1. Multitarget Stool DNA Testing for Colorectal-Cancer Screening NEJM April 3, 2014 Vol 3 Imperiale, T.F. et al Presented by Melissa Spera, MD

2. Background Colon cancer is a major cause of death in the US Simple, non invasive screening with high sensitivity could improve outcomes Colorectal cancer arises from genetic and epigenetic alterations These changes are the basis for analyzing stool to identify tumor specific changes

3. Background Previous stool DNA tests had low sensitivity for detecting advanced carcinomas Moderate sensitivity for colorectal cancers Advances have since been made Previously evaluation of the stool DNA test was performed on archived specimens with a diagnosis

4. Primary Aim To determine the performance characteristics of the DNA test in the detection of colorectal cancer.

5. Secondary Aim To compare the performance of the stool DNA test to the commercially available fecal immunochemical test (FIT) for human hemoglobin in the detection of both colorectal cancer and advanced precancerous lesions

6. Methods-Study Design Cross sectional study Participants enrolled from Jun 2011-Nov 2012 From 90 sites in the US and Canada Private Practice and academic settings Study design by the authors Funded by Exact Sciences Health Decisions-gathered/monitored data

7. Methods-Study Population Asymptomatic people-ages 50-84 years old Average risk for colorectal cancer Scheduled for a colonoscopy Enrollment weighted towards people > 65 Many were excluded

8. Methods-Clinical Procedures Participants provided stool specimen screening colonoscopy w/in 90 days Required to describe: the extent of examination cecal visualization quality of prep Size and location of lesion

9. Methods-Clinical Procedures The most advanced lesion present and its location were used to categorize participants If two similar lesions found, the larger lesion was the index lesion

10. Methods-Clinical Procedure Proximal Colon- the splenic flexure and all colon proximal to it, insertion depth > 60cm, or the phrase “right colon” was used Distal Colon-all other segments than above, insertion depth < 60cm or the phrase “left colon” was used

11. Methods-Clinical Procedures All biopsies and surgical specimens were evaluated at the lab typically used by the institution Concerning lesions were re reviewed Disagreements were resolved by a consensus of at least 2 pathologists

12. Methods-Lab Procedures 3 labs processed all stool samples Exact Sciences (Madison, WI) *Funded Study* Mayo Medical Lab (Rochester, Mn) Molecular Pathology Lab Network (Knoxville, Tn) Labs were blinded to clinical information The same stool sample was used for both tests

13. Multitarget stool DNA test Molecular assays for Aberrantly methylated BMP3 NDRG4 promoter regions Mutant KRAS B actin(reference gene for human DNAquality) IC assay for human hemoglobin

14. FIT(Fecal Immunochemical Test) Performed according to manufacturer instructions All samples sent to a single, blinded lab Samples with > 100ng of hemoglobin per milliliter were considered to be positive

15. Statistical Analysis Study designed to have a power of 90% Hypothesis: DNA test would have sensitivity of 65% or more to detect CR cancer 2 nd Hypothesis: R/O a 5% non inferiority margin for sensitivity for detection of CR cancer with the DNA test compared with FIT

16. Enrollment and Outcomes. Imperiale TF et al. N Engl J Med 2014;370:1287-1297

17. Sensitivity and Specificity of the Multitarget Stool DNA Test and the Fecal Immunochemical Test (FIT) for the Most Advanced Findings on Colonoscopy. Imperiale TF et al. N Engl J Med 2014;370:1287-1297

18. Sensitivity of the Multitarget Stool DNA Test and the Commercial Fecal Immunochemical Test (FIT), According to Subgroup. Imperiale TF et al. N Engl J Med 2014;370:1287-1297

19. Numbers of Persons Who Would Need to Be Screened with Colonoscopy, Multitarget DNA Test, and FIT to Detect One Colorectal Cancer and One Advanced Precancerous Lesion. Imperiale TF et al. N Engl J Med 2014;370:1287-1297

20. Extrapolation of Findings to an Expanded Population of 10,000 Persons at Average Risk for Colorectal Cancer Undergoing Screening with Colonoscopy, Multitarget Stool DNA Test, and FIT. Imperiale TF et al. N Engl J Med 2014;370:1287-1297

21. Discussion Sensitivity of DNA test exceeded FIT by 20% Difference attributed to the DNA markers and algorithm components of the test Both tests hemoglobin component were = FIT was more specific in detecting CRC by 6- 8%

22. Sensitivity Most important characteristic for screening tests Primary role to rule out cancer DNA test exceeded that of FIT in sensitivity A negative result on DNA testing reduced the chance of having CRC to a greater extent than a negative result on FIT

23. Specificity Also very important Affects the number of people who have a positive result A majority of the number with a false positive would be due to the low prevalence of cancer The specificity of FIT was superior to the DNA test

24. Specificity and false positivity Specificity of FIT 94.9-96.4% False positivity of FIT 3.6%-5.1% Specificity of DNA test 86.6-89.8% False positivity of DNA test 10.2-13.4%

25. Two points regarding the specificity of DNA testing The analysis of specificity included those with non advanced adenomas These could cause a positive DNA test But a negative colonoscopy Among patients with a negative colonoscopy the specificity was 90% FIT was still superior at > 96%

26. Second point Specificity correlated inversely with age Among patients with findings other than advanced neoplasia: Specificity of 91.5% for ages 50-64 yo Specificity of 83.7% for ages 65 yo and older But patients 50-64 yo with a negative colonoscopy the specificity was 94% and similar to that of FIT

27. Second point Age related variation in specificity could be due to missed lesions on colonoscopy These are more prevalent among those over 70 This could also be due to age related DNA methylation

28. Conclusions In asymptomatic persons at average risk for colorectal cancer, multitarget stool DNA testing detected significantly more cancers with a higher sensitivity than did FIT but had more false positive results and a lower specificity.