Protocol. This trial protocol has been provided by the authors to give readers additional information about their work.

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1 Protocol This trial protocol has been provided by the authors to give readers additional information about their work. Protocol for: Imperiale TF, Ransohoff DF, Itzkowitz SH, et al. Multitarget stool DNA testing for colorectal-cancer screening. N Engl J Med 2014;370: DOI: /NEJMoa

2 Page 1 of 52 Multi-Target Colorectal Cancer Screening Test for the Detection of Colorectal Advanced Adenomatous Polyps and Cancer: DeeP-C Study Investigational Plan Protocol Study Title: Study Sponsor: DeeP-C Study Exact Sciences 441 Charmany Drive Madison, WI Sponsor Contact: Nada Mlinarevich, MPH, RN Director, Clinical Operations Exact Sciences 441 Charmany Drive Madison, WI (608) January 2012 FOR INVESTIGATIONAL USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. The materials and procedures that are the subject of this clinical study protocol, and any analytical results obtained as a result of execution of this protocol, are for use in product development and regulatory submissions by Exact Sciences Corporation. These materials and procedures and any analytical results obtained cannot be used for diagnostic or prognostic purposes in humans. Use of these materials, procedures, or analytic results obtained from them for any other purpose than product development research is a violation of federal law. The information in this document is provided to you for review as an investigator or potential investigator. By accepting this document, you agree that the information contained herein will not be disclosed to others without written authorization from Exact Sciences Corporation per the separate Non-Disclosure Agreement and/or Materials Transfer Agreement. Results obtained from the execution of this protocol are for use by Exact Sciences Corporation in product development activities. Any other use requires the consent of Exact Sciences Corporation.

3 Page 2 of 52 SIGNATURE PAGE UNIT: Clinical Operations PROTOCOL NO.: Exact Protocol PROJECT NAME: The Exact CRC screening test DATE: 25 January 2012 TITLE: Multi-Target Colorectal Cancer Screening Test for the Detection of Colorectal Advanced Adenomatous Polyps and Cancer: DeeP-C Study SPONSOR: Exact Sciences 441 Charmany Drive Madison, WI INVESTIGATIONAL DEVICE: The Exact CRC screening test APPROVALS: Thomas F. Imperiale, MD Principal Study Investigator Date Graham Lidgard, PhD Chief Scientific Officer Date Nada Mlinarevich, MPH,RN Director, Clinical Operations Date Julie Kesler, MBA Project Manager Date Philip Lavin, PhD Statistician Date

4 Page 3 of 52 I have read the protocol and agree to: INVESTIGATOR S SIGNATURE PAGE Conduct the investigation in accordance with the agreement, the investigational plan, Part 812 and other applicable FDA regulations, applicable state and local laws and regulations, and conditions of approval imposed by the reviewing IRB and FDA; Supervise all testing of the device involving human subjects; Ensure that the requirements for obtaining informed consent are met; Provide sufficient and accurate financial disclosure information and update information if any relevant changes occur during the investigation and for one year following the completion of the study; Maintain the confidentiality of all information received or developed in connection with this protocol; Adhere to the publication policy of Exact Sciences for data collected during this trial. Not initiate this study without approval from the appropriate Institutional Review Board (IRB) and understand that any changes to the protocol must be approved in writing by the Sponsor and the IRB before they can be implemented, except where necessary to eliminate immediate risks to the subject. Signature of Principal Investigator Date Printed Name of Principal Investigator Please return the signed original to: Nada Mlinarevich, Director of Clinical Operations Exact Sciences 441 Charmany Drive Madison, WI PLEASE RETAIN A COPY FOR YOUR TRIAL RECORDS

5 Page 4 of 52 Section 1. Table of Contents Page SIGNATURE PAGE... 2 INVESTIGATOR S SIGNATURE PAGE List of Abbreviations Study Synopsis Introduction Current Approaches to Colorectal Cancer Screening Science of the Exact CRC Screening Test Prior Clinical Evaluation of the Exact CRC Screening Test Study Objectives Primary Objective Secondary Objective Description of the Device Study Population Inclusion Criteria Exclusion Criteria Study Design and Procedures Subject Selection and Enrollment Sample Collection Sample Assays Colonoscopy and Histopathology Procedure Data Collection and Entry Sample Preservation and Banking Study Schedule Study Materials and Device Tracking Materials Provided by Sponsor Materials Provided by the Investigative Site Device Tracking Procedures Statistical Considerations Study Endpoints Definitions Primary Endpoint Secondary Endpoints: Advanced Adenomas Secondary Endpoints: FIT Comparison Sample Size Calculations Statistical Analysis Plan: Primary Objective Statistical Analysis Plan: Secondary Objectives Additional Analyses Other Pre-Specified Analyses Ethical Considerations Risk Analysis... 40

6 Page 5 of Risks to the Subject Benefits to the Subject Justification of the Study Subject Population Scientific Soundness and Analysis of Data Monitoring Procedures Subject Confidentiality Subject Withdrawal from the Study Institutional Review Board Informed Consent InvestigatorObligations Record Retention Compliance Reporting of Adverse Events Serious Adverse Event Protocol Deviation Product Complaint References... 50

7 Page 6 of List of Abbreviations AA ACG ACR ACS AE AGA ASGE CIMP CMS CRA CRO CRC CT DNA EDC EDTA ELISA EUS FAP FDA FIT FN FP FRET GCP HNPCC ICF IRB IFU ITT IVD MSTF NCCN NLR NPV OR PLR PPV QuARTS sdna Se SEER SOP Sp Advanced Adenoma American College of Gastroenterology American College of Radiology American Cancer Society Adverse Event American Gastroenterological Association American Society of Gastroenterological Endoscopy CpG island methylator phenotype Center for Medicare and Medicaid Services Clinical Research Associate Contract Research Organization colorectal cancer computerized tomography deoxyribonucleic acid Electronic Data Capture ethylene diamine tetra-acetic acid Enzyme-linked immunosorbent assay Endoscopic ultrasound familial adenomatous polyposis Food and Drug Administration fecal immunochemical test False-negative False-positive fluorescence resonance energy transfer Good Clinical Practice hereditary non-polyposis CRC syndrome Informed Consent Form institutional review board Instructions for Use intention-to-treat In-vitro diagnostic multi GI-society task force (AGA, ACG, ASGE) National Comprehensive Cancer Network Negative likelihood ratio Negative predictive value Odds ratio Positive likelihood ratio Positive predictive value Quantitative Allele-Specific Real-time Target and Signal amplification stool DNA sensitivity Surveillance, Epidemiology and End Results Standards of Practice specificity

8 Page 7 of 52 TN TNM TP USPSTF True-negative International classification staging system for malignant tumors True-positive US Preventative Services Task Force

9 Page 8 of 52 Study Synopsis Sponsor: Primary Objective: Secondary Objective: Sites: Population: Investigational Device: Primary Endpoint: Exact Sciences Corporation 441 Charmany Drive Madison, WI The primary objective of this study is to determine the sensitivity and specificity of the Exact CRC screening test for colorectal cancer (CRC), using colonoscopy as the reference method. Lesions will be confirmed as malignant by histopathologic examination. The secondary objective of this study is to compare the performance (sensitivity and specificity) of the Exact CRC screening test to a commercially available FIT (OC FIT-CHEK, PolyMedco) ( Polymedco FIT ), both with respect to cancer and advanced adenoma (AA). Lesions will be confirmed as malignant or precancerous by colonoscopy and histopathologic examination. This screening study will be performed at approximately 100 clinical sites. Subjects between the ages of 50 and 84 inclusive, who are at average risk of developing colorectal cancer. Subjects are considered enrolled after understanding and signing the ICF and a determination is made by the Investigator that the subject is eligible to participate according to the inclusion/exclusion criteria. Enrollment will be stopped when a sufficient number of confirmed cases of colorectal cancer have been diagnosed among the study population. Under the current assumptions, approximately 10,500 to 12,000 subjects will need to be enrolled. To increase the point prevalence of colorectal cancer in this study, the study population will be enriched for older subjects; an effort will be made to enroll at least 75% of subjects age Exact CRC screening test, Exact Sciences Corporation The co-primary endpoints for this study are the sensitivity and specificity of the Exact CRC screening test for colorectal adenocarcinoma, with comparison to the colonoscopy result and histopathologic diagnosis of all lesions discovered during colonoscopy

10 Page 9 of 52 and either biopsied or removed during or subsequently removed after colonoscopy. Study Procedures: Study Duration: Subjects at average risk of developing colorectal cancer who are eligible for colorectal cancer screening will be invited to participate. After providing written consent to participate in the study, subjects will be provided with a stool collection kit and will collect stool samples for the Exact CRC screening test and for the commercially-available OC FIT-CHEK assay, distributed by Polymedco Inc. and run exclusively on the OC-Auto Micro 80 machine (referred to as the Polymedco FIT throughout the remainder of this document). The stool samples for the Exact CRC screening test will be sent to a central biorepository for batch testing at one of three central laboratories. The stool sample for the Polymedco FIT assay will be sent to a single central laboratory for testing. Subjects will undergo colonoscopy within 90 days of enrollment. Samples will be assayed by laboratory technicians blinded to the results of colonoscopy and the comparison test, the Polymedco FIT. Representative histopathology slides from tissue biopsied or excised during colonoscopy and representative histopathology slides resulting from subsequent definitive surgery may be retrieved in order to be evaluated by central pathology to confirm diagnosis and staging. Subjects and physicians will remain blinded to the results of the Exact CRC screening test and the Polymedco FIT, which will not be used in clinical management. Participation for each subject will not exceed 90 days between time of enrollment and colonoscopy. Subjects will not be evaluated after colonoscopy; however, if the subject is diagnosed with an advanced adenoma or colorectal cancer and undergoes resection, the surgical/procedure and pathology report will be collected and representative post-surgical histopathology slides may be collected and centrally reviewed. Subjects may be contacted by the investigative site personnel for up to five (5) years following the completion of the study to gather additional information.

11 Page 10 of 52 Introduction 4.1 Current Approaches to Colorectal Cancer Screening Colorectal cancer (CRC) is the second leading cause of death from cancers affecting both men and women in the United States. One in 17 Americans will suffer from CRC during his/her lifetime; the lifetime risk is 35% higher for men than for women. (Jemal, Siegel, Ward, et al, 2010.) Early detection by screening has been shown to reduce CRC mortality (Mandel, Bond, Church, et al, 1993; Hardcastle, Chamberlain, Robinson, et al, 1996; Kronberg, Fenger, Olsen, et al, 1996.) Current guidelines for CRC screening in the average-risk population recommend initiation of screening at age 50, as the incidence of both CRC and premalignant lesions increases sharply after this age. (Levin, Lieberman, McFarland, et al, 2008; SEER database; see table 1.1) Table 4.1. Incidence of CRC per 100,000* Age cohort Incidence/100, *includes all races and both sexes African-Americans have been found to have increased incidence of CRC, with poorer outcomes. Therefore, the recommended age, by the American College of Gastroenterology, to begin screening in this group is 45. (Rex, Johnson, Anderson, et al, 2009.) The US Preventative Services Task Force (USPSTF) recommends against routine screening in subjects aged 76 to 84, with the caveat that judgment should be exercised for healthy elderly subjects. Screening above age 85 is not recommended. (Whitlock, Lin, Liles, et al, 2008.) Detection of precancerous lesions, also known as advanced adenomas, is essential for CRC prevention. Relevant lesions include (Legett, Whitehall, 2010; Kim, Pickhardt, Taylor, 2007; Noffsinger, 2009; Hussain, Yab, Harrington, et al, 2010.): adenoma with carcinoma in situ or high grade dysplasia, any size adenoma, villous growth pattern (> 25%), any size adenoma > 1.0 cm in size, or serrated lesion, > 1.0 cm in size. Serrated lesions (sessile serrated polyps/sessile serrated adenoma) are typically found in the ascending colon, occur more frequently in the elderly, are often flat and inconspicuous endoscopically, and may have a more aggressive natural history than

12 Page 11 of 52 other common adenomas. (Noffsinger, 2009) Conventional screening for CRC includes both invasive and non-invasive options. Invasive tools include flexible sigmoidoscopy, barium enema, CT colonography, and colonoscopy. Colonoscopy is considered to be the most accurate screening tool; its use for screening has expanded rapidly in the past decade. (Whitlock, Lin, Liles, et al, 2008.) Non-invasive CRC screening tools have been limited to fecal occult blood testing. Among these tools, the guaiac-based fecal occult blood test (gfobt) is not specific for human blood, requiring the patient to limit his/her diet for at least two days prior to the test to avoid false positive results. This test has limited sensitivity for CRC and use of this test in large screening studies has yielded only a modest reduction in mortality due to CRC. (Mandel, Bond, Church, et al, 1993; Mandel, Church, Bond, et al, 2000.) A further disadvantage of the gfobt test is the requirement that this test be performed on three separate stool samples. An immunochemical test for fecal occult blood (FIT) offers several advantages over the gfobt test. Because it is based on an antibody reaction specific to human hemoglobin protein rather than on a peroxidase reaction to heme, this test does not require diet restrictions, and is less affected by non-specific upper gastrointestinal bleeding. (Morikawa,Kato,Yamaji,et al, 2005.) However, each manufacturer's anti-hemoglobin antibody identifies a different epitope on the hemoglobin molecule, leading to differences in the performance characteristics between various tests. Many of the manufacturers recommend testing a single stool sample rather than three separate samples as with the gfobt test. In recent comparisons on selected patients or in the screening setting, the sensitivity for CRC of fecal immunochemical tests (FIT) has been higher than that of traditional gfobt tests at comparable or higher specificity. (Whitlock, Lin, Liles, et al, 2008.) FIT has been endorsed for CRC screening in recent multisociety guidelines. (Levin, Lieberman, McFarland, et al, 2008.) and the USPSTF (Whitlock et al 2008.) 4.2 Science of the Exact CRC Screening Test Exact Sciences Corporation (Sponsor) has designed a next-generation stool-based test that detects complementary DNA biomarkers known to be associated with CRC and its precursor lesions as well as fecal hemoglobin. We expect the sensitivity of this test for CRC across all stages to be at least equivalent to currently marketed FIT. (Ahlquist, Zou, Domanico,et al, 2010.) Colonic epithelial DNA exfoliation is a continuous process, in contrast to bleeding, which tends to be intermittent. (Ahlquist, Harrington, Burgart, Roche, 2000.) An abundant variety of DNA biomarkers are generated by malignant and premalignant lesions, providing an opportunity for early CRC and pre-cancer detection with a non-invasive

13 Page 12 of 52 test, using a single stool sample. Although exfoliated tumor markers are present in minute quantities, selective enrichment and amplification techniques permit reliable detection. A panel of complementary informative biomarkers increases the likelihood of detection of cancerous or precancerous lesions, given the molecular heterogeneity of colorectal neoplasia. The Exact CRC screening test utilizes three families of biomarkers that are independent, exhibiting an additive association with CRC and premalignant neoplasms. The first DNA family targets epigenetic changes in the form of gene promoter region methylation. The second DNA family targets specific point mutations. The third family of biomarkers is non-dna based and detects hemoglobin (Exact FIT) in the fecal sample. Promoter region methylation (CpG island methylator phenotype, CIMP) is associated with colorectal carcinogenesis, and provides the basis for the most informative biomarker detected by the Exact CRC screening test. (Markowitz, Bertagnolli, 2009.) For example, early work by Chen et al demonstrated that aberrantly-methylated sequences of the non-expressed vimentin gene can be identified in 53-83% of CRC tissue specimens, as well as in the stool of patients with CRC and in colon cancer cell lines. (Chen, Han, Skoletsky, et al, 2005). This association has been confirmed by others. (Itzkowitz, Jandorf, Rabanek, et al, 2007; Itzkowitz, Brand, Jandorf, et al, 2008.) Other methylated biomarkers considered for Exact s next-generation CRC screening test have been identified in tissues from CRC and pre-malignant lesions including NDRG4, BMP3, and TFP12 (Kim, Lee, Sidransky, 2010; Zou, Harrington, Abdirashid, et al, 2007; Melotte, Lentjes, van den Bosch, et al, 2009; Glockner, Dhir, Yi, McGarvey et al, 2009). Exact demonstrated that these methylated markers could also be identified in stool samples from subjects with CRC and AA. These data were presented at a special meeting of the American Association of Cancer Research (Ahlquist, Zou, Domanico, et al, 2010) detailed below (see section 4.3). Two of these markers, NDRG4 and BMP3 are included in Exact s CRC screening test, which is the subject of this pivotal study. Similarly, KRAS mutations have been described in approximately 35% of CRC (Markowitz, Bertagnolli, 2009.) and DNA with these mutations can also be isolated from the stool of subjects with CRC and AA. (Imperiale, Ransohoff, Itzkowitz,et al, 2004). Seven KRAS mutations in KRAS Exon 2 are included in Exact s CRC screening test, which is the subject of this pivotal study. We have demonstrated that mutant KRAS is complementary to methylation markers for detection of colorectal neoplasia (Ahlquist, Zou, Domanico, et al 2010). Finally, the Exact CRC screening test combines these discriminate methylated DNA and

14 Page 13 of 52 mutation markers with a quantitative test for fecal hemoglobin in a single result. This combination should provide a maximally sensitive screening test. Such an approach was presented at AACR (Ahlquist. Zou, Domanico, et al, 2010), which included four methylation markers (NDRG4, BMP3, TFPI2, and vimentin), KRAS mutations and a determination of fecal hemoglobin using Hemoquant, an assay which measures stool porphyrins (see section 4.3 below). Further analysis of the data presented at AACR led to a final marker panel for the Exact CRC screening test, which is the subject of this pivotal study and which includes two methylation markers (NDRG4, BMP3), high frequency mutations on KRAS, Exon 2 (7) plus a quantitative test for hemoglobin detection using an immunologically based approach (FIT). The Exact CRC screening test is not intended to distinguish between CRC and AA. If any marker or combination of markers is found to be elevated in the stool sample, the subject should be referred to colonoscopy for evaluation. 4.3 Prior Clinical Evaluation of the Exact CRC Screening Test First generation Exact CRC screening tests were evaluated in two large multicenter studies on average risk subjects. In the first study, a prototype multi-marker DNA test detected 52% of invasive cancer, compared with 13% detected by conventional gfobt testing at comparable specificities (p=0.003). (Imperiale, Ransohoff, Itzkowitz, et al, 2004.) In an NIH-funded study, a 3-marker assay performed on single stool samples detected 46% of advanced adenomas, compared with 10% detected by conventional gfobt testing on 3 stool samples (p<0.001), however, specificity was lower with the stool DNA (sdna) test. Stool DNA test results were not affected by neoplasm site, in contrast to conventional gfobt tests. (Ahlquist, Sargent, Loprinzi, et al, 2008.) These first generation sdna tests were performed on un-buffered stool samples, with suboptimal marker panels and assays lacking sufficient analytical sensitivity. Marker stabilization has been achieved by incorporation of a stabilizing buffer in the collection kit, blocking bacterial DNA-ase and preventing DNA degradation. (Olson, Whitney, Durkee, Shuber, 2005.) Various combinations of methylation and mutational markers now achieve essentially 100% of coverage at the tissue level (Zou, Cao, Domanico, et al, 2010). Median stool level of any mutant gene biomarker is about 0.5% of the total gene copies present in the isolated DNA (Diehl, 2008). First generation tests had less sensitive thresholds of around 1% (1 mutant copy per 100 total copies) (Imperiale, Ransohoff, Itzkowitz, et al, 2004). Refined analytic techniques that will be used in this protocol

15 Page 14 of 52 detect biomarker levels well below that of the first generation tests. Current assays detect methylated DNA down to <10 copies and have the ability to discriminate methylated targets within an unmethylated population at 0.01% (1:10,000). (Zou, Cao, Domanico, et al, 2010). Data generated by a prototype assay using these refined analytic techniques were recently presented (Ahlquist. Zou, Domanico, et al, 2010) using four methylation markers (NDRG4, BMP3, TFPI2,vimentin), seven KRAS mutations and the Hemoquant assay for fecal hemoglobin. This assay achieved high discrimination for both CRC and adenomas >1 cm in Training, Test, and Combined Sets. At a specificity cutoff of 90%, the assay achieved sensitivities of 85.3% for CRC and 63.8% for adenomas >1 cm in the Combined Set. CRC detection rate was 87% for curable stages (Stages I-III). The assay was equally effective at detecting right-sided (proximal) and left sided (distal) colorectal neoplasms. The detection rate for CRC proximal to the splenic flexure was 87.2% versus 83.9% for distal CRC (p=0.5). The assay was equally effective in detecting adenomas >1 cm in the proximal versus distal colon (p=1.0). Detection rates were significantly affected by adenoma size p<0.0001, with sensitivity for adenomas >/= 1.0 cm of 54%, >1 cm of 64%, > 2 cm of 79% and > 3 cm of 91%. The risk for malignant progression increases significantly with adenoma size and the assay presented was highly sensitive for the highest risk adenomas. Subject age, gender, race had no effect on neoplasm detection rate (data not presented). Currently, based on the published performance of the first generation stool DNA test, the American Cancer Society (ACS), the Multi-GI Society Task Force (MSTF), and the American College of Radiology (ACR), added sdna testing as a standard of care for early detection of CRC to the national colorectal cancer screening guidelines in 2008.(Levin, Lieberman, McFarland, et al, 2008.) The USPSTF deferred a final recommendation on sdna tests, concluding that additional evidence should be accumulated. (Whitlock, Lin, Liles, et al, 2008.) The 2009 CRC screening guidelines from the American College of Gastroenterology (ACG) supported the ACS/MSTF guideline, considering sdna an alternative CRC detection test with a recommended frequency of every 3 years. (Rex, Johnson, Anderson, et al, 2009.)

16 Page 15 of Study Objectives 5.1 Primary Objective The primary objective of this study is to determine the sensitivity and specificity of the Exact CRC screening test for colorectal cancer (i.e. adenocarcinoma), using colonoscopy as the reference method. Lesions will be confirmed as malignant by histopathologic examination. The primary objective will form the basis for the clinical claim to be used in regulatory submissions. If established, the findings pertaining to the primary objective will be reported to the clinical community in a peer-reviewed publication. 5.2 Secondary Objective The secondary objective of this study is to compare the performance (sensitivity and specificity) of the Exact CRC screening test to a commercially available FIT (OC FIT- CHEK, PolyMedco) ( Polymedco FIT ), both with respect to cancer and advanced adenoma (AA) (see Section ). Lesions will be confirmed as malignant or precancerous by colonoscopy and histopathologic examination. 6 Description of the Device The Exact CRC screening test consists of a stool collection kit and proprietary assay methodologies for detection of the following markers in stool: NDRG4 promoter DNA region hypermethylation BMP3 promoter DNA region hypermethylation KRAS gene DNA point mutations (7), and quantitative hemoglobin. The Exact CRC screening test collection kit includes: Instructions for Use Stool collection container Bracket for stool collection container Bottle of liquid preservative Stool hemoglobin assay tube with stool collection stick with buffer solution (for quantitative hemoglobin component of Exact CRC screening test), Referred to as White Capped Sample Bottle in the Instructions for Use Polymedco FIT stool hemoglobin assay tube with stool collection stick and buffer

17 Page 16 of 52 solution, Referred to as the Green Capped Sample Bottle in the Instructions for Use Return Shipping Box Samples are sent to a centralized biorepository and subsequently to a certified laboratory for processing. Using purified stool DNA, the Exact CRC screening test assay platform uses Quantitative Allele-Specific Real-time Target and Signal amplification (QuARTS ) to identify the biomarkers which exhibit an association with CRC and premalignant neoplasms (advanced adenomas). The QuARTS technology combines the routinely used molecular biology techniques of real-time PCR and Invader chemistry to perform amplification and detection of methylated targets (NDRG4, BMP3) and specific point mutations (KRAS). A quantitative ELISA technique is used to analyze the level of hemoglobin present in the stool sample. This panel of complementary informative markers increases the likelihood of detection of cancerous or precancerous lesions, given the molecular heterogeneity of colorectal neoplasia. 7 Study Population Subjects to be included in this study will include men and women, years (inclusive), of average risk for development of colorectal cancer and asymptomatic of gastrointestinal symptoms warranting diagnostic colonoscopy. Enrollment will be stopped when a sufficient number of confirmed cases of colorectal cancer have been diagnosed among the study population to achieve 80% power for a one-sided test to rule out an Exact CRC screening test disadvantage vs. Polymedco FIT sensitivity; this is estimated to require between 49 and 56 CRC cases based on an assumed difference between Exact CRC and Polymedco FIT sensitivity as well as the percent discordant between the two tests. Based on the assumptions, approximately 10,500 to 12,000 subjects will be enrolled prospectively after meeting all inclusion and exclusion criteria and informed consent is signed, in order to detect and document the targeted number of confirmed colorectal cancer cases. Colorectal cancers will be documented by histopathology following colonoscopy. Subjects will be enrolled at approximately 100 sites. To increase the point prevalence of CRC in subjects recruited for the study, subject enrollment will be age-weighted toward a slightly older population. Enrollment of at least 75% of subjects aged will be targeted. The study population will be drawn from patients who are at average risk for colorectal cancer and who are candidates for colorectal cancer screening. The exclusion criteria

18 Page 17 of 52 listed below include the situations which place a candidate at higher than average risk of colorectal cancer. The investigative sites will include both colonoscopy centers and primary care sites. Because the proportion of subjects at primary care sites eligible for colon cancer screening is relatively low, it is expected that the majority of subjects will be enrolled at colonoscopy centers. 7.1 Inclusion Criteria Subjects enrolled into the study must meet all of the following criteria: Subject is average risk for development of colorectal cancer (as defined by the inclusion and exclusion criteria below) Subject able and willing to undergo a screening colonoscopy within 90 days of enrollment Subject is 50 to 84 years of age inclusive Subject is able to comprehend, sign, and date the written informed consent document to participate in the study Subject is able and willing to provide stool samples according to written instructions provided to them 7.2 Exclusion Criteria Subjects will be excluded from enrolling into the study if any of the following criteria are met: Subject has any condition which, in the opinion of the investigator should preclude participation in the study Subject has undergone colonoscopy within the previous nine (9) years Subject has undergone any double-contrast barium enema, virtual (CT-based) colonoscopy, or flexible sigmoidoscopy within the previous five (5) years Subject has a history of colorectal cancer or adenoma Subject has a history of aerodigestive tract cancer Subject has had a positive fecal occult blood test or FIT within the previous six (6) months Subject has had a prior colorectal resection for any reason other than sigmoid diverticular disease Subject has had overt rectal bleeding, e.g., hematochezia or melena, within the

19 Page 18 of 52 previous 30 days. (Blood on toilet paper, after wiping, does not constitute rectal bleeding) Subject has a diagnosis or personal history of any of the following high-risk conditions for colorectal cancer: Inflammatory bowel disease (IBD) including chronic ulcerative colitis (CUC) and Crohn's disease. > 2 first-degree relatives who have been diagnosed with colon cancer. (Note: first-degree relatives include parents, siblings and offspring). One first-degree relative with CRC diagnosed before the age of 60. Familial adenomatous polyposis (also referred to as "FAP", including attenuated FAP). Hereditary non-polyposis colorectal cancer syndrome (also referred to as "HNPCC" or "Lynch Syndrome"). Other hereditary cancer syndromes including but are not limited to Peutz Jeghers Syndrome, MYH-Associated Polyposis (MAP), Gardner's Syndrome, Turcot's (or Crail s) Syndrome, Cowden's Syndrome, Juvenile Polyposis, Cronkhite-Canada Syndrome, Neurofibromatosis and Familial Hyperplastic Polyposis Subject has a family history of: Familial adenomatous polyposis (also referred to as "FAP"). Hereditary non-polyposis colorectal cancer syndrome (also referred to as "HNPCC" or "Lynch Syndrome") Participation in any interventional clinical study within the previous 30 days in which an experimental treatment is administered or might be administered through a randomized assignment of the subject to one or more study groups. 8 Study Design and Procedures This is a prospective, cross-sectional, multi-center study to determine the sensitivity and specificity of the Exact CRC screening test for malignant lesions and advanced adenomas (AA) of the colon and rectum in the average-risk screening population. Prior to initiation of bowel preparation for their screening colonoscopy, all subjects will capture a single stool sample into a plastic collection container that is fitted over the toilet seat. Subjects will then follow collection instructions for properly gathering stool specimens for the Polymedco FIT and the Exact CRC screening test, as outlined in the subject Instruction for Use (IFU). The subject will undergo a colonoscopy within 90 days of enrollment into the study and the results of the Exact CRC screening test will be

20 Page 19 of 52 compared with the results of the colonoscopy and with any corresponding diagnostic histopathology results from clinically significant biopsied or subsequently excised lesions. The sensitivity and specificity of the Exact CRC screening test will also be compared with the results of the Polymedco FIT. Samples collected by the subject for the Exact CRC screening test will be sent to a biorepository and subsequently to one of three laboratories, each of which will assay specimens from approximately 1/3 of the study subjects. The stool sample for the Polymedco FIT will be sent to a single testing laboratory. Laboratory personnel will be blinded to the results of the colonoscopy and the comparator FIT test, and physicians performing the colonoscopy will be blinded to the results of the Exact CRC screening test and Polymedco FIT. Investigators and subjects will remain blinded to the Exact CRC screening test result and the Polymedco FIT result, and the results of these tests will not be used for clinical management of study subjects. Subject enrollment will continue until the study achieves 80% power to test the noninferiority hypothesis to rule out a 5% disadvantage in sensitivity for the Exact CRC screening test vs. Polymedco FIT according to a test with one-sided 5% Type I error. This is estimated to require between 49 and 56 CRC cases based on an assumed difference between Exact CRC and Polymedco FIT sensitivity as well as the percent discordant between the two tests. Based on the assumptions, approximately 10,500 to 12,000 subjects will be enrolled prospectively to achieve a sufficient number of confirmed colorectal cancers; the final count depends on the underlying assumptions for the comparison between the Exact CRC screening test and Polymedco FIT. Although no long-term follow-up is planned, subjects may be contacted by the investigative site personnel up to five (5) years following the completion of the study to gather additional information. 8.1 Subject Selection and Enrollment Sites will be expected to draw subjects through their normal patient referral channels. Subjects will be identified by site personnel and approached for participation in the study. After the study has been described by the clinical investigator or his/her designee and all questions answered, written informed consent will be obtained from those patients who have agreed to participate. Every enrolled subject at each investigative site will be assigned a unique subject identification number (subject ID). All reports and other documents, samples or histopathology slides collected during the study will be de-identified. The subject s initials and ID will be placed on all de-identified subject documents.

21 Page 20 of 52 Prior to subject recruitment into this study, Institutional Review Board (IRB) approval of this protocol, informed consent form (ICF), and any information supplied to the subject will be obtained. Subjects will be provided with the most currently IRB approved ICF and will have the opportunity to read, understand and have their questions answered prior to signing the ICF. The subject must sign and date the ICF prior to any studyrelated procedures being performed. The person reviewing the ICF with the subject will also sign and date the ICF. The subject will be provided with a copy of the signed ICF while the original ICF will be retained on site with the subject s records. Subjects are considered enrolled after understanding and signing the ICF and a determination is made by the Investigator that the subject is eligible to participate according to the inclusion/exclusion criteria. There will be no waivers or exceptions for any subject attempting to participate in this study. If the Investigator and site should have any questions regarding the eligibility of a subject to participate, they are to contact the Director of Clinical Operations at Exact Sciences, Nada Mlinarevich, by telephone ( ). If allowed by the investigative site and/or the IRB, subjects will be modestly compensated for their time, inconvenience and expenses associated with their participation in this study. 8.2 Sample Collection After a subject has signed the ICF, been qualified and enrolled into the study, the subject will be provided with all materials necessary to collect stool samples for the Exact CRC screening test, and for the Polymedco FIT. The obtaining of this specimen must be completed before the subject has initiated bowel preparation for their colonoscopy. Collection materials will be labeled with a unique barcode number which will be linked to the subject when dispensed. Detailed instructions are located in the Study Manual. Per the subject s Instructions for Use (IFU), stool samples will be collected by the subject at their convenience, prior to the initiation of the colon prep for the colonoscopy procedure. Once the sample collection is complete, per the IFU the subject is instructed to immediately contact the Call Center. Subjects will inform the Call Center operator their stool is ready for pick-up. The subject will be asked a series of questions referencing the date and time of the stool collection and the subject s experience with the collection process. The Call Center will arrange for a designated courier to pick-up the specimen. Following courier pick-up, the samples collected for the Exact CRC screening test will be sent to a central biorepository for cryopreservation and will subsequently be sent to

22 Page 21 of 52 one of the three designated Exact Study testing laboratories. The Polymedco FIT will be sent to and tested by a single, central, commercial laboratory. The shipment and arrival to Exact Sciences of the sample collection kit will be tracked using a database server provided by the Contract Research Organization (CRO). Once the sample arrives at Exact Sciences, it will be assessed for evaluability. If the stool sample is found to not meet sponsor requirements as defined in Sponsor s stool processing manual for this study, the site will be immediately notified. The site will be asked to contact the subject to request another stool specimen if collection can occur within the protocol defined window period and prior to the initiation of bowel preparation for their scheduled colonoscopy. At that time, another stool collection kit will be dispensed to the subject by the investigative site. If the subject encounters any issues with the stool collection kit, (including missing components, broken pieces, etc.),which would preclude them from successfully collecting their stool specimen, the subject will be instructed per the IFU to contact the Call Center to arrange for a pick-up of the defective kit. The investigative site should not dispense another collection kit until the original defective kit is out of the subject s possession. The same rules should apply in the event any of the stool collection kit components are lost or misused by the subject. 8.3 Sample Assays All samples will be assayed, following manufacturer's instructions for use, by the designated study laboratories. Exact CRC screening test samples may be stored at - 80 C and tested in batches at various time points during the study. Trained technicians at the designated study laboratories will be blinded to results of the colonoscopy. Different technicians will perform the Polymedco FIT and the Exact CRC Screening Test. The trained technicians performing the Polymedco FIT will be blinded to the results of the Exact CRC screening test, and the trained technicians performing the Exact CRC screening test will be blinded to the results of the Polymedco FIT. Care will be taken to ensure that the proper subject ID is associated with all results. Samples which are insufficient for testing (e.g., not enough stool, sample damaged in transit, etc.) will be tallied and reported by the sponsor. For samples that are processed, assay results for the Exact CRC screening test will be recorded as 'positive', 'negative', or sample processed, no result for each individual DNA or hemoglobin target. A sample processed, no result occurs when a step in the analytic process of a target fails to meet the quality metrics of the manufacturer. (This may occur, for example, when the run controls fail. Such samples will not be included in respective analyses).

23 Page 22 of 52 The commercially available Polymedco FIT is quantitative. Results will be compared to a pre-determined threshold, according to manufacturer's instructions. If any of the stool samples received by the central biorepository are of insufficient quantity or not received in the appropriate condition, the site will immediately be notified. The site will contact the subject to attempt to have the subject obtain another stool collection prior to initiation of the bowel preparation for the colonoscopy procedure. If the subject needs to perform another stool collection, the entire collection process must be performed again since the stool collection for the Polymedco FIT and the Exact CRC screening test must be from the same stool sample. If this is not possible, the subject s data will be deemed unevaluable and will not be used in any analyses. If an event should occur where the Exact CRC screening test meets the requirement for testing but the commercially available Polymedco FIT does not, the sample will be assessed to be acceptable for the primary endpoint analysis but not for the comparative endpoint analysis; no further requests will be made of the investigative site and/or the subject. 8.4 Colonoscopy and Histopathology Procedure Every subject will undergo a colonoscopy within 90 days of signing consent. Bowel preparation procedures and colonoscopy will be performed following standard of care at each clinical site. Results of the colonoscopy examination and, if done, histopathology on any excised or biopsied lesion, will be recorded on the appropriate case report form, taking care to ensure that the correct subject s initials and study ID number are recorded. All findings (i.e. colonoscopy report, pathology report, any radiology reports) from the colonoscopy must be collected, de-identified, labeled with the subject ID (per the Study Manual) and sent to the CRO. Personnel performing the colonoscopy and producing the resulting report or the personnel performing the histopathological review of tissue will remain blinded to the results of the Exact CRC Screening Test and the Polymedco FIT. If the Polymedco test cannot be run, the primary endpoint will still be analyzed. The colonoscopist will record the quality of the bowel preparation (after washing/suctioning) as follows: Excellent: minimal fecal residue and liquid, small volume of liquid easily aspirated, <5% of colonic surface covered; Good: small amounts of fecal residue, volume of clear liquid covering 5-25% of surface, easily aspirated; Fair: moderate amounts of fecal residue; stool limited examination but >90%

24 Page 23 of 52 could be examined; or Poor: large amounts of stool, <90% of surface could be examined. Cecal intubation will be documented with photographic evidence and/or documentation of cecal intubation; and colonoscope withdrawal time will be recorded. Withdrawal time is defined as the time from cecal identification to the time when the colonoscope is withdrawn across the anus (Barclay, Vicari, Doughty, et al, 2006.). A bowel preparation considered excellent, good, or fair will be acceptable. Subjects with no findings on the colonoscopy, and no photographic evidence of cecal intubation or no documentation of cecal intubation in the report, will be deemed to have a failed colonoscopy. These subjects will be deemed to be unevaluable and therefore will be excluded from analysis of all primary and secondary endpoints. Those subjects determined to have had a poor bowel preparation (after washing/suctioning), may repeat the colonoscopy again if the Investigator feels this is in the best interest for the subject. The subject may repeat the colonoscopy procedure as a study research subject only if the second procedure falls within the 90 day window period. A repeat stool specimen is not required. A completed colonoscopy procedure will be considered and defined as: reaching the cecum (or) reaching the junction between the small and large intestine (in the event the cecum has been resected) (or) reaching the neo-cecum. A colonoscopy that was limited in any way but wherein a colorectal cancer or advanced adenoma was identified or wherein a partially obstructing colorectal cancer or advanced adenoma was identified, will be included in the analyses. Colonoscopy findings will be recorded on the colonoscopy reports per site specific standard of practice. Histopathology interpretation of tissue samples will be conducted according to normal practice of each clinical site. Copies of all colonoscopy reports, histopathology reports and surgical reports and any other reports requested (e.g. radiology reports such as EUS) will be submitted to the CRO via a courier at specified times during the course of the study. Refer to the Study Manual for specifics. All subject identifying information on the documents will be obliterated, and labeled with the subject s initials and ID number as described in the Study Manual. Reports of histopathologic interpretation of endoscopic biopsies, polypectomy specimens, and excisional surgical pathology specimens should be available on all CRCs and advanced adenomas (AAs). Lesion location within the colon (site), size (cm), and TNM stage and overall CRC Stage (I-IV) will be recorded for all CRC s and the subject will be classified as category (i), regardless of the presence of any other

25 Page 24 of 52 adenomas or hyperplastic polyps. Similarly, in the absence of CRC, a subject with AA will be categorized as category (ii) regardless of the presence of any other adenomas or hyperplastic polyps. With category (ii), subjects will be categorized by the most clinically significant AA. For this study, the hierarchy of clinical significance in descending order of significance is 2.1 (highest clinical significance), then 2.2, then 2.3, and then 2.4 (lowest clinical significance). In summary, subjects will be placed in a single category for this study, based on the most clinical significant lesion confirmed on histopathologic analysis. The most clinically significant lesion is hereafter referred to as the index lesion and is the lesion upon which the subject categorization is made. Below is the listing of categories that the central pathologist will use when assessing and determining final categorization: i. CRC, all stages (I-IV); ii. Advanced adenoma, including the following subcategories: adenoma with carcinoma in situ or high grade dysplasia, any size (subcategory 2.1) adenoma, villous growth pattern (> 25%), any size (subcategory 2.2) adenoma > 1.0 cm in size (subcategory 2.3) serrated lesion, > 1.0 cm in size (subcategory 2.4); iii. 1 or 2 adenoma (s), > 5 mm in size, or < 10 mm/1.0 cm size, non advanced; iv. > 3 adenomas, < 1 cm in size, non-advanced; v. 1 or 2 adenoma(s), < 5 mm in size, non-advanced; vi. negative no neoplastic findings. For cases without CRC, the location within the colon (site) and size (cm) of the most clinically significant lesion, the index lesion, will be recorded. Subjects with adenomas with advanced features will be classified as category (ii) and subcategorized by the most advanced feature present in the lesion in the following hierarchy; adenoma highgrade dysplasia/cis any size (subcategory 2.1); adenoma, villous component >/= 25% any size (subcategory 2.2); adenoma size >/= 1.0 cm without high grade dysplasia or villous component >/= 25% (subcategory 2.3); serrated lesion >/= 1.0 cm (subcategory 2.4). The presence of multiple advanced adenomas does not change the classification of a patient as category (ii). Subjects with pathologically confirmed non-advanced adenoma(s) but without CRC (i) or advanced adenoma (ii) will be classified by the reviewing pathologists into category iii, iv, or v based on the size and number of lesions. The largest of the non-advanced adenomas will be the index lesion for the case. In the case of subjects with both small (<10 mm) non-advanced adenomas and hyperplastic polyps < 10 mm, they will be classified based on the non-advanced adenomas only and will be classified into category iii, iv, or v.

26 Page 25 of 52 All subjects who do not have CRC (i), advanced adenoma (ii), small, or non-advanced adenoma (iii, iv, or v) will be classified as negative (vi). Category vi includes subjects with any number of hyperplastic polyps <10mm and the largest hyperplastic polyp is the index lesion In the absence of hyperplastic polyps, subjects in category vi will either have no index lesion or will have no pathology submitted. Subjects who have lesions identified on colonoscopy that are neither biopsied initially nor excised subsequently or that are lost during lesion retrieval and, therefore, not submitted for histopathologic analysis, will be identified by the Sites and the colonoscopy report will be sent to the study pathologists for review. Because of the absence of confirmatory histopathologic diagnosis, these subjects will be excluded from the study based on the fact that they cannot be not be categorized for the study analysis. Sites with subjects diagnosed with CRCs or AAs may be required by the Central Pathologist to provide representative histology slides. Refer to Pathology Study Manual for details. Several measures will be taken to ensure high quality colonoscopy in this study. All colonoscopists will be experienced and board-certified, be advised to view an ASGE instructive video addressing refinements in the diagnostic approach to detection of flat proximal lesions, and be monitored with respect to their withdrawal times. As above, cecal intubation rates and recording of preparation quality will also be tracked. 8.5 Data Collection and Entry For the purposes of this study, all subject data represented by the subject s initials and identification (ID) number will be entered in an electronic data capture (EDC) database. Properly trained investigative site personnel will enter all data onto electronic case report forms, utilizing their assigned user names and passwords. Detailed instructions for data entry may found in the Study Manual. An independent, central pathologist(s) will be provided with de-identified colonoscopy histopathology and any other available reports requested by the pathologist that may assist in CRC staging (e.g. EUS) associated with the colonoscopy or subsequent lesion excision for all subjects that have had endoscopic biopsies and/or polypectomies and/or definitive excisional surgery. The central pathologist(s) will extract the required data from these reports and enter data into the EDC, following detailed instructions in the icrf instructions. Reports of histopathologic interpretation should be available on tissue removed during the colonoscopy (i.e. biopsies, polypectomies, etc.). For subjects that undergo surgical removal of CRC and/or non-crc lesions, surgical