Stampfer MJ, Majeed N, et al.
A prospective study of plasma Insulin-like growth factor and binding protein-3
and risk of colorectal neoplasia in women. Cancer Epidemiol Biomarkers Prev.
2000;9(4):345-9. 

66.Giovannucci E, Pollak M, Platz EA, Willet WC, Stampfer MJ, Majeed N, et al.
Insulin-like growth factor I (IGF-I), IGF-binding protein-3 and the risk of
colorectal adenoma and cancer in the Nurses’ Health Study. Growth Horm IGF Res.
2000;10 Suppl A:S30-1. 

67.Palmquist R, Stattin P, Rinaldi S, Biessy C, Stenling R, Riboli E, et al.
Plasma insulin, IGF- binding proteins-1 and -2 and risk of colorectal cancer: a
prospective study in northern Sweden. Int J Cancer. 2003;107(1):89-93. 

68.Cruz-Correa M, Cui H, Giardiello FM, Powe NR, Hylind L, Robinson A, et al.
Loss of imprinting of insulin growth factor II gene: a potential heritable
biomarker for colon neoplasia predisposition. Gastroenterology.
2004;126(4):964-70. 

69.Cui H, Onyango P, Brandenburg S, Wu Y, Hsieh CL, Feinberg AP. Loss of
imprinting in colorectal cancer linked to hypomethylation of H19 and IGF2.
Cancer Res. 2002;62(22):6442-6. 

70.Cui H, Cruz-Correa M, Giardiello FM, Hutcheon DF, Kafonek DR, Brandenburg S,
et al. Loss of IGF2 imprinting: a potential marker of colorectal cancer risk.
Science. 2003;299(5613):1753-5. 

71.Woodson K, Flood A, Green L, Tangrea JA, Hanson J, Cash B, et al. Loss of
insulin-like growth factor-II imprinting and the presence of screen-detected
colorectal adenomas in women. J Natl Cancer Inst. 2004;96(5):407-10. 

72.Tsushima H, Kawata S, Tamura S, Ito N, Shirai Y, Kiso S, et al. High levels
of transforming growth factor beta 1 in patients with colorectal cancer:
association with disease progression. Gastroenterology. 1996;110(2):375-82. 

73.Tsushima H, Ito N, Tamura S, Matsuda Y, Inada M, Yabuuchi I, et al.
Circulating Transforming Growth Factor beta-1 as a predictor of liver metastasis
after resection in colorectal cancer. Clin Cancer Res. 2001;7:1258-62. 

74.Narai S, Watanabe M, Hasegawa H, Nishibori H, Endo T, Kubota T, et al.
Significance of Transforming growth factor beta 1 as a new tumor marker for
colorectal cancer. Int J Cancer. 2002;97(4):508-11. 

75.Broll R, Erdmann H, Duchrow M, Oevermann E, Schwandner O, Markert U, et al.
Vascular endothelial growth factor (VEGF) -- a valuable serum tumour marker in
patients with colorectal cancer? Eur J Surg Oncol. 2001;27(1):37-42. 

76.Takeda A, Shimada H, Imaseki H, Okazumi S, Natsume T, Suzuki T, et al.
Clinical significance of serum vascular endothelial growth factor in colorectal
cancer patients : correlation with clinicopathological factors and tumor
markers. Oncol Rep. 2000;7(2):333-8. 

77.Shimoyama S, Yamasaki K, Kawahara M, Kaminishi M. Increased serum angiogenin
concentration in colorectal cancer is correlated with cancer progression. Clin
Cancer Res. 1999;5(5):1125-30. 

78.Feldman AL, Alexander HR, Jr, Bartlett DL, Kranda KC, Miller MS, Costouros
NG, et al. A prospective analysis of plasma endostatin levels in colorectal
cancer patients with liver metastases. Ann Surg Oncol. 2001;8(9):741-5. 

79.Simpson RA, Dickinson T, Porter KE, London NJ, Hemingway DM. Raised levels of
plasma big endothelin 1 in patients with colorectal cancer. Br J Surg.
2000;87(10):1409-13. 

80.Peeters CF, Thomas CM, Sweep FC, Span PN, Wobbes T, Ruers TM. Elevated serum
endothelin-1 levels in patients with colorectal cancer; relevance for prognosis.
Int J Biol Markers. 2000;15(4):288-93. 

81.Pellegrini P, Contasta I, Berghella AM, Gargano E, Mammarella C, Adorno D.
Simultaneous measurement of soluble carcinoembryonic antigen and the tissue
inhibitor of metalloproteinase TIMP 1 serum levels for use as markers of
pre-invasive to invasive colorectal cancer. Cancer Immunol Immunother.
2000;49(7):388-94. 

--------------------------------------------------------------------------------

25

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

82.Yukawa N, Yoshikawa T, Akaike M, Sugimasa Y, Takemiya S, Yanoma S, et al.
Plasma concentration of tissue inhibitor of matrix metalloproteinase 1 in
patients with colorectal carcinoma. Br J Surg. 2001;88(12):1596-601. 

83.Barozzi C, Ravaioli M, D’Errico A, Grazi GL, Poggioli G, Cavrini G, et al.
Relevance of biologic markers in colorectal carcinoma: a comparative study of a
broad panel. Cancer. 2002;94(3):647- 57. 

84.Holten-Anderson MN, Christensen IJ, Nielsen HJ, Stephens RW, Jensen V,
Nielsen OH, et al. Total levels of tissue inhibitor of metalloproteinases 1 in
plasma yield high diagnostic sensitivity and specificity in patients with colon
cancer. Clin Cancer Res. 2002;8(1):156-64. 

85.Alexiou D, Karayiannakis AJ, Syrigos KN, Zbar A, Kremmyda A, Bramis I, et al.
Serum levels of E-selectin, ICAM-1 and VCAM-1 in colorectal cancer patients:
correlations with clinicopathological features, patient survival and tumour
surgery. Eur J Cancer. 2001;37(18):2392-7. 

86.Hayes DF, Smerage JB. Circulating tumor cells. Prog Mol Biol Transl Sci.
2010;95:95-112. 

87.Wicha MS, Hayes DF. Circulating tumor cells: not all detected cells are bad
and not all bad cells are detected. J Clin Oncol. 2011;29(12):1508-11. 

88.Lim SH, Becker TM, Chua W, Ng WL, de Souza P, Spring KJ. Circulating tumour
cells and the epithelial mesenchymal transition in colorectal cancer. J Clin
Pathol. 2014;67(10):848-53. 

89.Zhang Z, Nagrath S. Microfluidics and cancer: are we there yet? Biomed
Microdevices. 2013;15(4):595-609. 

90.Stott SL, Lee RJ, Nagrath S, Yu M, Miyamoto DT, Ulkus L, et al. Isolation and
characterization of circulating tumor cells from patients with localized and
metastatic prostate cancer. Sci Transl Med. 2010;2(25):25ra3. 

91.Murlidhar V, Zeinali M, Grabauskiene S, Ghannad-Rezaie M, Wicha MS, Simeone
DM, et al. A radial flow microfluidic device for ultra-high-throughput
affinity-based isolation of circulating tumor cells. Small.
2014;10(23):4895-904. 

92.Cooper DN. Galectinomics: finding themes in complexity. Biochim Biophys Acta.
2002;1572(2- 3):209-31. 

93.Dudas SP, Yunker CK, Sternberg LR, Byrd JC, Bresalier RS. Expression of human
intestinal mucin is modulated by the beta-galactoside binding protein galectin-3
in colon cancer. Gastroenterology. 2002;123(3):817-26. 

94.Mazurek N, Conklin J, Byrd JC, Raz A, Bresalier RS. Phosphorylation of the
beta-galactoside- binding protein galectin-3 modulates binding to its ligands. J
Biol Chem. 2000;275(46):36311-5. 

95.Schoeppner H, Raz A, Ho S, Bresalier R. Expression of an endogenous
galactose-binding lectin correlates with neoplastic progression in the colon.
Cancer. 1995;75:2818-26. 

96.Sanjuan X, Fernandez PL, Castells A, Castronovo V, van den Brule F, Liu FT,
et al. Differential expression of galectin 3 and galectin 1 in colorectal cancer
progression. Gastroenterology. 1997;113(6):1906-15. 

97.Lotz MM, Andrews CW, Jr., Korzelius CA, Lee EC, Steele GD, Jr., Clarke A, et
al. Decreased expression of Mac-2 (carbohydrate binding protein 35) and loss of
its nuclear localization are associated with the neoplastic progression of colon
carcinoma. Proc Natl Acad Sci U S A. 1993;90(8):3466-70. 

98.Levi Z, Rozen P, Hazazi R, Vilkin A, Waked A, Maoz E, et al. A quantitative
immunochemical fecal occult blood test for colorectal neoplasia. Ann Intern Med.
2007;146(4):244-55. 

99.Shastri YM, Stein J. Quantitative immunochemical fecal occult blood test for
diagnosing colorectal neoplasia. Ann Intern Med. 2007;147(7):522-3; author reply
3. 

100.Rabeneck L, Rumble RB, Thompson F, Mills M, Oleschuk C, Whibley A, et al.
Fecal immunochemical tests compared with guaiac fecal occult blood tests for
population-based colorectal cancer screening. Can J Gastroenterol.
2012;26(3):131-47. 

101.Whitlock EP, Lin JS, Liles E, Beil TL, Fu R. Screening for colorectal
cancer: a targeted, updated systematic review for the U.S. Preventive Services
Task Force. Ann Intern Med. 2008;149(9):638-58. 

102.Auge JM, Pellise M, Escudero JM, Hernandez C, Andreu M, Grau J, et al. Risk
stratification for advanced colorectal neoplasia according to fecal hemoglobin
concentration in a colorectal cancer screening program. Gastroenterology.
2014;147(3):628-36 e1. 

103.Ahlquist DA, Shuber AP. Stool screening for colorectal cancer: evolution
from occult blood to molecular markers. Clin Chim Acta. 2002;315(1-2):157-68. 

104.Osborn NK, Ahlquist DA. Stool screening for colorectal cancer: molecular
approaches. Gastroenterology. 2005;128(1):192-206. 

105.Ahlquist DA, Sargent DJ, Loprinzi CL, Levin TR, Rex DK, Ahnen DJ, et al.
Stool DNA and occult blood testing for screen detection of colorectal neoplasia.
Ann Intern Med. 2008;149(7):441-50, W81. 

106.Imperiale TF, Ransohoff DF, Itzkowitz SH, Turnbull BA, Ross ME. Fecal DNA
versus fecal occult blood for colorectal-cancer screening in an average-risk
population. N Engl J Med. 2004;351(26):2704-14. 

107.Force USPST. Final Research Plan: Colorectal Cancer Screening 2014
[Available from:
http://www.uspreventiveservicestaskforce.org/Page/Document/ResearchPlanFinal/colorectal-
cancer-screening2. 

108.Chen WD, Han ZJ, Skoletsky J, Olson J, Sah J, Myeroff L, et al. Detection in
fecal DNA of colon cancer-specific methylation of the nonexpressed vimentin
gene. J Natl Cancer Inst. 2005;97(15):1124-32. 

109.Zou H, Harrington JJ, Shire AM, Rego RL, Wang L, Campbell ME, et al. Highly
methylated genes in colorectal neoplasia: implications for screening. Cancer
Epidemiol Biomarkers Prev. 2007;16(12):2686-96. 

--------------------------------------------------------------------------------

26

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

110.Itzkowitz S, Brand R, Jandorf L, Durkee K, Millholland J, Rabeneck L, et al.
A simplified, noninvasive stool DNA test for colorectal cancer detection. Am J
Gastroenterol. 2008;103(11):2862-70. 

111.Itzkowitz SH, Jandorf L, Brand R, Rabeneck L, Schroy PC, 3rd, Sontag S, et
al. Improved fecal DNA test for colorectal cancer screening. Clin Gastroenterol
Hepatol. 2007;5(1):111-7. 

112.Zackular JP, Rogers MA, Ruffin MTt, Schloss PD. The human gut microbiome as
a screening tool for colorectal cancer. Cancer Prev Res (Phila).
2014;7(11):1112-21. 

113.Kostic AD, Chun E, Robertson L, Glickman JN, Gallini CA, Michaud M, et al.
Fusobacterium nucleatum potentiates intestinal tumorigenesis and modulates the
tumor-immune microenvironment. Cell Host Microbe. 2013;14(2):207-15. 

114.Montrose DC, Zhou XK, Kopelovich L, Yantiss RK, Karoly ED, Subbaramaiah K,
et al. Metabolic profiling, a noninvasive approach for the detection of
experimental colorectal neoplasia. Cancer Prev Res (Phila). 2012;5(12):1358-67. 

115.Ahmed FE, Ahmed NC, Vos PW, Bonnerup C, Atkins JN, Casey M, et al.
Diagnostic microRNA markers to screen for sporadic human colon cancer in stool:
I. Proof of principle. Cancer Genomics Proteomics. 2013;10(3):93-113. 

116.Link A, Balaguer F, Shen Y, Nagasaka T, Lozano JJ, Boland CR, et al. Fecal
MicroRNAs as novel biomarkers for colon cancer screening. Cancer Epidemiol
Biomarkers Prev. 2010;19(7):1766-74. 

117.Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA, Jr., Kinzler
KW. Cancer genome landscapes. Science. 2013;339(6127):1546-58. 

118.Lieberman DA, Williams JL, Holub JL, Morris CD, Logan JR, Eisen GM, et al.
Race, ethnicity, and sex affect risk for polyps >9 mm in average-risk
individuals. Gastroenterology. 2014;147(2):351-8; quiz e14–5. 

119.Lieberman DA, Holub JL, Morris CD, Logan J, Williams JL, Carney P. Low rate
of large polyps (>9 mm) within 10 years after an adequate baseline colonoscopy
with no polyps. Gastroenterology. 2014;147(2):343-50. 

120.Dominitz JA, Robertson DJ. Tailoring colonoscopic screening to individual
risk. Gastroenterology. 2014;147(2):264-6. 

121.Moinova H, Leidner RS, Ravi L, Lutterbaugh J, Barnholtz-Sloan JS, Chen Y, et
al. Aberrant vimentin methylation is characteristic of upper gastrointestinal
pathologies. Cancer Epidemiol Biomarkers Prev. 2012;21(4):594-600. 

--------------------------------------------------------------------------------

27

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

EXHIBIT B

 

Specimen Volumes for Volition

Doc 1_Page_4.jpg [f10k123119ex10z224.jpg] 

--------------------------------------------------------------------------------

28

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

EXHIBIT C

 

Statement of Work (for University of Michigan Grant Activity) Volition

 

Aims

 

Aim 1: Complete GLNE 007, a trial designed to train and test and circulating
biomarkers for early detection of colorectal adenocarcinoma.

 

Aim 2: To perform phase 1 validation trials (training and test set designs) of
promising biomarkers discovered by EDRN Biomarker Validation Laboratories,
external academic collaborating institutions, and collaborating EDRN industrial
partners for the early detection of colorectal cancer.

 

Aim 3: To enhance and curate an archive of appropriately preserved stool, serum,
plasma, urine, tissue and DNA biospecimens to be used by EDRN investigators for
future validation and biomarker discovery research.

 

Work Plan

 

As part of the overarching EDRN project, adults age 50 or older undergoing a
screening or surveillance colonoscopy will be enrolled as Study Subjects for
this Project. Samples obtained from these Study Subjects will be sent to the
GLNE Central Laboratory for preparation for storage/shipment to Volition
(Laboratory). There, the samples will be tested for blood-based, cell-free
circulating biomarkers on their proprietary Nu.QTM platform.

 

Laboratory will provide a copy of all test results for the Clinical Study to
Principal Investigator’s designated Data Management Coordinating Center (DMCC)
following the completion of their services. The DMCC will collect and store all
such test results, and shall share test results with Institutions for
collaborative analysis.

--------------------------------------------------------------------------------

29

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

GLNE0l0

VALIDATION AND COMPARISON OF BIOMARKERS FOR THE EARLY

DETECTION OF COLORECTAL ADENOCARCINOMA

 

Great Lakes New England Clinical Validation Center

NCI Early Detection Research Network

2  UOl CA086400-16

 

Dean E. Brenner, M.D.1

John A. Baron, M.D. 2

Hermann Brenner, M.O.3

Robert Bresalier, M.D. 4

Jan Buckner, M.D.6

Timothy Church, PhD5

Seth Crockett, M.O.2

Mack Ruffin, M.D. 7

Sapna Syngal, M.D. 8

Ananda Sen, Ph.D.1

Ziding Feng, Ph.D.4

Margaret Pepe, Ph.D.9

Melissa Tuck, M.S.1

 

1 University of Michigan Medical Center, Ann Arbor, MI

2 University of North Carolina, Chapel Hill, NC

3German Cancer Research Center (DKFZ), Heidelberg, Germany

4MD Anderson Cancer Center, Houston, TX

5University of Minnesota, Minneapolis, MN

6Members of the Alliance through CTSU

7Pennsylvania State University/Hershey Medical Center, Hershey, PA

8Dana-Farber Harvard Cancer Center, Boston, MA

9Fred Hutchinson Cancer Research Center

 

Contact information for Great Lakes-New England CVC:

2150 Cancer Center

University of Michigan Medical Center

Ann Arbor, MI 48109-0930

Telephone: (734) 647-1417 Fax: (734) 764-2566

Email: dbrenner @umich.edu (Pl)  mtuck@umich.edu (Lead CRA)

 

Contact information for the Data Management Coordinating Center (DMCC)

Jackie Dahlgren

EDRN DMCC Project Director

1100 Fairview Ave N, M3-A306

PO Box 19024

Seattle, WA 98109-1024

Phone: 206-667-3438 Fax: 206-667-5964 Email: jdahlgre@fredhutch.org

 

EDRN Biomarker Reference Laboratories (BRL)

University of Maryland-PI Sanford Stass

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

Alliance for Clinical Trials in Oncology (Alliance)

 

Alliance- GLNE 010

Validation and Comparison of biomarkers for the Early Detection of Colorectal
Adenocarcinoma

 

For any communications regarding this protocol,

please call the protocol resource person on the following page.

 

Study Chair:Jan Buckner, M.D  

Mayo Clinic

200 First St. SW

Rochester, MN 55905

 

 

 

Alliance Protocol Resources

 

Questions:

Contact Name:

Protocol document, consent form,

Jacqueline M. Latky

Regulatory issues

Research Base Research Protocol Specialist

 

Phone: (507) 538-4633

 

Fax: (507) 284-5280

 

E-mail: lafky.jacqueline@mayo.edu

 

* No waivers of eligibility per NCI

 

CLINICAL TRIALS SUPPORT UNIT (CTSU) ADDRESS AND CONTACT INFORMATION

 

To submit site

registration documents:

For patient enrollments:

To Submit Study Data:

 

CTSU Regulatory Office

1818 Market Street, Suite 1100

Philadelphia, PA 19103

Phone - 1-866-651-CTSU

Fax - 215-569-0206

 

Refer to Appendix H for specific instructions.

 

All Groups must submit data via the EDRN’ s Validation Study Information
Management System (VSIMS) . To obtain access for data entry, sites will be
trained by webinar and given their own user name and password and access to the
system.

 

For additional information about VSIMS, refer to the Manual of Operations,
Appendix 13. For assistance with VSIMS or other data entry questions, call the
VSIMS Helpline: 206-667-3438.

 

Do not submit study data or forms to CTSU Data Operations. Do not copy the CTSU
on data

submissions.

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

Patient enrollments at all participating sites will use the GLNE’s Validation
Study Information Management System (VSIMS). Refer to Appendix H for specific
enrollment details. 

 

Data management will be performed as follows: 

 

All participating institutions will enter their data directly into the Early
Detection Research Network (EDRN) supported VSIMS system of the Data Management
and Coordinating Center (DMCC) as discussed in Section 4 of the protocol.

 

►Do not send study data or case report forms to the CTSU Data Operations. DO NOT
copy the CTSU on data submissions.

 

Data query and delinquency reports will be sent directly to the enrolling site
by the EDRN DMCC Operations Office. Please send query responses and delinquent
data to the DMCC and do not copy the CTSU Data Operations. 

 

CTSU sites should follow procedures outlined in Appendix H for Site
Registration, Patient Enrollment, Adverse Event Reporting, and Data Submission. 

 

For patient eligibility or treatment-related questions: Missy Tuck
(734-763-1141 or mtuck@umich.edu) 

 

For questions unrelated to patient eligibility, treatment, or data submission
contact the CTSU Help Desk by phone or e-mail: 

 

CTSU General Information Line- 1-888-823-5923, or ctsucontact@westat.com. All
calls and correspondence will be triaged to the appropriate CTSU representative.

 

For detailed information on the regulatory and monitoring procedures for CTSU
sites please review the CTSU Regulatory and Monitoring Procedures policy located
on the CTSU members’ website https://www.ctsu.org 

 

CTSU Web site is located at http s://www.ctsu.org 

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

TABLE OF CONTENTS

 

1.0

SUMMARY OF STUDY

1

2.0

SCHEMA USA  Germany/Canada

2

3.0

OBJECTIVES

3

4.0

BACKGROUND AND SIGNIFICANCE

3

4.1

Current State of the Art: Recommended Early Detection

3

4.2

Current State of the Art: Serum Based Biomarkers for Colorectal Neoplasia

4

4.3

Rationale and Current State of the Art: Stool Based Biomarkers for Detection of
Colorectal Neoplasia

6

4.4

Key Issues Driving Research Questions in CRC Early Detection Biomarkers

8

4.5

Rationale for Food Frequency Assessment and the Use of the NIH DHQ JI Food
Frequency Questionnaire

9

5.0

STUDY DESIGN

9

5.1

Subject Recruitment

9

5.2

Eligibility

9

5.3

Study Procedures

10

5.4

Study Definitions

11

5.5

Biological Sample and Data Collection

13

5.6

Disclosure  of results to subjects

14

5.7

Biomarker Analytical Approach

14

5.8

Data Collection, Management and Monitoring

15

6.0

STUDY CALENDAR (Table 1)

15

7.0

ANALYTICAL PROCEDURES

16

7.1

Vimentin methylation

16

7.2

Fecal immunochemical Test (FIT)

16

7.3

Galectin-3 Ligand

16

7.4

Circulating methylated genes BCATl/IKZFl  (Clinical Genomics)

16

7.5

Hypomethylated LINEl  from circulating cell free DNA (VolitionRx)

16

8.0

DATA ANALYSIS PLAN, SAMPLE SIZE JUSTIFICATION, AND STATISTICAL POWER.

16

8.1

Secondary Analyses

18

9.0

PROJECT MANAGEMENT PLAN

19

9.1

Strategies to Ensure Completion of Milestones

19

9.2

Timeline for Completing GLNE 010

19

9.3

Endpoint Event Justification and Milestones

20

9.4

Endpoint Event Monitoring

21

9.5

Data Safety and Monitoring

21

9.6

Adverse Event Reporting

22

10.0

DATA MANAGEMENT

23

10.1

Registration

23

10.2

Timeliness

23

10.3

Completeness and Accuracy

23

10.4

Accuracy--Revisions and Corrections

23

10.5

On Site Data Audits

23

10.6

Sample Tracking

24

10.7

Confidentiality

24

10.8

Security

24

11.0

ETHICAL & REGULATORY CONSIDERATIONS

24

11.1

Institutional Review

24

12.0

REFERENCES

25

13.0

APPENDICES

 

13.1

Appendix A -  Case Report Forms (separate document)

 

13.2

Appendix B -  Recruitment Materials (separate document)

 

13.3

Appendix C -  Blood SOP (separate document)

 

13.4

Appendix D -  Stool SOP (separate document)

 

13.5

Appendix E-   Colonoscopy SOP (separate document)

 

13.6

Appendix F -Model Informed  Consent (separate document)

 

13.7

Appendix G-   Urine SOP (separate document)

 

13.8

Appendix H -Alliance Site Logistics (separate document)

 

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

Abbreviations and Definitions

 

Early Detection Research Network (EDRN)

Biomarker Reference Laboratory (BRL)

Standard operating procedures (SOPs)

University of Michigan (UM)

German Cancer Research Center (Deutsches Krebsforschungszentrum, DKFZ),
Heidelberg, Germany

Deoxyribonucleic acid (DNA)

FIT test (sampling bottle provided by Polymedco for testing two different stool
samples)

Data Management and Coordinating Center (DMCC)

Participant Identification Number (PIO)

Clinical Research Associate or study nurse (CRA)

Inflammatory Bowel Disease (IBD)

Hereditary non-polyposis colon cancer (HNPCC)

Familial Adenomatous Polyposis (FAP)

Validation Study Information Management System (VSIMS)

Fecal Occult Blood Test (Guaiac-based) FOBT

Immunoassay fecal occult blood test (FIT)

Department of Transportation (DOT)

Personal Health Information (PHI)

Data Safety Monitoring Committee (DSMC)

National Cancer Institute (NCI)

National Institute of Health Diet History Questionnaire II (DHQII)

Colorectal adenocarcinoma or adenomas with high grade dysplasia or adenomas
greater than or equal to1 cm (Screen Relevant Neoplasia-{SRN})

Colorectal adenocarcinoma OR adenomas with high grade dysplasia (CRC/ HGD)

Cancer Trials Support Unit (CTSU)

Clinical Ligand Assay Satellite Services (CLASS)

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

1.0 SUMMARY OF STUDY 

 

The goal of this trial is to estimate the sensitivity and specificity of stool
vimentin methylation, serum galectin-3 ligand, and fecal immunochemical testing
for 1) colorectal adenocarcinoma, or 2) screen relevant neoplasms (high-grade
dysplasia or adenoma with 2:25% villous histologic features or adenoma measuring
2:1 cm in the greatest dimension or sessile serrated polyps measuring 1 cm or
more in diameter) as single markers and in combination. Four thousand
asymptomatic subjects aged 60 and older undergoing a first ever routine
colonoscopic screening for colorectal cancer from U.S. community and major
medical center outpatient settings across multiple centers and consortia will be
recruited. An additional five thousand subjects age 50 and older undergoing
routine colonoscopic screening for colorectal cancer will be recruited in
Germany and Canada (non-US sites). Up to 9,000 subjects will be recruited in
this protocol, adding to the 4,677 confirmed and evaluable subjects already
recruited. Subjects will meet with research staff prior to initiation of any
colonoscopic preparative procedure. After completing informed consent, they will
complete Early Detection Research Network (EDR.i’-J) data element forms. Blood
and urine will be obtained following EDR.i”‘J standard operating procedures
(SOPs). Subjects will be provided with kits to collect stool samples for fecal
immunochemical test (FIT) and stool tests. The collected samples will be shipped
to the Central Laboratory at the University of Michigan or German Cancer
Research Center (Deutsches Krebsforschungszentrum , DKFZ), Heidelberg, Germany
where the stool will be homogenized , aliquoted, and stored at the Umiversity of
Michigan CLASS laboratories. The FIT tests will be sent to the Central
Laboratory at the University of Michigan or to DKFZ for quantitative analysis
following standard operating procedures provided by Eiken Chemical Company. Data
from the screening colonoscopy will be obtained. One year after colonoscopy,
subjects will be contacted to determine if they have had a neoplastic colorectal
diagnosis or other neoplastic events. Data management and protocol coordination
will be performed by the Data Management and Coordinating Center (DMCC) of the
EDRN along with the GLNE Prevention Research Base at the University of Michigan
and will include a Web-based front end and relational database backend, with
biosample tracking (VSIMS). Biosamples will be managed in a high quality
repository facility at the University of Michigan until shipment to the EDR.”I\T
repository at NCI at Frederick Central Repository and to analytic partners.

 

We will estimate sensitivities and specificities and the corresponding
confidence intervals of the stool DNA tests and serum/plasma tests for detection
of invasive colorectal neoplasms and for screen relevant neoplasias (Aim 1). We
will then test the primary hypothesis to confirm the clinical accuracy of a
particular biomarker test or panel (Aim 2). The specific primary hypothesis will
be defined prior to data analysis based on state of the art information
available at that time about candidate biomarkers and tests. Several specific
examples of potential primary hypotheses are given to justify study sample size.
Finally, several alternative tests and multi-marker panels will be evaluated.
(Aim 3). In secondary analysis, we will (a) provide measures of diagnostic
accuracy standardized to the age and gender distribution of US population and
(b) assess the effect of subject heterogeneity on the marker performance. A
primary objective is to establish an archive of appropriately preserved stool,
serum, plasma and DNA human biospecimens to be used by ED -approved
investigators for future validation and biomarker discovery research (Aim 4).

--------------------------------------------------------------------------------

1

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

2.0 SCHEMA USAGermany/Canada 

 

 

Adults, age 60 and older who are undergoing

their first ever screening co1onoscopy and are

wi11ing to participate

 

Adults, age 50 and older who are undergoing

their first ever screening colonoscopy and

are willing to participate

 

 

 

 

 

 

 

 

 

  Eligible

 

Willing to sign Informed Consent document 

Able to tolerate removal of 50 ml of blood (5 tubes or 3.5 tablespoons) 

Willing to collect 2 stool samples 

Never had a full colonoscopy for screening purposes 

 

 

 Ineligible

 

Inability to provide informed consent 

History of Inflammatory Bowel Disease 

Overt rectal bleeding within 1 month 

Positive FOBT or FIT in the past 12 months 

Undergone resection of the colon for any indication 

Subjects with known HIV or chronic viral hepatitis (Hepatitis B and C) 

Subjects with known or suspected HNPCC (Lynch Syndrome) or FAP 

Any cancer within 5 years prior to enrollment except squamous cell carcinoma of
the skin or Basal cell carcinoma of the skin 

Prior history of Colon Cancer or Rectal Cancer. 

 

 

 

 

BASELINE OR PRE-COLONOSCOPY

 

Signed Informed Consent 

Blood Collection (50 ml) 

Urine Collection(100 ml) 

Complete Questionnaires 

Stool collection and FIT (x2) and shipped to UM/DKFZ 

Compensation sent to subject 

 

 

 

 

 

 

 

 

COMPLETE COLONOSCOPY

 

Endoscopy and pathology reports collected 

If eligible, compensation for enrollment sent to site/or credits earned 

Central pathology review of invasive colorectal neoplasms 

Central pathology review of screen relevant neoplasias 

 

 

 

 

 

 

 

 

 

Follow up phone call one year after colonoscopy & provide any additional

Surgery/Pathology data

 

 

 

 

 

 

 

 

Review of relevant  medical  records from follow-up events

 

 

--------------------------------------------------------------------------------

2

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

3.0OBJECTIVES 

 

We propose a prospective cross-sectional PRoBE-compliant validation trial of
stool-based and serum-based tests for the detection of colorectal neoplasia (3).
The trial is powered to evaluate tests for detecting early stage colorectal
adenocarcinoma. This is the most stringent, conservative approach to the early
diagnosis of colonic neoplasia and addresses the most important endpoint of
identifying individuals with curable, early stage cancer.

 

Aim 1: To estimate the sensitivity and specificity for 1) colorectal
adenocarcinoma or 2) screen relevant neoplasms (high-grade dysplasia or adenoma
with 2’.:25% villous histologic features or adenoma measuring 2’.:1 cm in the
greatest dimension or sessile serrated  polyps measuring 1 cm or more in
diameter) of the following individual colorectal neoplasia early detection
biomarkers using colonoscopy as the gold standard:

 

stool vimentin methylation 

serum galectin-3 ligand 

fecal immunochemical tests (FIT) 

Circulating methylated genes BCATl /IKZF l (Clinical Genomics) 

Hypomethylated LINEl from circulating cell free DNA (VolitionRx) 

Other currently unspecified biomarkers 

 

Aim 2 (primary objective): To assess the accuracy and potential clinical value
of a test for detection of colorectal adenocarcinoma. The specific test and
relevant hypothesis are not defined now but will be chosen when all samples have
been collected. This will allow the primary hypothesis to incorporate all
information about markers and clinical practice that is available at the time of
analysis and will ensure that the most compelling and timely hypothesis is
tested Statistical power calculations demonstrate that the study is well powered
for these hypotheses (Section 8.0).

 

(Secondary objective): While the study will be powered for the primary
objective, we shall also carry out a similar assessment of potential utility of
a clinical test for SRN.

 

Aim 3

To validate, or to construct, a combined early detection biomarker panel using
the above individual biomarkers (stool vimentin methylation, serum galectin-3
ligand, FIT, circulating methylated genes BCAT l /lKZFl , hypomethylated LINEl
from circulating cell free DNA), and other unspecified future biomarkers, and
describe its performance for 1) colorectal adenocarcinoma and for 2) screen
relevant neoplasms.

 

Aim 4

To establish an archive of appropriately preserved stool, serum, plasma and DNA
human biospecimens to be used by EDR: -approved investigators for future
validation and biomarker discovery research.

 

4.0BACKGROUND AND SIGNIFICANCE 

 

4.1Current State of the Art: Recommended Early Detection 

 

Randomized controlled trials have shown that annual or biennial fecal occult
blood tests (FOBT) reduce colorectal cancer (CRC) mortality by 15% to 33% (4-6).
The reduction is durable over 3 decades (7). Population based cohort studies of
colonoscopic screening demonstrate reduced CRC mortality, primarily in distal
but not in the proximal colon (8-10). This discrepancy has been attributed to
endoscopic quality issues, the technical difficulties in detecting lesions in
the right colon, and the more frequent occurrence of flat and depressed
dysplastic lesions in the right colon (11-14). In tandem colonoscopy studies, a
subset oflarge polyps may be missed by a single examiner. Shorter withdrawal is
time linked to a lower adenoma detection rate (15, 16).

 

Flat and depressed lesions are more challenging to detect and have been
described with a relatively high prevalence in a US colonoscopy cohort (17).
While colonoscopic removal of adenomatous polyps reduces CRC mortality (18),
prospective, randomized controlled trials of screening colonoscopy have been
initiated by the VA and in Europe (18-20). Over-diagnosis (i.e. early detection
of indolent invasive neoplasms that do not cause mortality) or lead-time bias in
early detection of colorectal neoplasms do not degrade the efficacy of screening
and early detection for colorectal cancers (21).

--------------------------------------------------------------------------------

3

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

Current screening guidelines for average risk individuals vary world-wide. In
the United States the American Gastroenterology Association recommends testing
for early detection of adenomas and cancer (structural examination) or of cancer
(non-invasive stool tests) beginning at age 50 (22). The United States
Preventive Services Task Force (USPSTF) recommends fecal occult blood testing
(FOBT) every two years with optional endoscopic screening with either flexible
sigmoidoscopy or colonoscopy (23). The majority of developed countries recommend
fecal occult blood testing every two years but do not support endoscopic
screening (24); albeit with some exceptions (e.g. Germany (24, 25)). In 2012,
65.1% of the United States adults adhered to USPSTF colorectal screening
guidelines with colonoscopy the commonly used screening method (61.7%) followed
by FOBT (10.4%) (26) whereas colonoscopic screening adherence in Germany is 16%
(25). Over 20 years of SEER data (1991 to 2011), United States CRC incidence
(all races, males, females) has fallen from 59.5 cases in 1991 to 39.3 cases per
100,000 in 2011 (35% reduction) with a corresponding mortality reduction over
the same time period from 24.0 to 15.1 deaths per 100,000 (37% reduction) (27).
Widespread adherence to screening guidelines in the United States may be driven
by the profound changes in the organization of medical care including enhanced
access via the Affordable Care Act, rigid guideline enforcement by payers with
physician performance incentives and disincentives, and the rapid adaptation of
electronic medical record systems enabling ease referrals for screening,
compliance reminders, and management tracking of compliance to care guidelines
(28).

 

4.2Current State of the Art: Serum Based Biomarkers for Colorectal Neoplasia 

 

Reasons for non-adherence with stool based or colonoscopic based CRC screening
include the volume of bowel preparation, inadequate analgesia, no recommendation
from primary physician, embarrassment (29) or cultural taboos surrounding
collection or manipulation of stool provide rationale for discovery and
validation of circulating biomarkers for early detection of colorectal
neoplasia. Circulating signatures may be detected from neoplasm generated
genetic products, antigens, antibodies, glycans, circulating tumor cells.

 

4.2.1Genetic Products 

 

In a recent study of 24 CRC patients, mutant DNA fragments (circulating tumor
DNA, ctDNA) are found in at relatively high concentrations in the circulation of
most patients with metastatic cancer and at were detected in ~70% of patients
with localized cancers (30). The direct detection of aberrant genes or genetic
material specific to colorectal neoplasms (e.g. APC, -catenin, K-ras, DCC, and
p53) has been limited by the technical challenge of DNA recovery, the large
number of potential underlying genetic mutations, and by the limited sensitivity
of any single genetic alteration due to the extremely low abundance gene
mutations in circulating plasma or serum (30-35). DNA hypermethylation, in
contrast, affects residues in regulatory portions of genes and provides major
advantages in designing biomarker assays (34, 36-38). Digital based quantitative
technologies improving upon bisulfite conversion while minimizing bisulfite
associated DNA fragmentation and single molecule detection technologies (39)
permit cost effective development of DNA hypermethylated gene biomarkers. Such
technology detected circulating methylated vimentin with 59% sensitivity (39).
Septin9, a methylated gene discovered in tissues with array technology (40, 41),
detects CRC with 50% sensitivity and 92% specificity in a large (7941
participants) prospective colonoscopy verified screening trial (1). For early
stage CRC, Septin9 sensitivity decreased to 35%. While circulating methylated
CpG DNA promoter sites appear to have higher CRC detection performance than
other genetic detection strategies, they substantially lag behind stool based
detection of blood DNA markers or endoscopy. Nevertheless, for individuals
refusing to use stool based screening, detection sensitivity of circulating
methylated DNA markers appears equivalent to guaiac based stool screening and
has the potential advantage of capturing the 35% of the population refusing
stool screening. miRNAs are stable and detectable in serum and plasma. As in
stool, numerous up and down regulated miRNA stool signatures discovered using
unsupervised array technology may be useful as CRC detection biomarkers. A
recent review identifies 19 miR..”l’,,JAs as individual or groups in panels as
candidates for detection markers; but, insufficient clinical validation renders
the data generated to date using small convenience sets confusing and not
mechanism driven (42).

 

4.2.2Proteins 

 

Antigens: Approximately 50% of all proteins are estimated to be glycosylated
(43). Glycan abundance and their micro- and macro-heterogeneity can be changed
in a disease-specific manner (44). Glycoprotein screening studies, many EDR.”N s
upported , have relied on immunoprecipitation or lectin affinity capture of
whole glycoproteins and mass spectrometry identification of the de-glycosylated
protein portion or probed with lectins in an array format containing up to a few
hundred antibodies (45-49). Sialylated Lewis A and Lewis X moieties carrying
proteins identify panels of potential markers. The Lampe EDRN laboratory has
found seven such proteins (B3GNT5, CD44, HSPG2, IL6, INHBC, NOTCH4 and VWF)
which, when combined in discovery set plasma samples ROC AUC of 0.83 (50). GLNE
discovered glycan ligand, galectin-3 ligand is circulating glycan biomarker in
large population based prospective validation (51).

--------------------------------------------------------------------------------

4

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

Antibodies: Serum antibodies recognizing multiple colon cancer antigens can be
detected in colorectal adenocarcinoma patients’ markers (52-54). Preliminary
validation of single or small autoantibody panels have been disappointing (55).
For example, antibodies to the Fas receptor have 17% sensitivity when 100%
specific for CRC detection (56). Experience with p53, Hsp60, and nucleobindin 1
(Calnuc) autoantibodies has been better (~50% sensitivity/70 to 90% specific);
but, they are not specific to CRC (55, 57, 58) and cannot be used as a colon
specific screening tool. Discovery sets that include a miniarray of
autoantibodies with other markers have reported improved detection accuracy
(sensitivity 83%/specificity 90%) (59) but require clinical validation.

 

Cytokines/growthfactors: High serum concentrations of insulin-like growth
factors (IGF) and low levels of their binding proteins have been shown to
correlate with CRC risk in large cohort studies (60-63) but have low
sensitivities with high specificities for CRC detection. Other cytokines or
angiogenesis factors such as TGF-P 1 (64-70), VEGF (71, 72), angiogenin (73),
endostatin (74), and endothelins (75, 76) also have low sensitivity in small
convenience sets and have not proceeded to clinical validation.

 

Other proteins: Of the matrix metalloproteinases (77-79), plasma TIMPl is
elevated in CRC but has not had sufficient sensitivity in larger validation
trials to merit development as a detection biomarker (80). Cell adhesion
molecules (81) have low sensitivities for detection of early stage CRC.

 

4.2.3Circulating Tumor Cells 

 

Circulating tumor cells (CTCs) entering the vascular space from primary
neoplasms have been considered to be initiators of metastases (82-84) and can be
detected in early stage invasive neoplasms (85, 86). CTC isolation from
epithelial cancers initially used antibody capture technology dependent upon
epithelial adhesion (EpCAM) and cytokeratins (82).

 

This technology limits CTC detection of early stage neoplasms because CTCs are
thought to undergo epithelial to mesenchymal transition (EMT), epithelial traits
are lost and epithelial marker such as EpCAM and cytokines are downregulated.
CTCs present in as few as 1 cell in 5 x 109 red cells, and up to 5-10 x 106
white blood cells, are rare events (84). Newer microfluidic or centrifugation
devices appear to more efficiently capture CTCs (85, 87). The inclusion of
mesenchymal/EMT-specific antibodies, for example, vimentin, PLS3 may improve CTC
capture and/or expansion (84). With the emergence of ex-vivo expansion protocols
of CTCs and the increased ability to detect stem like or stem progenitor cells,
CTCs are of future interest as an early cancer detection diagnostic (85, 87),
but remain in the technology development phase.

 

4.2.4Special consideration-EDRN discovered and preliminarily validated
circulating biomarker: Galectin-3 Ligand ELISA as a Serum Biomarker for the
Detection of Colorectal Neoplasia 

 

The galectins are widely distributed and evolutionarily conserved carbohydrate
binding proteins characterized by their binding affinity for p-galactosides and
by conserved sequence elements in the carbohydrate-binding region (88).
Galectin-3 is the galectin that is of most interest in regard to colon cancer
because of its demonstrated role and cancer progression and metastases and
interaction with mucins(89-93). Galectin-3 ligands include laminin, LAMP-1 and
2, LPS and colon cancer mucin. The major galectin-3 ligand detected in serum is
a 40 kDa band distinct from MUC2 and other mucins CEA, and Mac- 2-BP. We
reported a true positive rate for the detection ofCRC of 91% and false positive
rate of 18% using preliminary data using quantitative Western blot technology on
a convenience set of GLNE serum (51).

 

We developed a sensitive, reproducible ELISA assay for galectin-3 using a new
antibody we created. This was used to assay the GLNE colorectal reference set
(50 colorectal adenocarcinomas/50 adenomas/50 endoscopically normal controls).
The ROC analyses for galectin-3 ligand combined with FOBT (fecal occult blood
test-guaiac based) for detection of colorectal adenocarcinoma versus controls
who had normal colonoscopy shows an area under the ROC curve of 0.91, while
galectin-3 ligand detection of colorectal adenocarcinoma alone versus controls
who had normal colonoscopy shows an area under the curve of 0.84. The true
positive rate of galectin-3 ligand with FOBT for detection of CRC is 64% with a
false positive rate of 5%. Without FOBT, true positive rate of galectin- 2
ligand was 72% with a false positive rate of 20%.

--------------------------------------------------------------------------------

5

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

4.3Rationale and Current State of the Art: Stool Based Biomarkers for Detection
of Colorectal Neoplasia 

 

4.3.1Occult blood tests 

 

Stool testing as a screening approach offers the potential advantages of
noninvasiveness, low cost, avoidance of cathartic preparation, and minimal
impact on work time or daily activities. guaiac based FOBT is not specific for
human blood, and consequently it has a high false positive rate for colorectal
neoplasia. The fecal immunochemical test (FIT) detects human hemoglobin, thus
eliminating the false positives caused by non­ human hemoglobin in the diet (94,
95). FIT tests are more sensitive at detecting CRCs (sensitivity range 61% to
91%) and adenomas (sensitivity range 16% to 31%) than classical unrehydrated
guaiac FOBT (Hemoccult II) (sensitivity range 25% to 38% for CRC; 16% to 31% for
advanced adenomas) (96, 97). A recent meta-analysis that analyzed data from 19
prospective randomized trials or cohorts using 8 different commercially
available FIT tests with colonoscopy or 2 year observation endpoints reported an
overall sensitivity for detection of CRC of79% (95% CI = 0.69-0.86), specificity
of 94% (95% CI = 0.92- 0.95) and overall accuracy (defined as hierarchical
summary receiver operating characteristic (ROC) curve) of 95% (95% CI= 93% -
97%) (Figure 1). Differences in performance characteristics among FIT brands
were small, particularly between the two major brands used OC-Light (Eiken
Chemical) and QC-Micro/Sensor (Polymedco + Eiken Chemical). The Polymedco
product is widely used in the USA. Quantitative FIT (Eiken OC-SENSOR) >177 µg/gm
stool combined with age and sex predicts 11.46 fold risk of a large adenoma over
lower risk groups (98).

 

4.3.1.jpg [f10k123119ex10z225.jpg] 

Fig 1 from Lee et al (2): Hierarchical ROC curve of the sensitivity versus
specificity of FIT. The diamond= summary point of the curve to which the pooled
sensitivity and specificity correspond. Dashed line = 95% Cl for summary point;
dotted line = 95% confidence area of FIT diagnostic accuracy. AUC = area under
the curve; SENS = sensitivity; SPEC = specificity.

 

4.3.2Stool DNA tests 

 

Since the neoplastic transformation process of the colonic epithelium results in
cells shedding into the stool, collection of fecal material is likely to yield
detectable molecular and biochemical events associated with cellular
transformation (99, 100). First generation multi-marker stool DNA tests detected
52-73% of CRCs, 41-49% of CRCs plus adenomas with high grade dysplasia, and
15-46% of adenomas 2:1 cm, with specificities of 84-95% (101, 102). Stool DNA
test performance in both studies was compromised by failure to use stabilization
buffer with stool collection, inefficient marker recovery from stool, and
relatively insensitive analytical methods. Exact Sciences modified their
previously published stool DNA panel (102) and now uses a panel consisting of
methylated BMP3 and NDRG4 promoter regions, mutant  K-ras (7 point mutations,
Exon 2, codons 12,13), and a proprietary FIT test). In a recently published
cross sectional validation study of 9,989 patientsundergoing screening
colonoscopy, the panel performed with a sensitivity of 92% for CRC; 84% for CRC
+ high grade dysplasia; and 42% for advanced adenomas (Figure 2) (103).

 

The specificity was 87% for CRC, the ROC AUC for the Exact Sciences DNA stool
panel for the detection of colorectal cancer is 0.94. FIT alone (Polymedco FIT)
performed with sensitivity of 73.8% and specificity of 94.9% for detection of
CRC and sensitivity of 23.8% for screen relevant neoplasia.  Stool DNA component
of the panels adds ~20% sensitivity to FIT. The USPSTF is currently assessing
the role and contribution of fecal DNA panels such as the Exact Sciences panel
to CRC screening (104).

--------------------------------------------------------------------------------

6

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

4.3.2 graph.jpg [f10k123119ex10z226.jpg] 

Fig 2 from lmperiale et al [lmperiale 2014 #5977) sensitivity for detection of
CRC by Exact Sciences stool DNA panel + FIT (ight blue) vs Fit alone (dark

 

 

4.3.3Vimentin Methylation as a Stool DNA Test 

 

Aberrant methylation of vimentin exon 1 was initially described as a highly
frequent biomarker of colorectal cancers and adenomas by Markowitz and
co-workers (105). In reproducible studies, aberrant methylation of vimentin has
been detected in 72%-83% of colon cancers and 70%-84% of colon adenomas (105,
106).  The current assay for detection of vimentin exon 1 methylation is based
on using methylation specific PCR (MSP). Adaptation of the vimentin MSP to
testing fecal DNA is accomplished by recovery ofvimentin DNA sequences from
human stool using hybrid capture to vimentin specific oligonuclotides(l05).
Initial study showed that MSP assay of vimentin purified from feces (fecal
vimentin DNA) detected methylated fecal vimentin DNA in 46% of cancer patients
(N=94) at a specificity of 90% (N=198)(105). This initial study involved
collaboration between the Markowitz laboratory who had discover d the methylated
vimentin DNA marker, and Exact Sciences, who implemented detection of this
marker in fecal DNA. This initial study was limited by use of samples that had
suffered problems of DNA degradation during sample collection and shipping
(102). A recently published two stage followup study lead by Itzkowitz et al in
collaboration with Exact Sciences and the Markowitz laboratory showed markedly
improved results with the use of a DNA stabilizing buffer added to stools at the
time of collection (107). Detection of methylated fecal virnentin

 

DNA was found in 77% of cancers (N=82) at 83% specificity (N=363). Six of 7
adenomas with high-grade dysplasia were also detected. This assay has
successfully detected 55% (N=22) of adenomas that were greater or equal to 1cm
in size (107). This is a published assay of capture of fecal vimentin DNA and
then MSP detection of methylated vimentin exon 1 sequences (105, 107, 108).

 

4.3.4Other Stool Based Biomarkers Under Investigation 

 

Considerable interest in fecal microbiome populations has triggered EDRN
supported investigators into identifying unique bacterial species that are
associated with colonic carcinogenesis and suggests that a microbiome signature
may be a useful stool biomarker for CRC risk (109, 110). Metabolome signatures
promise to identify amino acid or fatty acid profiles associated with colorectal
cancer or high risk (111) have been preliminarily developed in EDRN supported
research. Micro-RNAs (miR._NA) have both oncogenic and suppressor properties,
can be detected in stool, and have been explored as stool based early detection
biomarkers (112, 113). Studies published to date have used small convenience
samples and array technologies that have identified diverse and non-reproducible
miRNAs as classifiers for colonic neoplasms.

--------------------------------------------------------------------------------

7

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

4.3.5Urine base Biomarkers 

 

We demonstrated previously that human urine contains circulation-derived DNA[<
300 base pairs (bp), designated as low molecular weight (LM\V) DNA] and that
LM\V urine DNA can be used to detect colorectal cancer (CRC) associated k-ras
mutations from patients with CRC. A quantitative MethylLight PCR-based assay
targeting a 39-bp template of the hypermethylated vimentin gene (mVIM) was
developed to detect circulation derived mVIM DNA. A blinded concordance study
was performed using matching tissue and urine DNA samples from patients with
CRC. The 20 CRC tissue samples and 20 urine samples from patients with CRC were
provided with barcodes. LMW urine DNA and tissue DNA were isolated, bisulfite
converted and assayed for mVIM. The mVIMwas detectable in 85% (17/20) of the CRC
tissue DNA samples and 75% ofLMW urine DNA. As control, LMW urine DNA isolated
from 20 subjects with no known neoplasm was also tested for the mVIM DNA. Two of
20 (10%) normal control LMW urine DNA contained detectable mVIM DNA. After all
of the samples were tested, the urine and tissue ID numbers were unblinded and
matched The concordance value between the mV/J\1-positive CRC tissue and matched
urine DNA samples was 71% (12/17). We thus conclude that CRC-associated mVIM DNA
can be detected in the urine of patients with CRC with a concordance of 71%
between marker-positive tissue and matched urine samples with a sensitivity of
75% (Su Y-H, personal communication). These results support further development
of a urine test for CRC screening.

 

4.4Key Issues Driving Research Questions in CRC Early Detection Biornarkers 

 

Until therapeutic agents with much greater potency and minimal side effects are
developed, the current best strategy for reducing cancer morbidity and mortality
is early detection of neoplastic disease (114). Key opportunities in the current
state of colorectal screening and early detection include:

 

1.Enhancing adherence to current screening guidelines: Screening and early
detection reduce mortality from colorectal cancer; yet 35% of the population in
the USA remain non-adherent. Adherence is much lower in other countries (25).
The barriers to these recommendations (cost, discomfort, cultural taboos) may be
overcome with circulating biomarkers that provide individuals with persuasive
evidence that undergoing invasive screening procedures, i.e. colonoscopy, will
have important life-saving benefit that reduces mortality from CRC (8-10, 18).
Developing, validating and bringing circulating biomarkers to population
screening use remains a high priority that will likely increase adherence to
endoscopic screening. GLNE 010 addresses this priority by working closely with
EDRN and industry groups to clinically assess and validate circulating
biomarkers of CRC risk that might drive individuals who might decline to
endoscopic screening. 

 

2.Tailoring colonoscopic screening to individual risk: Recently published data
from the Clinical Outcomes Research Initiative found the prevalence of large
polyps higher in blacks than whites among both men and women (115). Tailoring
endoscopic screening to those at risk while limiting screening for those with
minimal or no risk (116, 117) will enhance screening adherence and eliminate
excess cost. Recommendations for tailoring were primarily population demographic
based (116, 117); yet, the translation of carcinogenesis biology and genetics
into biomarker panels with extremely high sensitivity (99%), i.e. no false
negative tests, promises precise tailored endoscopic screening. The current
state of art stool using based biomarker tools is coming close-92% sensitivity
(103) but insufficient to permit tailored or individualized risk. GLNE 010
addresses the priority ofbiomarker driven tailored risk by completing the
ongoing phase 3 validation trial of stool and circulating biomarkers and using
the extensive repository created by this and other GLNE protocols to rapidly
identify new markers that may enhance sensitivity of the current biomarker
panels. 

 

3.Persistently positive stool DNA tests with negative colonoscopic screening:
The stool methylated DNA panels report 5% false positives (103, 108). A positive
stool DNA test with a negative screening colonoscopy could potentially arise
from neoplasia in the upper gastrointestinal tract or from occult and missed
lesions in the colorectum. The latter is a particular concern in the right
colon, where flat lesions and/or sessile serrated adenomas are more prevalent.
Preliminary data from the Case Western EDRN BDL found near 100% vimentin
methylation in gastric dysplasia while no methylation in adjacent gastric mucosa
(S. Markowitz, Personal Communication).  In Barrett’s esophagus (BE), 7 of 7
high grade dysplasias (HOD), and 15 of 18 esophageal adenocarcinomas (EAC) and
even in some squamous cancers (SCC) had methylated vimentin , whereas it was
absent in all 9 normal squamous mucosa (118). A “false positive” stool DNA test
may detect dysplasia or invasive neoplasms in the upper GI tract. The GLNE will
propose to address this priority in the future in a future project. This
project, to be submitted as a separate proposal will propose a longitudinal
study of participants registered in an ongoing cross sectional Phase 2 colon
biomarker validation trial with a positive stool test and negative colonoscopy
registered in the current 

--------------------------------------------------------------------------------

8

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

4.5Rationale for Food Frequency Assessment and the Use of the NIH DHQ II Food
Frequency Questionnaire 

 

Numerous epidemiologic studies have controversially implicated high total fat
and saturated fat, alcohol, inadequate calcium, vitamin D, dietary fiber,
vitamin B6, folate, methionine, antioxidant vitamins such as C, and E, and lack
of fruits and vegetables as dietary risk factors for colon carcinogenesis
(119-127). The causal contribution of these dietary factors to risk of colon
cancer has been difficult to assess and compare in meta analyses due to
different instruments used to assess diet, including diet records, 24 hr.
dietary recalls and food frequency questionnaires (128, 129). However, because
there is significant epidemiologic support for dietary variables affecting
cancer risk in populations (130-132), it is important to collect dietary
information along with human biosamples to allow future study of the
relationship of selected dietary variables and their impact upon biomarkers of
cancer risk, for early detection, or post­ diagnosis prognosis. For example,
vimentin methylation is a key stool DNA marker we propose to validate as an
early detection tool in this trial. Since the methylation reaction requires
methyl tetrahydrofolate (133-135), it is conceivable that dietary folate may
impact the methylation status of this and other future methylated biomarkers for
cancer risk and detection.

In a large validation biosample and annotated data set such as the one in this
trial, diet intake among different subjects is likely to be an important source
of bias, thus an adjustable variable in the analysis of validated biomarkers. We
have chosen to administer a food frequency questionnaire to the subjects emolled
in this trial because it assesses dietary exposures over time (typically 6
months). We recognize the weaknesses of a food frequency instrument, including
recall bias, but the instrument has value when used as a semi-quantitative
measure to rank order individuals according to their intake of a given nutrient
rather than a continuous variable (136, 137). We propose to use the computerized
self-administered user-friendly National Institute of Health Diet History
Questionnaire II (DHQII). The DHQ I was developed specifically to study dietary
risk factors for cancer and has been validated to adequately assess dietary
intake over time against established FFQs such as the Block and Willett FFQ
(136, 138). The questionnaire assesses supplement use in addition to dietary
intake data and has been used in multiple cancer studies to date. The DHQ II is
a refinement of the validated DHQ I with improved separation of some food sub
categories to enhance detail of data intake.

 

5.0STUDY DESIGN 

 

5.1Subject Recruitment 

 

The clinical research associate or study nurse (hereafter “CRA”) at each
clinical site will identify subjects with appointments for colonoscopy via
!RB-approved HIPAA-compliant site-specific methods (Appendix B-tailored to each
site). Recruitment methods could include letters from the primary care
physicians and gastroenterologists, direct referrals to the study team by
physicians, in-clinic recruitment advertisements, use of navigator programs,
county or statewide screening programs, and other !RB-approved means of
identifying and contacting subjects. Interested subjects will be asked to
participate in a baseline visit prior to initiation of colonoscopy preparative
procedures, either at the local Center or during a visit to the subject’s home
by a CRA. Advertisements (e.g., newspapers, AARP Magazine, Clinicaltrials.gov)
may also be used to recruit subjects from the surrounding communities.

 

5.2Eligibility 

 

5.2.1Inclusion Criteria (at time of consent) 

 

Subjects at US Sites

 

Adults 60 and older 

Never had a full colonoscopy for screening purposes 

Willing to sign informed consent 

Able to physically tolerate removal of about 50 ml of blood 

Willing to collect 2 stool samples 

 

Subjects at Sites in Germany/Canada

 

Adults 50 and older 

Never had a full colonoscopy for screening purposes 

Willing to sign informed consent 

Able to physically tolerate removal of about 50 ml of blood 

Willing to collect 2 stool samples 

--------------------------------------------------------------------------------

9

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

5.2.2Exclusion Criteria (at time of consent)-All subjects 

 

Inability to provide informed consent 

History of Inflammatory Bowel Disease 

Overt rectal bleeding within 1 month (30 days) (including due to suspected
hemorrhoids) 

Positive guaiac-based occult blood or fecal immunochemical test (e.g. FOBT,
FIT) in the past 12 months (365 days) 

Undergone resection of the colon for any indication 

Subjects with known HIV or chronic viral hepatitis (Hepatitis Band C) 

Subjects with known or suspected HNPCC (Lynch Syndrome) or FAP 

Any cancer within 5 years prior to enrollment except squamous cell carcinoma of
the skin or Basal cell carcinoma of the skin. 

Prior history of Colon Cancer or Rectal Cancer. 

 

5.3Study Procedures 

 

5.3.1Enrollment and Registration Procedure 

 

Subjects who meet the eligibility criteria will be scheduled for a baseline
visit. The baseline visit must occur prior to any preparative regimen for
colonoscopy (e.g. PEG {Golytely, Halflytely}, Miralax/Gatorade, Suprep, etc.)
and within 16 weeks of the scheduled colonoscopy procedure. At this baseline
visit, subjects will provide informed consent (see model consent, appendix F)
for analysis of stool, urine and blood samples for biomarkers; medical record
review, including colonoscopy and pathology reports; and for completion of
questionnaires.

 

The subject will be enrolled and given a unique participant identification
number (PID) generated randomly by the DMCC. The sites will subsequently link
the PID to the specimen collection kits once specimens are collected.

 

5.3.2Demographic and Other Data Collection 

 

The subject will be asked to provide data to complete EDRN demographic and
medical history questionnaires. Clarification or additional information may be
obtained from the medical records. Case report forms (CRFs) will also be used to
collect information on concomitant medications, colonoscopy outcomes, resection
information, any new cancer treatment, and new diagnostic tests. Long term data
collection (medical records review and follow up data) will be prompted by
information gathered at a phone call with the subject at one-year post
colonoscopy. Data may be collected via face-to-face interviews, via phone or
email interviews, or returned by mail dependent on subject preference. Subjects
(U.S and Canada only) will be asked to complete a NCI DHQ II food frequency
questionnaire (Appendix A) at home after the baseline visit as defined in
Section 5.3.1. The NCI DHQII can be done online through a secure web-based
system ( http://riskfactor.cancer.gov/dhq2 ). If subjects chose to report diet
data on paper forms, sites will be responsible for entering the data into the
web-based system.

 

5.3.3Sample Collection: Blood 

 

Baseline blood samples will be obtained according to standard operating
procedures (Appendix C). The blood will be collected during or after the
baseline visit but prior to any preparative regimen or procedure as detailed in
section 5.3.1 and the Operations Manual. Samples must be collected within 16
weeks prior to the qualifying colonoscopy (detailed in the operations manual).
Blood may not be collected at or after the colonoscopy.

 

5.3.4Sample Collection: Urine Sample 

 

A baseline urine sample will be obtained according to standard operating
procedures (Appendix G). The urine will be collected during or after the
baseline visit, but prior to any preparative regimen or procedure as detailed in
section 5.3.1 and the Operations Manual. The urine specimen must be collected
within 16 weeks prior to the qualifying colonoscopy. Urine may not be collected
at or after colonoscopy.

--------------------------------------------------------------------------------

10

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

5.3.5Sample Collection: Stool Sample and FIT #1 

 

Subjects will be required to collect stool samples prior to any preparative
regimen or procedure within 16 weeks prior to colonoscopy. Women will be asked
to avoid collection during heavy menses if applicable. Subjects will be asked to
collect their stool in the collection bucket (hat) provided. Subjects will be
given detailed instructions and complete kits to collect the stool samples at
home. They will prepare an OC_SENSOR FIT (Eiken Chemical Company) (FIT #1) from
the stool sample. Subjects will also collect scoops of stool into a container
with an EDTA-based buffer (“buffered stool”) and additional scoops of stool into
tubes provided to be sent on ice packs (“native stool”) The subjects will then
package both the stool and the FIT for shipping per provided instructions. The
US and Canadian subjects will ship the stool sample to the Central Laboratory at
the University of Michigan using pre-paid DOT (Department
ofTransportation)-compliant packaging.

 

German subjects will send their stool samples to the German Cancer Center (DKFZ)
(Dr. H. Brenner).

 

5.3.6Sample Collection: FIT #2 

 

Subjects will be asked to collect another bowel movement (ideally the next one)
for a second FIT only (FIT #2). The subject will use the 2nd FIT to collect
another sample from the stool collected on paper provided. The subject will mail
the FIT using provided self­ addressed postage-paid envelopes. The US and
Canadian subjects will ship the FIT #2sample to the Central Laboratory at the
University of Michigan and the German subjects will send their stool samples to
the German Cancer Center (DKFZ) (Dr. H. Brenner).

 

5.3.7Subject Compensation 

 

To compensate for the inconvenience and cost of driving and parking, $25 will be
provided to each subject once blood samples, urine samples, stool samples and
questionnaires are completed. If the research coordinator visits the subject at
home, no payment will be offered at the site’s discretion. U.S. and Canadian
recruiting sites will receive gift cards to distribute to subjects that complete
the requirements to receive payment. Gift cards will be to places like Target,
Walmart, or other similar stores in the specific region, purchased by UM
Prevention Research Base staff and distributed to sites. Sites are required to
account for distribution of gift cards to subjects. German subjects will be paid
according to local policies.

 

5.3.8Colonoscopy Standards 

 

Colonoscopy standards for inclusion of the data into GLNE 010 will include
verification of insertion to cecum, photos of all lesions (available at the
site), size, histology and location in colon of all suspected colorectal
cancers, adenomas, or other polyps. Case report forms will capture some of this
information; the rest will be reviewed directly at site monitoring visits or by
review of redacted reports.

 

5.3.9Sample Collection: Tissue Samples 

 

One H & E slide from clinical tissue blocks of all detected colorectal
adenocarcinomas , high grade dysplasia and advanced adenomas will be obtained
(given, shared digitally or borrowed), sent to the lead site and reviewed by a
reference GI pathologist at the University of Michigan. Up to ten slides (lOum
thick sections) from clinical tissue blocks of all detected colorectal
adenocarcinomas, high grade dysplasia and advanced adenomas will be obtained
whenever possible and sent to the lead site for storage. Specific details will
be worked out with each site depending on costs and standard practices.

 

5.3.10Sample Labeling and Tracking 

 

All samples will be labeled with a unique specimen ID (embedded barcodes or
other labels) managed by the DMCC. The site will subsequently associate the
specimen IDs to the PID. The bar codes will be scanned at each step of the
procedure (collection, on-site processing, shipment, receipt, and storage in a
repository). All biosamples are property of the EDRN.

--------------------------------------------------------------------------------

11

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

5.4Study Definitions 

 

5.4.1Assessing Inclusion Criteria-Definition of “Full Colonoscopy” 

 

Prior colonoscopy eligibility requirements for screening (versus surveillance)
indications are defined per AGA guidelines (22, 139) and are used to define the
study group. A subject who has had a flexible sigmoidoscopy is eligible. An
incomplete colonoscopy is one where the prep was considered “poor” or more than
15% obscured (see SOP, Appendix E) or the entire colon could not be visualized
or the scope did not reach the cecum (unless an obstructing mass was the reason
the scope didn’t reach the cecum). Subjects having a repeat colonoscopy due to a
previous “incomplete” colonoscopy are eligible if they otherwise meet
inclusion/exclusion criteria because incomplete colonoscopies are not a “full
colonoscopy”.

 

5.4.2Minimum Requirements for Subject Enrollment 

 

a.Two FIT tests shipped properly per SOPs (within tolerance) 

b.Stool samples shipped properly per SOPs (within tolerance) 

c.Blood: minimum 18 aliquots of serum, 18 aliquots of plasma, and 2 huffy coats
processed per SOPs 

d.Complete colonoscopy to cecum with good or better bowel preparation (per
colonoscopy SOPs) or an obstructing mass prohibiting insertion to cecum 

e.All data forms 

f.Four 5 ml vials of urine 

 

5.4.3Enrolled Subject 

 

An enrolled subject is one that has signed the informed consent, is eligible
based on inclusion and exclusion criteria at the time of consent (section 5.2)
AND has the minimum specimens required (5.4.2). Replacement samples or
additional visits before the screening colonoscopy are options to meet the
minimum requirements to enroll a subject. Once a subject meets the inclusion and
exclusion criteria and provides specimens within the 16- week window, the
qualifying colonoscopy for study purposes is the first one that is complete (to
cecum) with a good or better preparation (defined as less than 15% of mucosa
obscured). An otherwise eligible subject may need a repeat colonoscopy due to
poor prep, poor sedation , or some other technical or logistical issue. These
subjects would be considered enrolled as long as the colonoscopy is done within
16 weeks of original specimen collection. Enrolled subjects are listed as
“pending” in VSIMS until confirmed. (See 5.4.5) or deemed ineligible (see
5.4.6).

 

5.4.4Protocol Deviations 

 

Subjects who do not meet the minimum requirements (5.4.2), do not have a
complete colonoscopy or have to provide replacement samples will not be reported
as protocol deviations.

 

5.4.5Evaluable Subjects 

 

Once an enrolled subject has completed their colonoscopy, and the recruiting
site has pathology and colonoscopy reports, the site should run “Confirm
Eligibility” in VSIMS. The “Confirm Eligibility” function will verify that the
subject met the inclusion/exclusion criteria, provided the required samples and
data (5.4.2), and count the subject in a final group or bin based on the
colonoscopy results (including no colonoscopy). Evaluable subject’s samples and
data will be used for analysis or building a reference set.

 

Bin #1 - Colorectal Cancer

Bin #2 - Carcinoma in Situ

Bin #3 - Adenoma with High-Grade Dysplasia

Bin #4 - Advanced Adenoma

Bin #5 - Adenoma

Bin #6 - Hyperplastic Polyps

Bin #7 - Polyp of Other & Unknown Types

Bin #11 - Normal Colon

--------------------------------------------------------------------------------

12

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

5.4.6Screen Failures, Not Eligible and Unevaluable Subjects 

 

Subjects who are approached to participate via a face-to-face visit and do not
meet the eligibility criteria in section 5.2 are “screen failures”. These
subjects will not be entered in VSIMS, should not be issued a PID, and will not
receive payment for samples ($25).

 

Screen Failures will not count as accruals.

 

Subjects who sign the informed consent, but end up not meeting the eligibility
criteria in section 5.2 (with samples already collected) will be “ineligible”.
Subjects who meet the inclusion/exclusion criteria but do not provide stool and
blood will be labeled ineligible. An “ineligible” CRF will be completed.
Ineligible subjects will be entered in VSISMs and may receive payment for stool
samples if provided before determined ineligible. Ineligible subjects will not
count as accruals toward the total 9000 subjects.

 

Subjects who are eligible, sign the consent form, and then do not meet the
minimum requirements for subject enrollment (section 5.4.2) are unevaluable.
Subjects that provide specimens but do not have a colonoscopy or subjects that
have a colonoscopy with poor prep, poor sedation or an otherwise incomplete
colonoscopy are considered unevaluable. The site should run “Confirm
Eligibility”. Unevaluable subjects will be entered in VSIMS and may receive
payment for stool samples if provided. Unevaluable subjects will count as
accruals toward the total 9000 subjects.

 

Bin #8 - Uncategorized Colonoscopy

Bin #9 - Incomplete Colonoscopy

Bin #10 - No Colonoscopy

 

5.4.7Off-Study 

 

A subject is off-study when the data, food frequency questionnaire, blood, urine
and stool samples (including both FIT tests) have been obtained, properly
processed and delivered to, the Central Laboratory at the University of
Michigan/DKFZ, a colonoscopy has been completed, eligibility confirmed and the
one year follow up contact has been conducted.

 

Data collection will continue on subjects that have findings of cancer or
require surgical excision of lesions (i.e. adenomas) in order to obtain staging,
treatment, and outcomes relevant to the use of the biomarkers, and these
subjects will not be off study until that data collection is complete. Adverse
events or serious adverse events will not be reported for subjects remaining on
study between the completion of their baseline visit and going “off­ study” as
this is a minimal risk, non-interventional study (section 9.6).

 

5.5Biological Sample and Data Collection 

 

5.5.1Blood Collection, Processing and Storage 

 

Subjects will provide 50 ml of blood as defined above. Blood samples will be
drawn in a specific order: 2 x 10 ml red top tubes and then 3 x 10 ml purple top
tubes. Purple tops tubes must be filled to manufacturer’s level to maintain
blood:EDTA ratio. Sufficient blood is needed to ensure that a minimum of 18
aliquots of serum, 18 aliquots of plasma and 2 huffy coats are collected.
Additional red and purple top tubes may be collected to get the full 50 mls
needed. Additional blood draws, prior to prepping for the colonoscopy may be
done to get to the necessary blood volume.

 

The serum samples (red top tubes) will sit at room temperature for a minimum of
30 minutes (maximum of 60 minutes) to allow the clot to form, and if not
processed immediately, they can be held at 4° C for a maximum of 4 hours after
collection. Plasma samples (purple {aka lavender} top tubes) will be held at 4°
C for a maximum of 4 hours after collection. The red top collection tubes will
be centrifuged at>1,300 x g at 4° C for 20 minutes. The serum will be removed,
transferred to pre-labeled polypropylene capped tubes, and frozen at - 70° C or
colder. The purple top collection tubes will be centrifuged at >1,300 x g at 4°
C for 10 minutes without the brake on the centrifuge. The plasma will be
transferred to a 15 ml conical tube for a second centrifugation step (>1,300 x g
at 4° C for 10 minutes) prior to aliquoting in pre-labeled polypropylene capped
tubes, and frozen at - 70° C or colder. The huffy coat, remaining in the purple
top tubes above the red blood cells, will be removed and placed into 2
pre-labeled vials, up to 1.2 mls of RNALater® (Sigma Chemical Corp, St. Louis,
MO) will be added and stored at -70° C or colder. All frozen samples will be
stored at - 70° C or colder at the collection site and shipped on dry ice to the
CLASS labs at the University of Michigan and stored at- 70° C or colder until
assayed. Detailed Standard Operating Procedures including shipping and sample
handling instructions are provided in Appendix C.

--------------------------------------------------------------------------------

13

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

5.5.2Urine sample collection, processing, storage 

 

At the baseline visit, subjects will be asked to provide a urine sample of at
least 25 mls. The urine specimen will be stabilized with IM EDTA, and held at
4°C for up to 4 hours until aliquoted. The urine will be aliquoted and stored
frozen at -70° C or colder. All frozen samples will be stored at - 70° C or
colder at the collection site and shipped on dry ice to the CLASS labs at the
University of Michigan and stored at - 70° C or colder until assayed. Detailed
Standard Operating Procedures including shipping and sample handling
instructions are provided in Appendix G.

 

5.5.3FIT Analysis 

 

Subjects will be provided with a standard collection kit including detailed
instructions on how to complete the FIT sampling (Appendix D). The first FIT
tube will be shipped inside the same shipping container with the stool sample
(see 5.3.5). The second FIT tube will be mailed (pre-paid) to the University of
Michigan or DKFZ at room temperature in the manufacturer’s DOT-compliant
envelope. The test will be analyzed at the Central Laboratory at the UM or DKFZ
using analytic equipment provided by Eiken Chemical Company. (OC-SENSOR Diana).

 

5.5.4Stool Sample Collection and Handling 

 

Subjects will be asked to collect their stool in the collection bucket (hat)
provided. Subjects will be given detailed instructions and complete kits to
collect the stool samples at home.

 

They will prepare a FIT tube (FIT #1) from the stool sample. Subjects will also
collect scoops of stool into a container with an EDTA-based buffer (“buffered
stool”) and additional scoops of stool into tubes provided to be sent on ice
packs (“native stool”) The subjects will then package both the stool and the FIT
for shipping per provided instructions. The US and Canadian subjects will ship
the stool sample to the Central Laboratory at the University of Michigan using
pre-paid DOT (Department of Transportation)-compliant packaging. German subjects
will send their stool samples to the German Cancer Center (DKFZ) (Dr. H.
Brenner). Buffered stool samples will be homogenized and frozen in four 5 ml
aliquots at -70° C or colder for batch shipment to the analytical labs. The
native stool will be placed at - 70° C or colder upon receipt.

 

5.5.5Follow up 

 

The CRA will contact the subject via phone or letter or email about one year
(window 11- 14 months) after their qualifying colonoscopy for additional follow
up data including changes in family history of cancers, significant personal
medical events such as hospitalizations or new medical diagnoses, and any
diagnosis or treatments for cancer or dysplastic lesions (e.g. adenomas). Data
will be collected on medical record review forms and follow up data forms
(Appendix A).

 

5.5.6Medical Records Documentation 

 

Medical records will be reviewed to collect information regarding the results of
the procedures, pathology analysis, surgery, treatment, history, or outcomes and
documented in the CRFs. The medical records will serve as the source documents
and will be maintained at the site enrolling the subject. Redacted copies
(identifiers blocked out) of colonoscopy reports and pathology reports may need
to be sent to the University of Michigan for review. Medical records and/or
source documents may be reviewed at the site during audits or monitoring visits.

 

5.6Disclosure of results to subjects 

 

Subjects will be informed as part of the consent process that neither they nor
their health care providers will receive any subject-specific results from
participation in this study including results of FIT, stool or blood sample
assay results. Subjects and their health care providers will be furnished with
published data (abstracts, published manuscripts) upon request.

 

5.7Biomarker Analytical Approach 

 

5.7.1Sample Shipment to Analytical Sites 

 

Samples will be shipped in batches to analytical sites. Shipment date and time
will be recorded in shipping logs in VSIMS. Date and time of receipt will be
recorded at the analytical laboratory. Laboratory staff will be blinded to all
subject data except specimen ID and relevant handling or processing information
. No diagnostic or additional demographic data will be provided. The analytical
laboratories will not have access to the database, and will not be able to link
the bar code to a specific PID.

--------------------------------------------------------------------------------

14

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

5.7.2Analytical Performance and Reporting Standard 

 

The laboratory will have 12 weeks (3 months) from the date of sample receipt to
complete the analytical task and report the data to the DMCC. The data will be
reviewed for quality control by the biostatistician (DMCC). If there are
concerns regardin g variance of the data, an on-site visit will occur to review
the methods of assay quality control and data manipulation.

 

5.8Data Collection, Management and Monitoring 

 

Data will be collected, managed and monitored through the EDRN supported VSIMS
system of the Data Management and Coordinating Center (DMCC). This system is a
fully featured, Good Computing Practice compliant (secure, audit trail, daily
backup) database system with biosample tracking capability. Data will be entered
via a Web-fronted interface at each collaborating clinical site. The data will
be subject to internal and external audits. The DMCC and GLNE Prevention
Research Base at the UM will organize and implement on site audit procedures.
Biosample tracking ·will be accomplished using a bar code reader and the VSIMS
system in real time for each step in sample management.

 

6.0 STUDY CALENDAR (Table 1) 

 

Procedures

Baseline1

Stool collection2

Colonoscopy

Follow up5

Informed Consent

X

 

 

 

Study Documentation CRF4

X

 

 

 

General Information CDE4

X

 

 

 

Medical History CDE4

X

 

 

 

Concomitant meds CRF

X

 

 

 

Hormone Replacement TherapyCRF

X*

 

 

 

Colonoscopy CRF

 

 

X

 

Surgery Report CRF

 

 

X*

X*

CRC treatment CRF

 

 

 

X*

Ineligible CRF

X*

 

 

 

Food Frequency Data Collection

X

 

 

 

Blood Collection

X

 

 

 

Urine Collection

X

 

 

 

FITxl

 

X

 

 

Protocol deviation CRF

 

X*

X*

X*

Stool Collection

 

X

 

 

Fixed Tissue3

 

 

 

X3

One Year Follow up

 

 

 

X5

Subject Expense Payment

 

X

 

 

Protocol deviation CRF

 

X

X

X

Study Termination CRF

 

 

 

X

 

1Visit prior to colonoscopy procedure; subject should not have started the colon
preparation procedures.

2Stool collection any time after first visit and subject returns home with kits.
All samples collected prior to beginning colon preparation procedures

3Fixed slides are obtained for pathology review.

4CDE=Common Data Elements; CRF=Case Report Fonn Note: CDEs and CRFs are in
Appendix A

5To be completed 12 months after the completion of the colonoscopy

* Only if needed/applicable

--------------------------------------------------------------------------------

15

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

7.0ANALYTICAL PROCEDURES 

 

7.1Vimentin methylation 

 

This assay will be performed at an EDRN-designated BRL according to previously
published methods described in the background. The assay will be run both
qualitatively and quantitatively for presence of and quantity of methylated
vimentin gene by the University of Maryland BRL (Pl Sandy Stass). The vimentin
methylation assay will be performed blinded without knowledge of clinical source
or results of other assays.

 

7.2Fecal immunochemical Test (FIT) 

 

The QC-SENSOR product will be used according to manufacturer’s instructions. The
threshold for a positive test is 100 ng/ml. The Central Laboratory at UM and
DKFZ will process the samples using equipment provided by Eiken Chemical
Company. Technicians will undergo tutorial and quality assessment with Eiken
Chemical Company support technicians prior to study launch. A quantitative
result will be generated and recorded and uploaded into VSIMS by the DMCC. If
either stool result is above the recommended cut­ off, that subject will be
called positive.

 

7.3Galectin-3 Ligand 

 

The analytically validated ELISA method described in the preliminary data will
be transferred to an EDRN Biomarker Reference Laboratory. Serum aliquots will be
provided to the analytical sites in a blinded fashion. The Bresalier laboratory
will assay 20% of the samples to ensure quality control. All of the samples will
be assayed by the UCLA Biomarker Reference Laboratory. Galectin-3 Ligand assay
result is a continuous variable. To facilitate comparison, a threshold
corresponding to the specificity of vimentin methylation, estimated from
controls in this study that do not have SR,N” from colonoscopy, will be used.

 

7.4Circulating methylated genes BCATl/IKZFl (Clinical Genomics) 

 

A Good Laboratory Practice validated bisulfite PCR assay developed by Clinical
Genomics will be used for this assay. Clinical Genomics will perform this assay
on blinded samples at their laboratory facility in Rutherford, NJ. Clinical
Genomics is not responsible for analysis of any other biomarkers other than
their BCATl/IKZFl product.

 

7.5Hypornethylated LINEl from circulating cell free DNA (VolitionRx) 

 

A Good Laboratory Practice validated assay developed by VolitionRx will be used
for this assay. VolitionRx will perform this assay on blinded samples at their
laboratory facility in Namur, Belgium. Volition is not responsible for analysis
of any other biomarkers other than their hypomethylated LINE I assay.

 

8.0DATA ANALYSIS PLAN, SAMPLE SIZE JUSTIFICATION, AND STATISTICAL POWER 

 

Aim 1: This aim proposes to estimate the sensitivity and specificity for 1)
colorectal adenocarcinoma or 2) screen relevant neoplasms (high grade dysplasia
or adenoma with 2:25% villous histologic features or adenoma measuring 2:1 cm in
the greatest dimension or sessile serrated polyps measuring 2:1 cm in diameter)
of the following individual colorectal neoplasia early detection biomarkers
using colonoscopy as the gold standard:

 

stool vimentin methylation 

serum galectin-3 ligand 

fecal immunochemical tests (FIT) 

Circulating methylated genes BCATl/JKZFI  (Clinical Genomics) 

Hypomethylated LINE! from circulating cell free DNA (Volition) 

Other currently unspecified biomarkers 

 

For each of the individual biomarkers, we will first verify through a receiver
operating curve (ROC) analysis the previously established thresholds for an
optimum sensitivity and specificity. For ties in area ofROC, we shall choose the
cut-off based on the highest percent agreement.

 

From this point on, we will treat the performance of the markers as a dichotomy.
We will calculate accuracy summaries such as sensitivity, specificity,
predictive values, as well as the likelihood ratios for each individual marker
along with the associated confidence intervals.

--------------------------------------------------------------------------------

16

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

Aim 2 (primary objective): Three potential scenarios of possible primary
hypotheses and corresponding plans for data analysis and statistical power are
presented. The choice of primary hypothesis will be finalized prior to data
analysis based on state of the art information about candidate biomarkers and
clinical practice at that time.

 

a.To determine if a blood based panel (for example, serum galectin-3 ligand,
CEA, methylated genes BCATl/lKZFl,  Hypomethylated LINEl  from circulating cell
free DNA), at the same sensitivity of that for fecal immunochemical testing
(FIT) for the detection of colorectal adenocarcinoma, has a specificity greater
than 0.55 with an anticipated specificity 2’.: 0.70. 

 

b.To determine if stool vimentin methylation and the blood based panel (serum
galectin-3 ligand, CEA, methylated genes BCATl/IKZFl, Hypomethylated LINEl from
circulating cell free DNA) when combined with fecal immunochemical testing (FIT)
will significantly improve the sensitivity of FIT for the detection of
colorectal adenocarcinoma, and maintain specificity greater than 0.80. 

 

c.To determine if stool vimentin methylation, the blood based panel (serum
galectin-3 ligand, CEA, methylated genes BCATl/IKZFl, Hypomethylated LINEl from
circulating cell free DNA), when combined will improve the detection of
colorectal adenocarcinoma: at sensitivity 2’.:0.98 it will have a specificity
significantly greater than 0.55. 

 

The statistical power analysis assumes 71 CRC cases (63 from the additional
9,000 subjects plus 10 CRC cases already recruited less 2 CRC cases in the
surveillance sub-cohort), and>13,000 non-CRC controls (assuming ~12,000 non-SRN
controls and ~1,300 SRNs).

 

Example a: A blood based panel (serum ga]ectin-3 ligand, or combined with other
blood based biomarker if necessary) will be defined and locked-down prior to
data analysis. At a cutoff with the same sensitivity as FIT (assumed here to be
0.75 but the value will be estimated from the study data) the specificity for
non-SRN controls and its 95% C.I. will be calculated. The I-sided hypothesis
that this specificity is significantly higher than 0.55 (Ho) will be tested with
an anticipated specificity 2:: 0.70 (H1), using the kernel method described by
Bantis and Feng (141). The kernel ROC estimate has been proven to have smaller
mean square error than that of the empirical ROC estimate (142).

 

Statistical power will be >85% or >90% with an anticipated specificity 2’.: 0.70
or 2’.: 0.75 respectively. Preliminary data from GLNE investigator Robert
Bresalier supports the performance assumptions made. The blood based panel used
Galectin 3 ligand, methylated genes BCATl/IKZFl, Hypomethylated LINEl  from
 circulating cell free DNA, CEA with four different modeling approaches using
GLNE data (94 normals, 50 small adenomas, 100 CRCs, and 51 advanced adenomas).
The specificity at 75-80% sensitivity is >70% for both negative colonoscopy
group and for negative colonoscopy plus small adenoma group.

 

Example b: To determine if stool vimentin methylation and the blood based panel
(serum galectin-3 ligand , CEA, methylated genes BCATl/IKZFl, Hypomethylated
LINEl from circulating cell free DNA) will significantly improve the sensitivity
of fecal immunochemical testing (FIT) for the detection of colorectal
adenocarcinoma, and maintain specificity greater than 0.80. From other training
samples, the cutoff points and the combination rules of vimentin methylation and
blood based panel will be detem1ined based on their ability to detect FIT
negative colon cancer and maintain high specificity, then combined with FIT
either by an “OR” rule or a linear combination. This decision rule will be
locked-down prior to GLNE10 protocol data analysis. The difference of the
sensitivities of this decision rule and FIT and the I-sided 95% confidence
interval of this difference will be calculated from 10,000 bootstrap samples to
accommodate the dependence between two tests. The null hypothesis will be
rejected if the lower bound of this confidence interval is above zero.

 

The statistical power will be >0.83 if the true difference in sensitivity is
2’:.0.16 (e . g. 0.75 for FIT, 0.91 for the new test) under the conservative
assumption of independence of the two tests. The actual power will be larger as
these two tests are expected to be positively correlated.

 

Example c: To determine if stool vimentin methylation, the blood based panel
(serum galectin-3 ligand, CEA, methylated genes BCAT I /IKZF l , Hypomethylated
LINEl from circulating cell free DNA), when combined will increase the detection
of colorectal adenocarcinoma: at sensitivity 2’: 0.98 it will have a specificity
significantly greater than 0.55, with an anticipated specificity 2’: 0.79. This
has significant clinical value as it has potential to spare more than 55% people
in US from colonoscopy screening, and improve screening rate for those who do
not want to have colonoscopy as first-line screening modality. From other
training samples, a panel will be built to achieve 2’: 0.98 in sensitivity in
detecting colorectal adenocarcinoma while maintaining specificity 2’: 0.79 for
subjects without SRN lesions. This is feasible if other markers can pick up
majority of FIT negative CRCs without reducing specificity by more than 15%. The
combination rule will be defined and locked-down prior to GLNEl O protocol data
analysis. To test the study hypothesis, at cutoff corresponding to 0.98
sensitivity, the specificity of this decision rule, its I­ sided 95% C.I., and
the I-sided hypothesis that this specificity is significantly higher than 0.55
(Ho) will be tested using the method using the kernel method described by Bantis
and Feng (141). Statistical power is > 0.90 if the true specificity 2: 0.79. The
1-sided 95% C.I. for sensitivity will also be reported.

--------------------------------------------------------------------------------

17

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

Secondary objective: While the study will be powered for the primary objective,
we shall also carry out a similar assessment of potential utility of a clinical
test for SRN.

 

Aim 3 To construct a combined early detection biomarker panel using the above
individual biomarkers (stool vimentin methylation, serum galectin-3 ligand, FIT,
the Exact Sciences stool DNA panel, circulating methylated genes BCATl /IKZFl ,
hypomethylated LINEI from circulating cell free DNA), and describe its
performance for 1) colorectal adenocarcinoma and for 2) screen relevant
neoplasms.

 

After the primary hypothesis (Aim 2) has been finalized, the examples described
in Aim 2 and are not chosen as the primary hypotheses will be tested in Aim 3 as
they are all clinically relevant hypotheses. The data analyses and their
statistical power for these hypotheses have been described under Aim 2 above and
so are not repeated here.

 

In addition, new panels could be constructed using the trial data as training
set but these panels will need to be validated on other independent cohorts.

 

Aim 4

To establish an archive of appropriately preserved stool, serum, plasma and DNA
human biospecimens to be used by EDRN-approved investigators for future
validation and biomarker discovery research.

 

8.1Secondary Analyses 

 

8.1.1Secondary Analyses for Screen Relevant Neoplasias 

 

Similar analyses as described above will be performed for the secondary endpoint
(SRN). The minimally acceptable performance used for setting null hypotheses
will differ: sensitivity=0.25 for secondary specific aim 2, and sensitivity=0.45
for secondary specific aim 3.

 

8.1.2Secondary Analyses for projecting biomarker performance in US population  

 

Since our enrollment is enriched with older subjects, in Aim la we will also
report age and gender-standardized accuracy summaries that is calculated with an
inverse probability weighted method in order to adequately reflect the
performance of biomarkers in the US screening population which may have a
different age distribution from that of our study population. The ratio of
observed proportion within each age by gender stratum versus the corresponding
proportion in the US population as reported by the Census data will be used as
the weights for subjects in the stratum.

 

Note that the weighted analyses may provide reasonable projections for
biomarkers’ performance in the US population, however we will treat this as a
secondary analysis and therefore our power calculation is based on unweighted
analyses from the observed sample.

 

8.1.3Additional Secondary Analyses 

 

The cohort will be characterized in terms of demographics and epidemiological
variables such as gender, education status, intake of dietary micro and
macronutrients, hormone replacement therapy, alcohol intake and smoking. We will
assess the relationship of each of these factors individually to each marker
under evaluation and risk of colorectal cancer in multiple regression models.
For factors that modulate the relationship between biomarkers and clinical
endpoints, we will further evaluate their effects on the accuracy parameters of
the marker, by evaluating for example the covariate-specific ROC curves. We will
also test directly covariate effects on the accuracy of markers in order to
identify subpopulations for which markers are mostly effective.

 

8.1.4Inclusion of New Biomarkers Discovered by EDRN Investigators over the Next
Two Years 

 

The design of this project including the collection of serum, DNA and tissue
samples permit the inclusion of new EDR._ discovered biomarkers into this panel.
Should EDR.i”“\J” investigators provide sufficient preliminary data to justify
inclusion in this panel; new biomarkers will be included in the validation
program using the procedures described above.

--------------------------------------------------------------------------------

18

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

9.0PROJECT MANAGEMENT PLAN 

 

9.1Strategies to Ensure Completion of Milestones 

 

Milestones set up and regularly reviewed: Milestones are set on a quarterly
schedule and managed by the principal investigator.

 

Conference calls: a) Investigator calls: All GLNE investigators communicate
every other week by scheduled telephone conference call organized and chaired by
the PI, Dr. Brenner (11 AM to 12 Noon, Thursdays). b) Coordinator calls: All
GLNE research coordinators and support staff communicate once per month by
scheduled telephone conference call organized and chaired by the Project
Manager, Mr. Kirk Herman and the DMCC.

 

Data and Safety Monitoring: The University of Michigan Cancer Center Prevention
Program’ s Data Safety and Monitoring Committee meets monthly and reviews
progress towards milestones. Accrual, endpoints, toxicity, and strategies to
ensure goals are met reviewed by this committee. Minutes are forwarded to the
supervising IRB (IRBMED) and to relevant regulatory agencies.

 

9.2Timeline for Completing GLNE 010 

 

Table 2, a Gantt diagram, outlines our milestones for the proposed CRC early
detection biomarker trial.

9.2 table.jpg [f10k123119ex10z227.jpg] 

Table 2: GLNE 010 (Colon Biomarker Validation Trial) Milestones

 

We have broken down this trial into the re-organizational phase (Months -6-0)
that began on April 6, 2016 and will be completed on April 1, 2017. Accrual
phase beginning April 1, 2017, will last 3.5 years with data assay and data
analysis being complete in Years 5. Milestones are outlined below and add detail
to the events depicted in Table 2. We estimate 250 participants/month accrual at
all sites (EDRN, Alliance/NCORP, and Germany, see Table 3).

 

Center

Accrue/mo

Pl

%Minority

Case Western

2

Coooer

40%

Columbia

2

Kastrinos

80%

Dana Farber

7

Syngal

10%

MD Anderson

8

Bresalier

20%

Minnesota

30

Allen/Church

3%

St Michaels/Toronto

15

Marcon

3%

Univ IL Chicaqo

1

Carroll

55%

Univ Michigan

12

Turgeon

3%

Univ North Carolina

13

Crockett

1%

Univ Washington

11

Gradv

1%

Alliance/NCORP-Mavo

61

Marshall

3%

Total USA

162

 

 

Heidelbera, Germanv

95

H Brenner

0%

Total USA-Germany

257

 

 

 

Table 3 (Left): Prior accrual experience, GLNE USA, limited to age .!:65 yrs.

DKFZ Germany documented accrual, all age groups over 7 years, BliTz (personal
communication, H. Brenner).

--------------------------------------------------------------------------------

19

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

9.3Endpoint Event Justification and Milestones 

 

9.3.1Required Endpoints 

 

In order to successfully complete this project a minimum of 70 invasive
colorectal neoplasms must be detected. To ensure we reach this endpoint, we
revised the protocol to 1. Expand the trial to include Germany; 2. Limit
recruitment in USA sites to participants 60 years or older undergoing first
colonoscopy.

 

9.3.2Justification for Requiring only Invasive Colorectal Neoplasms 

 

First, recent as well as older large validation trials have found that
dysplastic adenomas as endpoints degrade biomarker detection accuracy (102,
103). For example, in the recently published Exact Sciences stool DNA panel,
sensitivity for invasive colorectal neoplasms only was 92% which reduced to 84%
when high grade dysplastic adenomas were included with invasive colorectal
neoplasms (103). Second, a strategy which allows high grade dysplasia in
adenomas as primary endpoints reduces the likelihood of successful
identification of early detection biomarkers for colorectal neoplastic disease.
We run the risk oflosing biomarkers to failure requiring classifier performance
of single markers or panels that might be useful for the detection of curable
invasive colorectal neoplasms despite poorer performance for detection of high
grade but non-invasive neoplasms. We increase barriers for success to the very
biomarkers that the EDRN exists to discover and validate. Third, inclusion of
high grade dysplasia in adenomas with invasive colorectal neoplasms degrades the
usefulness of the repository samples generated by GLNE 010.

 

There will be insufficient invasive colorectal neoplasm events to use for large
cross sectional validation of future biomarkers. Our only other resource for
invasive colorectal neoplasms is a cross sectional reference set that is not
PRoBE compliant.

 

9.3.3Strategies to Ensure Sufficient Invasive Colorectal Cancer Events 

 

Expand GLNE to Germany: The rapid expansion of colonoscopic screening in the USA
to younger age groups (ages 50-59) (Table 4) with high adherence to colonoscopic
screening guidelines (61%), while reducing incidence and presumably mortality
from invasive colorectal neoplasms may be reducing the numbers of screen
detected invasive colorectal neoplasms in USA screening trials such as GLNE 010.
In GLNE 010, the detected rate of screen relevant (“advanced”) adenomas of 13%
exceeds the expected rates in recently published biomarker trials using
screening populations [Lieberman, 2014 #5685;Lieberman, 2014 #5684;Imperiale,
2014 #5977;Church, 2014 #4704]. In Germany, colonoscopic screening is provided
as a benefit to the population, but with much lower colonoscopic screening
adherence rates (16%) (25) as opposed to 61% adherence in the USA (19). The
higher invasive colorectal neoplasm case proportion of 0.7% in a large ongoing
prospective screening trial (Table 4) makes a primary colorectal neoplasm
endpoint feasible.

 

Age

Exact

PRESEPT

BliTz

GLNE

50-59

29%

35%

36%

42%

60-64

8%

27%

23%

18%

65-69

37%

21%

25%

21%

70-74

17%

11%

13%

21%*

?:.75

9%

6%

4%

---

CRC Event

0.7%

0.8%

0.7%

0.2%

HGD Event

0.4%

0.7%

0.7%

0.4%

 

Table 4: Enrollment Ages, Recent Large Cross Section CRC Screening Trials.

Exact Sciences (2014, (1)), PRESEPT (2014, (2)), BliTz

(ongoing, Heidelberg, H. Brenner, Personal Communication.)

 

Enrich North America Risk to Bring Endpoint Event Percentages to the Level of
Other Trials: The incidence of invasive colorectal neoplasm increases with age,
particularly at ?:.60 yrs. The recent published PRESEPT and Exact Sciences trial
enriched their screening populations to increase invasive colorectal neoplasm
event rates. PRESEPT restricted enrollment to first colonoscopic procedures.
 PRESEPT  did  not  limit  age  of  enrollment, two thirds of participants were
?:.60 yrs. Exact Sciences required two thirds of enrolled participants be ?:.65
yrs, but permitted prior colonoscopic screens ?:.9 yrs. BliTz has no age limits
or prior endoscopy limits, but the low screening adherence rate in Germany
suggests that the population represents first colonoscopic screening procedures.

--------------------------------------------------------------------------------

20

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

9.4Endpoint Event Monitoring 

 

9.4.1Endpoint Monitoring 

 

As part of weekly accrual monitoring, endpoints events (invasive cancers, high
grade dysplasia, screen relevant neoplasia, and adenoma) will be reported.

 

9.4.2Monitoring Primary Endpoint Events 

 

The primary endpoint, invasive colorectal cancer, will be monitored weekly.

 

9.4.3Primary Endpoint Expectations 

 

At an expected accrual rate of 1,500 per 6 months, we expect 10 invasive cancers
to be detected every 6 months. Because events do not occur at regularly spaced
intervals, but rather as randomly distributed events, to identify potential
problems in meeting our primary endpoint goal we need a sufficient number of
evaluable accruals to determine whether the current enrollment strategy will
meet the accrual goal of at least 70 invasive neoplasms.

 

For these reasons, we will wait until 1,000 subjects have been enrolled to
assess whether our case event rates will be sufficient to meet the current
primary study endpoint.

 

9.4.4Monitoring to Ensure Sufficient Cancer Endpoints 

 

The following procedure will be followed in collaboration with the DMCC: Review
every 1,000 evaluable subjects through the course of the project with the
expectation that 7±1 new invasive neoplasm cases will be detected for every
1,000 evaluable subjects. If <7 new invasive neoplasm cases are enrolled for
every 1,000 evaluable subjects , the project will be reviewed by statistical
consultants and coinvestigators and revision of study goals and design by
allowing FIT positive screens.

 

9.5Data Safety and Monitoring 

 

9.5.1Authority 

 

The University of Michigan Prevention Research Base Data Safety and Monitoring
Committee (DSMC) reviews, makes recommendations, and acts on the following:

 

a.All protocols being run through the GLNE EDRN will be monitored by the DSMC 

b.Progress towards completion of the study-recruitment and retention of study
participants 

c.Evaluation of interim new information 

d.Evaluation of toxicity events including reporting of adverse events, if
applicable 

e.Timeliness of data 

f.Quality of data 

g.Ethical conduct of research 

 

The DSMC is empowered with the authority to recommend a study be suspended or
terminated based upon concerns in any of the above areas ofreview. Monitoring
also considers factors external to the study, such as scientific or therapeutic
developments that may have an impact on the safety of the participants or the
ethics of the study.

 

Recommendations that emanate from monitoring activities are reviewed by the
principal investigator and addressed.

 

9.5.2Composition 

 

The current UM Prevention Research Base Data and Safety Monitoring Committee is
Chaired by one of the faculty members present at the meeting, usually the most
senior member who is not a principal investigator on studies being discussed.
Membership includes faculty members from Gastroenterology, Family Medicine, and
Hematology/Oncology. At least 3 faculty members must be present to have quorum.
If the DSMC cannot meet face-to-face, a conference call is acceptable.

--------------------------------------------------------------------------------

21

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

9.5.3Meeting Frequency 

 

The UM Prevention Research Base DSMC meets monthly by means of regularly
scheduled meetings. Prior to each meeting, the UM Prevention Research Base
Clinical Research Associate distributes a standard summary report detailing
accrual, biomarker modulations data, new publications or presentations relevant
to the ongoing project, quality control audit information, any ethical concerns,
patient-subject complaints and adverse events or serious adverse events of all
prevention protocols.

 

9.5.4Recommendations and Reporting 

 

Recommendations for action are sent to the Principal Investigator. The Principal
Investigator is responsible for reviewing and if necessary, implementing DSMC
recommendations.

 

9.6Adverse Event Reporting 

 

9.6.1Definition 

 

An adverse event (AE) is any condition, which appears or worsens after the
participant is enrolled in an investigational study. For this minimal risk,
sample collection study, we provide a definition of what would be considered
related to the study participation.

 

9.6.2AE Information 

 

No adverse events are expected, as there is no intervention for this study. Any
adverse events related (as judged by the site PI, overall PI or DSMC) to the
subject’s participation (sample or data collection) in this study will be
forwarded to the data coordinating center and reported to regulatory bodies per
study-specific guidelines. Adverse events or serious adverse events will not be
reported for subjects remaining on study between the completion of their
baseline visit and going off-study as this is a minimal risk, non-interventional
study. Subjects could be considered on study for over a year and have adverse
events completely unrelated to study participation. Examples of related adverse
events that could be reported could include problems with the blood draw
(bruising, fainting), loss of confidentiality of data, or lost samples. Examples
of events that would not be reported would include any complications from the
colonoscopy or events related to other medical conditions like colon cancer or
diabetes.

 

9.6.3Serious Adverse Events 

 

Some percentage of participants will have colorectal cancer identified during
colonoscopy by study design, and deaths due to disease progression or serious
adverse events due to cancer treatment are expected. The only procedures that
are part of this study are blood, urine and stool collection, so it is unlikely
that any deaths or hospitalizations will be related to the sample collection in
this study. Only Serious Adverse Events that are deemed to be directly related
to a study procedure (sample collection) by the DSMC will be reported to any
regulatory body.

 

A serious adverse event is defined (by ICH Guideline E2A and Fed. Reg. 62, Oct.
7, 1997) as an event, occurring at any dose, which meets any of the following
criteria:

 

Results in death 

Is immediately life threatening 

Requires inpatient hospitalization or prolongation of existing hospitalization 

Results in persistent or significant disability/incapacity 

Is a congenital anomaly/birth defect 

 

In addition, events that may not meet these criteria, but which the investigator
finds very unusual and/or potentially serious, will be reported in the same
manner.

--------------------------------------------------------------------------------

22

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

10.0DATA MANAGEMENT 

 

10.1Registration 

 

Institutional collaborators will enter IRB information into the secure database,
including IRB approval date, expiration date, and document versions. Subject
registrations will not be allowed without IRB approval. The DMCC will assign the
participant ID number (PID).

 

No exceptions to eligibility requirements will be permitted.

 

10.2Timeliness 

 

In collaboration with the DMCC, a data expectation system will be developed.
Detailed instructions are provided in the operations manual. For sampie
shipments, data from the shipping and receiving laboratory describing the date
of sample shipping and sample receipt are captured in the database.

 

10.3Completeness and Accuracy 

 

The DMCC will assure the completeness of the data by writing data entry programs
that will not allow for empty fields whenever possible. The accuracy of the data
will be checked by identifying appropriate parameters allowed to be entered in a
given data field. Periodic reviews of the paper forms and the database data will
be conducted by the lead CRA and a Site Monitor from the Coordinating Center.

 

10.4Accuracy--Revisions and Corrections 

 

All corrections to paper study documents will be initialed and dated. If
computer-readable data is corrected by replacement of a data set, the replaced
version of the data set will be retained in an archive. The collection of these
auxiliary data sets represents an audit trail of corrections to the database.

 

10.5On Site Data Audits 

 

All consortium sites will be subject to periodic on-site audits. The objective
of the on-site audit will be to conduct a general review of a random sample of
registered subjects from the selected protocol to assess overall protocol
adherence with respect to subject eligibility, appropriate procedure for
informed consent, registration process, general protocol adherence, sample
shipment process, follow-up and off-study process.

 

An On-Site Audit checklist will be developed which will contain all of the
essential elements of an On-Site audit. Each of the essential elements will be
reviewed and discussed with the clinical site. The Checklist will be signed by
the auditors and retained at the DMCC.

 

In preparation for a site audit, the study statistician will select the subjects
for review using a randomized selection procedure. Other cases may also be
selected at the discretion of the audit team. A minimum of 10% of the subjects
accrued since the last audit will be reviewed for the first year. The number of
subjects to be audited for the subsequent year will be determined based on
findings of the audits from prior years in order to have sufficient power to
identify important issues. The on-site audit team will audit additional
unannounced cases. The consortium site investigator and research coordinator
will be notified of the impending audit at least 3 weeks in advance. All data
and material pertinent to the subject will be reviewed including eligibility
criteria, informed consent, and sample shipment logs. All informed consent
documents for all subjects may be reviewed at the on-site audit. At the audit,
the data from the DMCC will be compared to the original data (source documents
and/or CRFs). On-site audit staff will review the documentation of IRB
approvals, for each audited protocol, any amendments or adverse events, and
consent forms.

 

Based on the findings of the audit, a follow-up schedule will be defined. A
report of the audit will be written and emailed or faxed to the consortium site
investigator. The site PI will have 30 days from receipt of the report to
respond in writing to the DMCC directly.

 

The DMCC will maintain a file containing the latest version of the On-Site Audit
guidelines, a listing of all consortium institutions reviewed to date, a copy of
the On-Site Audit results and all correspondence for each audit conducted. These
results will be reviewed by the DSMC, the DMCC, and others as needed and will be
made available to the NCI.

--------------------------------------------------------------------------------

23

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

 

 

10.6Sample Tracking 

 

CLASS labs must be notified via e-mail of a shipment due to arrive so if samples
are delayed or lost, tracking may be initiated by the sending site. Sample
shipment packing lists generated by the database are included with shipments.
The receiving site will evaluate the sample condition on arrival, scan the
bar-coded samples into database, verify samples shipped match samples sent and
store at appropriate conditions until shipment to analytical labs or
repositories.

 

10.7Confidentiality 

 

Subjects will be identified in the database by their unique PID only.
Information that could identify subjects, such as name, address, or medical
record number will be kept only by the enrolling site and will not be supplied
to the DMCC or GLNE Research Base. During an on-site audit or NCI site visit,
audit staff may review medical records and other information that contains PHI,
but this information will not be removed from the enrolling site. Neither the
DMCC nor the research base at UM will keep copies of signed informed consent
documents. No information, including copies of the informed consent unless
required by the institution, obtained during the study will be placed in a
subject’s medical record.

 

10.8Security 

 

All subject files will be stored under lock and key at all times. All computer
systems will be password-protected against intrusion; all network-based
communications between sites of confidential information are encrypted. An
on-going computer-virus-protection program is available and used, maintained,
and audited on all computers and pathways into the system, including good
practice policies, screening of data files, executable software, diskettes, text
macros, downloads, and other concerns as they arise. The DMCC will assist in
maintaining appropriate levels of network security.

 

11.0ETHICAL & REGULATORY CONSIDERATIONS 

 

11.1Institutional Review 

 

This study must be approved by an appropriate institutional review committee as
defined by Federal Regulatory Guidelines (Ref. Federal Register Vol. 46, No. 17,
January 27, 1981, part 56). The protocol and informed consent form for this
study must be approved in writing by the appropriate Institutional Review Board
(IRB). The IRB must be from an institution that has a valid Federal Wide
Assurance on file with the Office for Human Research Protections, Department of
Health and Human Services. The institution must comply with regulations of the
Food and Drug Administration and the Department of Health and Human Services.
Changes to the protocol, consent, as well as a changes to the investigator list
at each site, must also be approved by the IRB and documentation of this
approval provided to the Coordinating center.

 

Records of the Institutional Review Board review and approval of all documents
pertaining to this study must be kept on file by the investigator and are
subject to OHRP, FDA or NCI inspection at any time during the study. Periodic
status reports must be submitted to the Institutional Review Board at least
yearly, as well as notification of completion of the study and a final report
within 3 months of study completion or termination. The investigator must
maintain an accurate and complete record of all submissions made to the
Institutional Review Board, including a list of all reports and documents
submitted.

--------------------------------------------------------------------------------

24

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

12.0 REFERENCES 

 

1.Church TR, Wandell M, Lofton-Day C, Mongin SJ, Burger M, Payne SR, et al.
Prospective evaluation of methylated SEPT9 in plasma for detection of
asymptomatic colorectal cancer. Gut. 2014;63:317-25. PMCID: 3913123. 

2.Lee JK, Liles EG, Bent S, Levin TR, Corley DA. Accuracy of fecal
immunochemical tests for colorectal cancer: systematic review and meta-analysis.
Ann Intern Med. 2014;160:171. PMCID: 4189821. 

3.Pepe MS, Feng Z, Janes H, Bossuyt PM, Potter JD. Pivotal evaluation of the
accuracy of a biomarker used for classification or prediction: standards for
study design. J Natl Cancer Inst. 2008;100:1432-8. 

4.Mandel JS, Bond JH, Church TR, Snover DC, Bradley GM, Schuman LM, et al.
Reducing mortality from colorectal cancer by screening for fecal occult blood.
Minnesota Colon Cancer Control Study [published erratum appears in N Engl J Med
1993 Aug 26;329(9):672] [see comments]. N Engl J Med. 1993;328:1365-71. 

5.Kronborg 0, Fenger C, Olsen J, Jorgensen 0, Sondergaard 0. Randomised study of
screening for colorectal cancer with faecal-occult-blood test. Lancet.
1996;348:1467-71. 

6.Hardcastle JD, Chamberlain JO, Robinson MH, Moss SM, Amar SS, Balfour TW, et
al. Randomised controlled trial of faecal-occult-blood screening for colorectal
cancer. Lancet. 1996;348:1472-7. 

7.Shaukat A, Mongin SJ, Geisser MS, Lederle FA, Bond JH, Mandel JS, et al.
Long-term mortality after screening for colorectal cancer. N Engl J Med.
2013;369:1106-14. 

8.Baxter NN, Goldwasser MA, Paszat LF, Saskin R, Urbach DR, Rabeneck L.
Association of colonoscopy and death from colorectal cancer. Ann Intern Med.
2009;150:1-8. 

9.Singh H, Nugent Z, Demers AA, Kliewer EV, Mahmud SM, Bernstein CN. The
reduction in colorectal cancer mortality after colonoscopy varies by site of the
cancer. Gastroenterology. 2010;139:1128-37. 

10.Brenner H, Chang-Claude J, Seiler CM, Rickert A, Hoffmeister M. Protection
from colorectal cancer after colonoscopy: a population-based, case-control
study. Ann Intern Med. 2011;154:22-30. 

11.Muto T, Kamiya J, Sawada T, Konishi F, Sugihara K, Kubota Y, et al. Small
flat adenoma of the large bowel with special reference to its clinicopathologic
features. Dis Colon Rectum. 1985;28:847-51. 

12.Rex DK, Cutler CS, Lemmel GT, Rahmani EY, Clark DW, Helper DJ, et al.
Colonoscopic miss rates of adenomas determined by back-to-back colonoscopies.
Gastroenterology. 1997;112:24-8. 

13.Saitoh Y, Waxman I, West AB, Popnikolov NK, Gatalica Z, Watari J, et al.
Prevalence and distinctive biologic features of flat colorectal adenomas in a
North American population. Gastroenterology. 2001;120:1657-65. 

14.Samadder NJ, Curtin K, Tuohy TM, Pappas L, Boucher K, Provenzale D, et al.
Characteristics of missed or interval colorectal cancer and patient survival: a
population-based study. Gastroenterology. 2014;146:950-60. 

15.Barclay RL, Vicari JJ, Greenlaw RL. Effect of a time-dependent colonoscopic
withdrawal protocol on adenoma detection during screening colonoscopy. Clin
Gastroenterol Hepatol. 2008;6:1091-8. 

16.Barclay RL, Vicari JJ, Doughty AS, Johanson JF, Greenlaw RL. Colonoscopic
withdrawal times and adenoma detection during screening colonoscopy. N Engl J
Med. 2006;355:2533-41. 

17.Soetikno RM, Kaltenbach T, Rouse RV, Park W, Maheshwari A, Sato T, et al.
Prevalence of nonpolypoid (flat and depressed) colorectal neoplasms in
asymptomatic and symptomatic adults. JAMA. 2008;299:1027-35. 

18.Zauber AG, Winawer SJ, O’Brien MJ, Lansdorp-Vogelaar I, van Ballegooijen M,
Hankey BF, et al. Colonoscopic polypectomy and long-term prevention of
colorectal-cancer deaths. N Engl J Med. 2012;366:687-96. PMCID: 3322371. 

19.Salas D, Vanaclocha M, Ibanez J, Molina-Barcelo A, Hernandez V, Cubiella J,
et al. Participation and detection rates by age and sex for colonoscopy versus
fecal immunochemical testing in colorectal cancer screening. Cancer Causes
Control. 2014;25:985-97. 

20.Quintero E, Castells A, Bujanda L, Cubiella J, Salas D, Lanas A, et al.
Colonoscopy versus fecal immunochemical testing in colorectal-cancer screening.
N Engl J Med. 2012;366:697-706. 

21.Welch HG, Black WC. Overdiagnosis in cancer. J Natl Cancer Inst.
2010;102:605-13. 

22.Levin B, Lieberman DA, McFarland B, Andrews KS, Brooks D, Bond J, et al.
Screening and surveillance for the early detection of colorectal cancer and
adenomatous polyps, 2008: a joint guideline from the American Cancer Society,
the US Multi-Society Task Force on Colorectal Cancer, and the American College
of Radiology. Gastroenterology. 2008;134:1570- 95. 

23.Force USPST. Screening for colorectal cancer: U.S. Preventive Services Task
Force recommendation statement.[see comment][summary for patients in Ann Intern
Med. 2008 Nov 4;149(9):1-44; PMID: 18838719]. Annals of lnternal Medicine.
2008;149:627-37. 

24.Institute NC. International Cancer Screening Network Bethesda, MD2014.
Available from:
http://appliedresearch.cancer.gov/icsn/colorectal/screening.html. 

25.Pox CP, Altenhofen L, Brenner H, Theilmeier A, Von Stillfried D, Schmiege! W.
Efficacy of a nationwide screening colonoscopy program for colorectal cancer.
Gastroenterology. 2012;142:1460-7 e2. 

26.Centers for Disease C, Prevention. Vital signs: colorectal cancer screening
test use-- United States, 2012. MMWR Morb Mortal Wkly Rep. 2013;62:881-8. 

27.Surveillance E, and End Results Program. SEER Stat Fact Sheets: Colon and
Rectum Cancer: Centers for Disease Control; 2014 [cited 2014]. Available from:
http://seer.cancer.gov/statfacts/html/coIorect.html. 

--------------------------------------------------------------------------------

25

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

28.Green BB, Wang CY, Anderson ML, Chubak J, Meenan RT, Vernon SW, et al. An
automated intervention with stepped increases in support to increase uptake of
colorectal cancer screening: a randomized trial. Ann Intern Med.
2013;158:301-11. PMCID: 3953144. 

29.Harewood GC, Wiersema MJ, Melton LJ, 3rd. A prospective, controlled
assessment of factors influencing acceptance of screening colonoscopy. Am J
Gastroenterol. 2002;97:3186-94. 

30.Bettegowda C, Sausen M, Leary RJ, Kinde I, Wang Y, Agrawal N, et al.
Detection of circulating tumor DNA in early- and late-stage human malignancies.
Sci Transl Med. 2014;6:224ra24. PMCID: 4017867. 

31.Hibi K, Robinson CR, Booker S, Wu L, Hamilton SR, Sidransky D, et al.
Molecular detection of genetic alterations in the serum of colorectal cancer
patients. Cancer Res. 1998;58:1405-7 . 

32.Vlems FA, Diepstra JH, Cornelissen IM, Ligtenberg MJ, Wobbes T, Punt CJ, et
al. Investigations for a multi-marker RT-PCR to improve sensitivity of
disseminated tumor cell detection. Anticancer Res. 2003;23:179-86. 

33.Yamaguchi K, Takagi Y, Aoki S, Futamura M, Saji S. Significant detection of
circulating cancer cells in the blood by reverse transcriptase-polymerase chain
reaction during colorectal cancer resection. Ann Surg. 2000;232:58-65. 

34.Zou H, Yu B, Wang Z, Sun J, Cang H, Gao F, et al. Detection of aberrant pl6
methylation in the serum of colorectal cancer patients. Clin Cancer Res.
2002;8:188-91. 

35.Noh YH, Im G, Ku JH, Lee YS, Ahn MJ. Detection of tumor cell contamination in
peripheral blood by RT-PCR in gastrointestinal cancer patients. J Korean Med
Sci. 1999;14:623- 8. 

36.Grady WM, Rajput A, Lutterbaugh JD, Markowitz SD. Detection of aberrantly
methylated hMLHl promoter DNA in the serum of patients with microsatellite
unstable colon cancer. Cancer Res. 2001 ;61:900-2. 

37.Nakayama H, Hibi K, Taguchi M, Takase T, Yamazaki T, Kasai Y, et al.
Molecular detection of p16 promoter methylation in the serum of colorectal
cancer patients. Cancer Lett. 2002;188:115-9. 

38.Verma M, Srivastava S. Epigenetics in cancer: implications for early
detection and prevention. Lancet Oncol. 2002;3:755-63. 

39.Li M, Chen WD, Papadopoulos N, Goodman SN, Bjerregaard NC, Laurberg S, et al.
Sensitive digital quantification of DNA methylation in clinical samples. Nat
Biotechnol. 2009;27:858-63. 

40.Grutzmann R, Molnar B, Pilarsky C, Habermann JK, Schlag PM, Saeger HD, et al.
Sensitive detection of colorectal cancer in peripheral blood by septin 9 DNA
methylation assay. PLoS One. 2008;3:e3759. PMCID: 2582436. 

41.Lofton-Day C, Model F, Devos T, Tetzner R, Distler J, Schuster M, et al. DNA
methylation biomarkers for blood-based colorectal cancer screening. Clin Chem.
2008;54:414- 23. 

42.Hofsli E, Sjursen W, Prestvik WS, Johansen J, Rye M, Trano G, et al.
Identification of serum microRNA profiles in colon cancer. Br J Cancer.
2013;108:1712-9. PMCID: 3668463. 

43.Apweiler R, Hermjakob H, Sharon N. On the frequency of protein glycosylation,
as deduced from analysis of the SWISS-PROT database. Biochim Biophys Acta.
1999;1473:4-8. 

44.Adamczyk B, Tharmalingam T, Rudd PM. Glycans as cancer biomarkers. Biochim
Biophys Acta. 2012;1820:1347-53. 

45.Patwa TH, Zhao J, Anderson MA, Simeone DM, Lubman DM. Screening of
glycosylation patterns in serum using natural glycoprotein microarrays and
multi-lectin fluorescence detection. Anal Chem. 2006;78:6411-21. 

46.Yue T, Maupin KA, Fallon B, Li L, Partyka K, Anderson MA, et al. Enhanced
discrimination of malignant from benign pancreatic disease by measuring the CA
19-9 antigen on specific protein carriers. PLoS One. 2011;6:e29180. PMCID:
3248411. 

47.Zhao J, Patwa TH, Qiu W, Shedden K, Hinderer R, Misek DE, et al. Glycoprotein
microarrays with multi-lectin detection: unique lectin binding patterns as a
tool for classifying normal, chronic pancreatitis and pancreatic cancer sera. J
Proteome Res. 2007;6:1864-74. 

48.Zhao J, Qiu W, Simeone DM, Lubman DM. N-linked glycosylation profiling of
pancreatic cancer serum using capillary liquid phase separation coupled with
mass spectrometric analysis. J Proteome Res. 2007;6:1126-38. 

49.Qiu Y, Patwa TH, Xu L, Shedden K, Misek DE, Tuck M, et al. Plasma
glycoprotein profiling for colorectal cancer biomarker identification by lectin
glycoarray and lectin blot. J Proteome Res. 2008;7:1693-703. 

50.Rho JH, Mead JR, Wright WS, Brenner DE, Stave JW, Gildersleeve JC, et al.
Discovery of sialyl Lewis A and Lewis X modified protein cancer biomarkers using
high density antibody arrays. J Proteomics. 2014;96:291-9. PMCID: 3946870. 

51.Bresalier RS, Byrd JC, Tessler D, Lebel J, Koomen J, Hawke D, et al. A
circulating ligand for galectin-3 is a haptoglobin-related glycoprotein elevated
in individuals with colon cancer. Gastroenterology. 2004;127:741-8. 

52.Scanlan MJ, Welt S, Gordon CM, Chen YT, Gure AO, Stockert E, et al.
Cancer-related serological recognition of human colon cancer: identification of
potential diagnostic and immunotherapeutic targets. Cancer Res. 2002;62:4041-7. 

53.Lu H, Goodell V, Disis ML. Targeting serum antibody for cancer diagnosis: a
focus on colorectal cancer. Expert Opin Ther Targets. 2007;11:235-44. 

54.Nam MJ, Madoz-Gurpide J, Wang H, Lescure P, Schmalbach CE, Zhao R, et al.
Molecular profiling of the immune response in colon cancer using protein
microarrays: occurrence of autoantibodies to ubiquitin C-terminal hydrolase L3.
Proteomics. 2003;3:2108-15. 

--------------------------------------------------------------------------------

26

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

55.Zaenker P, Ziman MR. Serologic autoantibodies as diagnostic cancer
biomarkers--a review. Cancer Epidemiol Biomarkers Prev. 2013;22:2161-81. 

56.Reipert BM, Tanneberger S, Pannetta A, Bedosti M, Poell M, Zimmermann K, et
al. Increase in autoantibodies against Fas (CD95) during carcinogenesis in the
human colon: a hope for the immunoprevention of cancer? Cancer Immunol
Immunother. 2005;54:1038-42. 

57.He Y, Wu Y, Mou Z, Li W, Zou L, Fu T, et al. Proteomics-based identification
of HSP60 as a tumor-associated antigen in colorectal cancer. Proteomics Clin
Appl. 2007;1:336-42. 

58.Chen Y, Lin P, Qiu S, Peng XX, Looi K, Farquhar MG, et al. Autoantibodies to
Ca2+ binding protein Calnuc is a potential marker in colon cancer detection. Int
J Oncol. 2007;30:1137-44. 

59.Liu W, Wang P, Li Z, Xu W, Dai L, Wang K, et al. Evaluation of
tumour-associated antigen (TAA) miniarray in immunodiagnosis of colon cancer.
Scand J Immunol. 2009;69:57-63. 

60.Giovannucci E. Insulin-like growth factor- I and binding protein-3 and risk
of cancer. Horm Res. 1999;51:34-41. 

61.Giovannucci E, Pollak MN, Platz EA, Willet WC, Stampfer MJ, Majeed N, et al.
A prospective study of plasma Insulin-like growth factor and binding protein-3
and risk of colorectal neoplasia in women. Cancer Epidemiol Biomarkers Prev.
2000;9:345-9. 

62.Giovannucci E, Pollak M, Platz EA, Willet WC, Stampfer MJ, Majeed N, et al.
Insulin- like growth factor I (IGF-1), IGF-binding protein-3 and the risk of
colorectal adenoma and cancer in the Nurses’ Health Study. Growth Horm IGF Res.
2000;10 Suppl A:S30-l. 

63.Palmquist R, Stattin P, Rinaldi S, Biessy C, Stenling R, Riboli E, et al.
Plasma insulin, IGF-binding proteins-I and -2 and risk of colorectal cancer: a
prospective study in northern Sweden. Int J Cancer. 2003;107:89-93. 

64.Cruz-Correa M, Cui H, Giardiello FM, Powe NR, Hylind L, Robinson A, et al.
Loss of imprinting of insulin growth factor II gene: a potential heritable
biomarker for colon neoplasia predisposition. Gastroenterology.
2004;126:964-70. 

65.Cui H, Onyango P, Brandenburg S, Wu Y, Hsieh CL, Feinberg AP. Loss of
imprinting in colorectal cancer linked to hypomethylation of Hl 9 and IGF2.
Cancer Res. 2002;62:6442-6. 

66.Cui H, Cruz-Correa M, Giardiello FM, Hutcheon DF, Kafonek DR, Brandenburg S,
et al. Loss ofIGF2 imprinting: a potential marker of colorectal cancer risk.
Science. 2003;299:1753-5. 

67.Woodson K, Flood A, Green L, Tangrea JA, Hanson J, Cash B, et al. Loss of
insulin-like growth factor-II imprinting and the presence of screen-detected
colorectal adenomas in women. J Natl Cancer Inst. 2004;96:407-10. 

68.Tsushima H, Kawata S, Tamura S, Ito N, Shirai Y, Kiso S, et al. High levels
of transforming growth factor beta 1 in patients with colorectal cancer:
association with disease progression. Gastroenterology. 1996;110:375-82. 

69.Tsushima H, Ito N, Tamura S, Matsuda Y, Inada M, Yabuuchi I, et al.
Circulating Transforming Growth Factor beta-I as a predictor of liver metastasis
after resection in colorectal cancer. Clin Cancer Res. 2001;7:1258-62. 

70.Narai S, Watanabe  M, Hasegawa  H, Nishibori H,  Endo T, Kubota  T, et al.
Significance of Transforming gro’-¾1h factor beta 1 as a new tumor marker for
colorectal cancer. Int J Cancer. 2002;97:508-11. 

71.Broll R, Erdmann H, Duchrow M, Oevermann E, Schwandner 0, Markert U, et al.
Vascular endothelial growth factor (VEGF) -- a valuable serum tumour marker in
patients with colorectal cancer? Eur J Surg Oncol. 2001;27:37-42. 

72.Takeda A, Shimada H, Imaseki H, Okazumi S, Natsume T, Suzuki T, et al.
Clinical significance of serum vascular endothelial growth factor in colorectal
cancer patients : correlation with clinicopathological factors and tumor
markers. Oncol Rep. 2000;7:333-8. 

73.Shimoyama S, Yamasaki K, Kawahara M, Kaminishi M. Increased serum angiogenin
concentration in colorectal cancer is correlated with cancer progression. Clin
Cancer Res. 1999;5:1125-30. 

74.Feldman AL, Alexander HR, Jr, Bartlett DL, Kranda KC, Miller MS, Costouros
NG, et al. A prospective analysis of plasma endostatin levels in colorectal
cancer patients with liver metastases. Ann Surg Oncol. 2001;8:741-5. 

75.Simpson RA, Dickinson T, Porter KE, London NJ, Hemingway DM. Raised levels of
plasma big endothelin 1 in patients with colorectal cancer. Br J Surg.
2000;87:1409-13. 

76.Peeters CF, Thomas CM, Sweep FC, Span PN, Wobbes T, Ruers TM. Elevated serum
endothelin-1 levels in patients with colorectal cancer; relevance for prognosis.
Int J Biol Markers. 2000;15:288-93. 

77.Pellegrini P, Contasta I, Berghella AM, Gargano E, Mammarella C, Adorno D.
Simultaneous measurement of soluble carcinoembryonic antigen and the tissue
inhibitor of metalloproteinase TIMP 1 serum levels for use as markers of
pre-invasive to invasive colorectal cancer. Cancer Immunol Immunother.
2000;49:388-94. 

78.Yukawa N, Yoshikawa T, Akaike M, Sugimasa Y, Takemiya S, Yanoma S, et al.
Plasma concentration of tissue inhibitor of matrix metalloproteinase 1 in
patients with colorectal carcinoma. Br J Surg. 2001;88:1596-601. 

79.Barozzi C, Ravaioli M, D’Errico A, Grazi GL, Poggioli G, Cavrini G, et al.
Relevance of biologic markers in colorectal carcinoma: a comparative study of a
broad panel. Cancer. 2002;94:647-57. 

80.Holten-Anderson MN, Christensen IJ, Nielsen HJ, Stephens RW, Jensen V,
Nielsen OH, et al. Total levels of tissue inhibitor of metalloproteinases 1 in
plasma yield high diagnostic sensitivity and specificity in patients with colon
cancer. Clin Cancer Res. 2002;8:156-64. 

--------------------------------------------------------------------------------

27

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

81.Alexiou 0, Karayiannakis AJ, Syrigos KN, Zbar A, Kremmyda A, Bramis I, et al.
Serum levels of E-selectin, ICAM-1 and VCAM-1 in colorectal cancer patients:
correlations with clinicopathological features, patient survival and tumour
surgery. Eur J Cancer. 2001;37:2392-7. 

82.Hayes OF, Smerage JB. Circulating tumor cells. Prog Mol Biol Transl Sci.
2010;95:95- 112. 

83.Wicha MS, Hayes DF. Circulating tumor cells: not all detected cells are bad
and not all bad cells are detected. J Clin Oncol. 2011;29:1508-11. 

84.Lim SH, Becker TM, Chua W, Ng WL, de Souza P, Spring KJ. Circulating tumour
cells and the epithelial mesenchymal transition in colorectal cancer. J Clin
Pathol. 2014;67:848-53. 

85.Zhang Z, Nagrath S. Microfluidics and cancer: are we there yet? Biomed
Microdevices. 2013;15:595-609. PMClD: 4017600. 

86.Stott SL, Lee RJ, Nagrath S, Yu M, Miyamoto OT, Ulkus L, et al. Isolation and
characterization of circulating tumor cells from patients with localized and
metastatic prostate cancer. Sci Transl Med. 2010;2:25ra3. PMCIO: 3141292. 

87.Murlidhar V, Zeinali M, Grabauskiene S, Ghannad-Rezaie M, Wicha MS, Simeone
DM, et al. A radial flow microfluidic device for ultra-high-throughput
affinity-based isolation of circulating tumor cells. Small. 2014;10:4895-904. 

88.Cooper DN. Galectinomics: finding themes in complexity. Biochim Biophys Acta.
2002;1572:209-31. 

89.Dudas SP, Yunker CK, Sternberg LR, Byrd JC, Bresalier RS. Expression of human
intestinal mucin is modulated by the beta-galactoside binding protein galectin-3
in colon cancer. Gastroenterology. 2002;123:817-26. 

90.Mazurek N, Conklin J, Byrd JC, Raz A, Bresalier RS. Phosphorylation of the
beta- galactoside-binding protein galectin-3 modulates binding to its ligands. J
Biol Chem. 2000;275:36311-5. 

91.Schoeppner H, Raz A, Ho S, Bresalier R. Expression of an endogenous
galactose-binding lectin correlates with neoplastic progression in the colon.
Cancer. 1995;75:2818-26. 

92.Sanjuan X, Fernandez PL, Castells A, Castronovo V, van den Brule F, Liu FT,
et al. Differential expression of galectin 3 and galectin 1 in colorectal cancer
progression. Gastroenterology. 1997;113:1906-15. 

93.Lotz MM, Andrews CW, Jr., Korzelius CA, Lee EC, Steele GD, Jr., Clarke A, et
al. Decreased expression of Mac-2 (carbohydrate binding protein 35) and loss of
its nuclear localization are associated with the neoplastic progression of colon
carcinoma. Proc Natl Acad Sci US A. 1993;90:3466-70. 

94.Levi Z, Rozen P, Hazazi R, Vilkin A, Waked A, Maoz E, et al. A quantitative
immunochemical fecal occult blood test for colorectal neoplasia. Ann Intern Med.
2007;146:244- 55. 

95.Shastri YM, Stein J. Quantitative immunochemical fecal occult blood test for
diagnosing colorectal neoplasia. Ann Intern Med. 2007;147:522-3; author reply
3. 

96.Rabeneck L, Rumble RB, Thompson F, Mills M, Oleschuk C, Whibley A, et al.
Fecal immunochemical tests compared with guaiac fecal occult blood tests for
population-based colorectal cancer screening. Can J Gastroenterol.
2012;26:131-47. PMCID: 3299236. 

97.Whitlock EP, Lin JS, Liles E, Beil TL, Fu R. Screening for colorectal cancer:
a targeted, updated systematic review for the U.S. Preventive Services Task
Force. Ann Intern Med. 2008;149:638-58. 

98.Auge JM, Pellise M, Escudero JM, Hernandez C, Andreu M, Grau J, et al. Risk
stratification for advanced colorectal neoplasia according to fecal hemoglobin
concentration in a colorectal cancer screening program. Gastroenterology.
2014;147:628-36 el. 

99.Ahlquist DA, Shuber AP. Stool screening for colorectal cancer: evolution from
occult blood to molecular markers. Clin Chim Acta. 2002;3 l 5:157-68. 

100.Osborn NK, Ahlquist DA. Stool screening for colorectal cancer: molecular
approaches. Gastroenterology. 2005;128:192-206. 

101.Ahlquist DA, Sargent DJ, Loprinzi CL, Levin TR, Rex DK, Ahnen DJ, et al.
Stool DNA and occult blood testing for screen detection of colorectal neoplasia.
Ann Intern Med. 2008;149:441-50, W81. 

102.lmperiale TF, Ransohoff DF, Itzkowitz SH, Turnbull BA, Ross ME. Fecal DNA
versus fecal occult blood for colorectal-cancer screening in an average-risk
population. N Engl J Med. 2004;351:2704-14. 

103.Imperiale TF, Ransohoff DF, Itzkowitz SH, Levin TR, Lavin P, Lidgard GP, et
al. Multitarget Stool DNA Testing for Colorectal-Cancer Screening. N Engl J Med.
2014. 

104.Force USPST. Final Research Plan: Colorectal Cancer Screening 2014.
Available from: http://www.uspreventiveservicestaskforce
.org/Page/Document/ResearchPlanFina l/colorectal­ cancer-screening2. 

105.Chen WD, Han ZJ, Skoletsky J, Olson J, Sah J, MyeroffL, et al. Detection in
fecal DNA of colon cancer-specific methylation of the nonexpressed vimentin
gene. J Natl Cancer Inst. 2005;97:1124-32. 

106.Zou H, Harrington JJ, Shire AM, Rego RL, Wang L, Campbell ME, et al. Highly
methylated genes in colorectal neoplasia: implications for screening. Cancer
Epidemiol Biomarkers Prev. 2007;16:2686-96. 

107.Itzkowitz S, Brand R, Jandorf L, Durkee K, Millholland J, Rabeneck L, et al.
A simplified, noninvasive stool DNA test for colorectal cancer detection. Am J
Gastroenterol. 2008;103:2862-70. 

108.Itzkowitz SH, Jandorf L, Brand R, Rabeneck L, Schroy PC, 3rd, Sontag S, et
al. Improved fecal DNA test for colorectal cancer screening. Clin Gastroenterol
Hepatol. 2007;5:l 1l-7. 

109.Zackular JP, Rogers MA, Ruffin MTt, Schloss PD. The human gut microbiome as
a screening tool for colorectal cancer. Cancer Prev Res (Phila). 2014;7:1112-21.
PMCID: 4221363. 

--------------------------------------------------------------------------------

28

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

110.Kostic AD, Chun E, Robertson L, Glickman JN, Gallini CA, Michaud M, et al.
Fusobacterium nucleatum potentiates intestinal tumorigenesis and modulates the
tumor-immune microenvironment. Cell Host Microbe. 2013;14:207-15. PMCID:
3772512. 

111.Montrose DC, Zhou XK, Kopelovich L, Yantiss RK, Karoly ED, Subbaramaiah K,
et al. Metabolic profiling, a noninvasive approach for the detection of
experimental colorectal neoplasia. Cancer Prev Res (Phila). 2012;5:1358-67.
PMCID: 3518611. 

112.Ahmed FE, Ahmed NC, Vos P\V, Bonnerup C, Atkins JN, Casey M, et al.
Diagnostic microRNA markers to screen for sporadic human colon cancer in stool:
I. Proof of principle. Cancer Genomics Proteomics. 2013;10:93-113. 

113.Link A, Balaguer F, Shen Y, Nagasaka T, Lozano JJ, Boland CR, et al. Fecal
MicroR.”T\l”As as novel biomarkers for colon cancer screening. Cancer Epidemiol
Biomarkers Prev. 2010;19:1766-74. PMCID: 2901410. 

114.Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA, Jr., Kinzler
KW. Cancer genome landscapes. Science. 2013;339:1546-58. PMCID: 3749880. 

115.Lieberman DA, Williams JL, Holub JL, Morris CD, Logan JR, Eisen GM, et al.
Race, ethnicity, and sex affect risk for polyps >9 mm in average-risk
individuals. Gastroenterology. 2014;147:351-8; quiz e14-5. PMCID: 4121117. 

116.Lieberman DA, Holub JL, Morris CD, Logan J, Williams JL, Camey P. Low rate
of large polyps (>9 mm) within 10 years after an adequate baseline colonoscopy
with no polyps. Gastroenterology. 2014;147:343-50. 

117.Dominitz JA, Robertson DJ. Tailoring colonoscopic screening to individual
risk. Gastroenterology. 2014;147:264-6. 

118.Moinova H, Leidner RS, Ravi L, Lutterbaugh J, Barnholtz-Sloan JS, Chen Y, et
al. Aberrant vimentin methylation is characteristic of upper gastrointestinal
pathologies. Cancer Epidemiol Biomarkers Prev. 2012;21:594-600. PMCID: 3454489. 

119.Fedirko V, Bostick RM, Flanders WD, Long Q, Shaukat A, Rutherford RE, et al.
Effects of vitamin D and calcium supplementation on markers of apoptosis in
normal colon mucosa: a randomized, double-blind, placebo-controlled clinical
trial. Cancer Prev Res (Phila Pa). 2009;2:213-23. 

120.Weinstein SJ, Albanes D, Selhub J, Graubard B, Lim U, Taylor PR, et al.
One-carbon metabolism biomarkers and risk of colon and rectal cancers. Cancer
Epidemiol Biomarkers Prev. 2008;17:3233-40. PMCID: 2656360. 

121.Giovannucci E, Stampfer M, Golditz G, Hunter D, Fuchs C, Rosner B, et al.
Multivitamin use, folate, and colon cancer in women in the nurses’ health study.
Ann Intern Med. 1998;129:517-24. 

122.Le Marchand L, White KK, Nomura AM, Wilkens LR, Selhub JS, Tiirikainen M, et
al. Plasma levels of B vitamins and colorectal cancer risk: the multiethnic
cohort study. Cancer Epidemiol Biomarkers Prev. 2009;18:2195-201. 

123.Bruce WR, Giacca A, Medline A. Possible mechanisms relating diet and risk of
colon cancer. Cancer Epidemiol Biomarkers Prev. 2000;9:1271-9. 

124.English DR, Macinnis RJ, Hodge AM, Hopper JL, Haydon AM, Giles GG. Red meat,
chicken, and fish consumption and risk of colorectal cancer. Cancer Epidemiol
Biomarkers Prev. 2004;13:1509-14. 

125.Terry P, Giovannucci E, Michels KB, Bergkvist L, Hansen H, Holmberg L, et
al. Fruit, vegetables, dietary fiber, and risk of colorectal cancer. J Natl
Cancer Inst. 2001;93:525-33. 

126.Trock B, Lanza E, Greenwald P. Dietary fiber, vegetables, and colon cancer:
critical review and meta-analyses of the epidemiologic evidence. J Natl Cancer
Inst. 1990;82:650-6 1. 

127.Vogel V, McPherson R. Dietary epidemiology of colon cancer. Hematol Oncol
Clin North Am. 1989;3:35-63. 

128.Ribas-Barba L, Serra-Majem L, Roman-Vinas B, Ngo J, Garcia-Alvarez A.
Effects of dietary assessment methods on assessing risk of nutrient intake
adequacy at the population level: from theory to practice. Br J Nutr. 2009;101
Suppl 2:S64-72. 

129.Roman-Vinas B, Serra-Majem L, Ribas-Barba L, Ngo J, Garcia-Alvarez A,
Wijnhoven TM, et al. Overview of methods used to evaluate the adequacy of
nutrient intakes for individuals and populations. Br J Nutr. 2009;101 Suppl
2:S6-l 1. 

130.Lipworth L, Bender TJ, Rossi M, Bosetti C, Negri E, Talamini R, et al.
Dietary vitamin D intake and cancers of the colon and rectum: a case-control
study in Italy. Nutr Cancer. 2009;61:70-5. 

131.Jacobs ET, Thompson PA, Martinez ME. Diet, gender, and colorectal neoplasia.
J Clin Gastroenterol. 2007;41:731-46. 

132.Forte A, De Sanctis R, Leonetti G, Manfredelli S, Urbano V, Bezzi M. Dietary
chemoprevention of colorectal cancer. Ann Ital Chir. 2008;79:261-7. 

133.de Vogel S, Bongaerts BW, Wouters KA, Kester AD, Schouten LJ, de Goeij AF,
et al. Associations of dietary methyl donor intake with MLHl promoter
hypermethylation and related molecular phenotypes in sporadic colorectal cancer.
Carcinogenesis. 2008;29:1765-73. 

134.Powers HJ, Hill MH, Welfare M, Spiers A, Bal W, Russell J, et al. Responses
of biomarkers of folate and riboflavin status to folate and riboflavin
supplementation in healthy and colorectal polyp patients (the FAB2 Study).
Cancer Epidemiol Biomarkers Prev. 2007;16:2128- 35. 

135.Slattery ML, Potter JD, Duncan DM, Berry TD. Dietary fats and colon cancer:
assessment of risk associated with specific fatty acids. Int J Cancer.
1997;73:670-7. 

136.Suhar AF, Thompson FE, Kipnis V, Midthune D, Hurwitz P, McNutt S, et al.
Comparative validation of the Block, Willett, and National Cancer Institute food
frequency questionnaires: the Eating at America’s Table Study. Am J Epidemiol.
2001;154:1089-99. 

--------------------------------------------------------------------------------

29

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

137.Hu FB, Rimm E, Smith-Warner SA, Feskanich D, Stampfer MJ, Ascherio A, et al.
Reproducibility and validity of dietary patterns assessed with a food-frequency
questionnaire. Am J Clin Nutr. 1999;69:243-9. 

138.Bloch DA. Comparing two diagnostic tests against the same “gold standard” in
the same sample. Biometrics. 1997;53:73-85. 

139.Lieberman DA, Rex DK, Winawer SJ, Giardiello FM, Johnson DA, Levin TR.
Guidelines for colonoscopy surveillance after screening and polypectomy: a
consensus update by the US Multi-Society Task Force on Colorectal Cancer.
Gastroenterology. 2012;143:844-57. 

140.Ahlquist DA, Zou H, Domanico M, Mahoney DW, Yab TC, Taylor WR, et al. Next­
generation stool DNA test accurately detects colorectal cancer and large
adenomas. Gastroenterology. 2012;142:248-56; quiz e25-6. 

141.Bantis LE, Feng Z. Comparison of two correlated ROC curves at a given
specificity or sensitivity level. Stat Med. 2016;35:4352-67. 

142.Lloyd C, Yong Z. Kernel estimators of the ROC curve are better than
emipircal Statistics and Probability Letters. 1999;44. 

--------------------------------------------------------------------------------

30