Abstract CN13-03: Next-generation stool DNA testing
Authors: Ahlquist David A. 
Published: January 2011

Abstract
Prevention of colorectal cancer (CRC) by screening hinges on the effective detection of precancers. As no conventional screening method fully prevents cancer across the colorectum, there is justification and incentive to consider alternative and complementary approaches.
Precancers as Targets for Screening. In addition to curable stage CRC, precancers at greatest risk of progression are the critical screening targets. Adenomas likely account for 70-80% of CRC. While roughly 50% of Americans will acquire adenomas in their lifetime, only 6% develop CRC; so most adenomas do not progress. Advanced adenomas (those ≥1cm or containing high-grade dysplasia or prominent villous histology) appear to confer the highest risk and are generally considered to be the relevant subset for screening. Serrated polyps (sessile serrated adenomas, serrated adenomas, large hyperplastic polyps) are increasingly recognized as important precursors to CRC, especially on the right side. They account for 20-30% of CRCs, are often flat and endoscopically inconspicuous, harbor mutated BRAF and multiple hypermethylated genes, may have a more aggressive natural history, and have routinely been excluded or ignored as outcomes in screening studies1, 2. Flat dysplasia, as occurs sporadically and with inflammatory bowel disease, can be especially difficult to detect endoscopically and may often go undetected until CRC develops. Finally, field changes (molecular alterations occurring prior to phenotypic transformation) represent a largely unexplored target for screening.
Limitations of Conventional Screening Tools. The commonly used screening tools have proven to be insensitive for colorectal precancers, especially right-sided ones. Such proximal lesions are above the reach of sigmoidoscopy, which has no effect on incidence of proximal CRC3. Fecal blood tests miss most precancers irrespective of their location4,,5 and have little of no effect on CRC prevention6. Because hemoglobin degrades during colon transit, commonly-used fecal blood tests, such as guaiac cards or FIT, are proportionately less sensitive for right-sided CRC5, 7. And, based on surprising recent reports8,, 9 even colonoscopy as historically practiced may fail to reduce incidence and mortality of right-sided CRC; and this may, in part, be explained by missed serrated polyps.
Stool DNA Testing. As extensively reviewed,10 stool assay of exfoliated molecular markers from stool represents a noninvasive, biologically rational, and feasible strategy that continues to evolve technologically. Based on multicenter studies with early sDNA test approaches7,, 11 the American
Cancer Society and other organizations endorsed sDNA testing for CRC screening12. Next-generation stool DNA (sDNA) test methods incorporate sample stabilization, the requisite analytical sensitivity to detect pre-cancers at high rates, and broad marker coverage. In a recent tissue study, panels of just 3-4 methylated DNA markers provide complete discrimination between normal colon and CRC or adenomas13. Various sDNA test approaches using new technologies in selected patients have detected 81-100% of CRC.
Application of next generation sDNA testing for detection of three critical types of CRC precursors will be the major focus of this brief update. Adenomas. Based on recent reviews10,, 14 various nonoptimized stool DNA tests detect 44-86% of advanced adenomas at specificities of 85-96%. In a comparison on archival stools from a multicenter study,15 analytically sensitive next-generation methods detected significantly more advanced adenomas detection than did early sDNA tests. In a preliminary report using a multimarker next-generation assay,16 we observed that the detection rate increases with size and degree of dysplasia: 62% if ≥1cm, 78% if ≥2cm, and 90% if ≥3cm (comparable to CRC detection); and &gt;80% of adenomas with high-grade dysplasia were detected. In contrast to fecal blood tests which are less sensitive for proximal than distal neoplasms, lesion site does not affect stool DNA test results7. Thus, regardless of anatomic site, adenomas with highest risk of progression appear to be the ones most likely to be detected by sDNA testing.
Serrated Polyps. In a preliminary report,17 our group recently showed that advanced serrated polyps (i.e., those ≥1cm) can be detected noninvasively by sDNA testing. Using archived stools from a multicenter screening study, assay of mutated BRAF, methylated vimentin, or the combination detected 50%, 57%, and 71% of sessile serrated adenomas ≥1cm compared to just 7% by prospectively performed Hemoccult or HemoccultSensa (p&lt;0.01) at comparable specificities. Large prospective studies are clearly needed to corroborate and extend these early findings. IBD-related Dysplasia. Conventional colonoscopy surveillance in patients with inflammatory bowel disease (IBD) has limited sensitivity for both premalignant dysplasia and CRC. In a preliminary report,18 our group presented findings from an ongoing multicenter study evaluating sDNA testing for detection of IBD-related neoplasia. We found that stool assay of methylated EYA4 detected 5/5 (100%) of CRCs and 4/5 (80%) of dysplasia at a 90% specificity (using colonoscopy or resected surgical specimens as criterion standards). Thus, sDNA testing appears to be feasible for the noninvasive detection of neoplasia in setting of IBD and could have an important complementary role in surveillance. Further evaluation of sDNA testing for noninvasive screening of IBD-related neoplasia are warranted
Optimization and clinical validation of multimarker next-generation sDNA tests for detection of CRC, advanced adenomas, and serrated polyps in the screen setting are currently underway.
References:
1. Noffsinger AE. Serrated polyps and colorectal cancer: new pathway to malignancy. Annu Rev Pathol 2009;4:343-64.
2. Lu FI et al. Longitudinal outcome study of sessile serrated adenomas of the colorectum: an increased risk for subsequent right-sided colorectal carcinoma. Am J Surg Pathol;34:927-34.
3. Atkin WS et al. Once-only flexible sigmoidoscopy screening in prevention of colorectal cancer: a multicentre randomised controlled trial. Lancet;375:1624-33.
4. Ahlquist DA et al. Accuracy of fecal occult blood screening for colorectal neoplasia. A prospective study using Hemoccult and HemoQuant tests. Jama 1993;269:1262-7.
5. Morikawa T et al. A comparison of the immunochemical fecal occult blood test and total colonoscopy in the asymptomatic population. Gastroenterology 2005;129:422-8.
6. Heresbach D et al. Review in depth and meta-analysis of controlled trials on colorectal cancer screening by faecal occult blood test. Eur J Gastroenterol Hepatol 2006;18:427-33.
7. Ahlquist DA et al. Stool DNA and occult blood testing for screen detection of colorectal neoplasia. Ann Intern Med 2008;149:441-50, W81.
8. Baxter NN et al. Association of colonoscopy and death from colorectal cancer. Ann Intern Med 2009;150:1-8.
9. Brenner H et al. Protection from right- and left-sided colorectal neoplasms after colonoscopy: population-based study. J Natl Cancer Inst;102:89-95.
10. Ahlquist DA. Molecular detection of colorectal neoplasia. Gastroenterology 2010;138:2127-39.
11. Imperiale TF et al. Fecal DNA versus fecal occult blood for colorectal-cancer screening in an average-risk population. N Engl J Med 2004;351:2704-14.
12. Levin B 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. CA Cancer J Clin 2008.
13. Zou H et al. Sensitive quantification of methylated markers with a novel methylation specific technology. 2010.
14. Ahlquist DA. Next-generation stool DNA testing: expanding the scope. Gastroenterology 2009;136:2068-73.
15. Zou H et al. High detection rates of colorectal neoplasia by stool DNA testing with a novel digital melt curve assay. Gastroenterology 2009;136:459-70.
16. Zou H JX et al. Quantitative stool DNA testing for detection of both colorectal cancer and advanced adenoma. Presented at DDW 2009.
17. Hussain FT et al. Noninvasive Detection of Serrated Colorectal Polyps by Stool Assay of Methylated Vimentin and Mutant BRAF Genes. Presented at DDW 2010.
18. Kisiel JB et al. Noninvasive Detection of Colorectal Neoplasia in Inflammatory Bowel Disease by Stool DNA Testing: A Pilot Study. Presented at DDW 2010.
Citation Information: Cancer Prev Res 2010;3(12 Suppl):CN13-03.