Ahlquist, David A.

Patients with inflammatory bowel disease (IBD) are at increased risk for colorectal cancer (CRC). Despite weak supporting evidence, important logistic barriers and high cost, colonoscopy is currently the only recommended approach to CRC surveillance in patients with IBD. As such, there is imperative to explore alternative or complementary strategies with potential to improve the efficiency and effectiveness of surveillance in IBD. Given our increasing understanding of tumorigenesis in IBD and the accompanying cascade of molecular alterations, there is a strong rationale to pursue biomarker assays for this application. Stool-based DNA testing with advanced technology has been shown to be highly discriminatory for detection of sporadic colorectal cancer and advanced precancers. In early observations, stool DNA testing also shows promise for the accurate detection of IBD-associated colorectal neoplasms. These findings raise important clinical and translational questions about how to best evaluate and develop this technology, and devise clinical algorithms that will complement colonoscopy to improve patient outcomes.

SummaryBackgroundCurrent approaches to the detection of colorectal neoplasia associated with inflammatory bowel disease (IBD‐CRN) are suboptimal.AimTo test the feasibility of using stool assay of exfoliated DNA markers to detect IBD‐CRN.MethodsThis investigation comprised tissue and stool studies. In the tissue study, gene sequencing and methylation assays were performed on candidate genes using tissue DNA from 25 IBD‐CRNs and from 25 IBD mucosae without CRN. Mutations on p53, APC, KRAS, BRAF or PIK3CA genes were insufficiently informative, but several aberrantly methylated genes were highly discriminant. In the stool study, we evaluated candidate methylated genes (vimentin, EYA4, BMP3, NDRG4) in a prospective blinded study on buffered stools from 19 cases with known IBD‐CRN and 35 age‐ and sex‐matched IBD controls without CRN. From stool‐extracted DNA, target genes were assayed using quantitative allele‐specific real‐time target and signal amplification method.ResultsIBD‐CRN cases included 17 with ulcerative colitis (UC) and two with Crohn's disease (CD); nine had cancer and 10 had dysplasia. Controls included 25 with UC and 10 with CD. Individually, BMP3, vimentin, EYA4 and NDRG4 markers showed high discrimination in stools with respective areas under the ROC curve of 0.91, 0.91, 0.85 and 0.84 for total IBD‐CRN and of 0.97, 0.97, 0.95 and 0.85 for cancer. At 89% specificity, the combination of BMP3 and mNDRG4 detected 9/9 (100%) of CRC and 80% of dysplasia, 4/4 (100%) of high grade and 4/6 (67%) of low grade.ConclusionThese findings demonstrate the feasibility of stool DNA testing for non‐invasive detection of colorectal neoplasia associated with inflammatory bowel disease.

Abstract Aberrantly methylated genes represent attractive and broadly informative markers for colorectal cancer (CRC). As demographics, numerous ingestants, environmental exposures, and obesity variably influence both gene methylation rates and CRC incidence, it is important to understand the effects of such covariates on the clinical performance of CRC screening tests that incorporate assay of methylated gene markers. Based on a recent multicenter study (Ahlquist et al. Gastroenterology 2012 (in press)), stool assay of carefully selected methylated gene marker candidates (NDRG4, BMP3, vimentin, TFPI2), alone or in combination, yield high detection rates of both CRC and large adenomas. Aim: To assess the impact of demographic, exposure, body mass, and other patient variables on stool levels of the above 4 methylated gene markers. Methods: We studied buffered stools collected within 3 years of a normal colonoscopy from 500 patients undergoing average-risk screening or polyp surveillance (median age 64 (range 44-85); 53% women). Upon receipt, stools were promptly homogenized, aliquoted, and frozen at –80°C. On supernatants from thawed/spun aliquots, target genes were purified by hybrid capture, bisulfite treated, and assayed using the analytically-sensitive QuARTS method (quantitative allele-specific real-time target and signal amplification), as described (above citation). The reference gene β-actin was assayed along with methylation of NDRG4, BMP3, vimentin, TFPI2. Log-converted data were normalized to allow comparison of effects between markers, and effect size was expressed as % change relative to standard deviation for each marker (standardized relative change (SRβ)). Results: The only patient characteristic that significantly influenced all methylated marker levels in stool was age (p<0.0001 for each marker); SRΔ was greatest with TFPI2 at +91.3 (p<0.001 vs other markers), least with BMP3 at +29.7 (p<0.001 vs others), and intermediate with vimentin at +46.0 and NDRG4 at +45.1. In contrast to methylation markers, levels of β-actin did not change across age. The other demographic variables of sex, race, and geographic residence had no effect on methylation markers. Marker levels were also not affected by exposure variables (smoking, alcohol consumption, or analgesic use), by family history of CRC or polyps, or by personal history of polyps. Finally, neither overweight status (n=176) nor obesity (n=148) had an effect on marker levels. Conclusions: Age significantly affects stool levels of tumor-associated methylated gene markers in colonoscopy-normal patients, and the degree of this age effect differs across markers. In contrast, other key patient variables had no effect on stool marker levels. Comment: Optimal use of these methylation markers for stool screening of CRC may require age-adjustment of normal cutoff levels to maximize test specificity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3572. doi:1538-7445.AM2012-3572

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States, yet it is potentially the most treatable and preventable cancer with effective screening. Exact Sciences has developed a multi-biomarker CRC screening test that targets DNA methylation, DNA mutations, and hemoglobin in stool. The aim of this study was to evaluate the analytical accuracy of a novel assay for the detection of KRAS mutation sequences at Codons 12 and 13 in well-characterized colorectal tissues. Methodology: A multiplexed KRAS assay was designed utilizing QuARTS (Quantitative Allele-specific Real-time Target and Signal amplification), a highly sensitive technology that combines allele-specific DNA amplification with invasive cleavage chemistry to generate signal during each amplification cycle similar to real-time PCR. The assay, which detects seven KRAS mutations and the reference gene β-actin, was used to assess 87 colorectal tissue samples (52 CRCs, 16 adenomas ≥ 1cm, and 19 normal epithelia) as determined by Mayo Clinic Pathology. Samples were obtained by microdissection of fresh frozen tissue biopsies. DNA was extracted by Mayo Clinic using a standardized chloroform/phenol methodology. The genotypes of each sample were established using dye terminator dideoxy sequencing in both the forward and reverse orientations. Copy numbers of KRAS mutations and β-actin were determined by conventional comparison against standard curves. KRAS data are reported as percent mutation and calculated by dividing mutant copies by β-actin copies and multiplying by 100. Results: Based on sequencing data: the 52 CRC samples contained 22 KRAS mutations and 30 wild-type genotypes, the 16 adenomas ≥ 1cm contained 8 mutations and 8 wild-type genotypes, and the 19 normal tissues contained all wild-type genotypes. The QuARTS assay detected 100% of the KRAS mutations in the CRC and adenomas and provided excellent differentiation between wild-type and mutation, with the highest percent KRAS mutation of normal wild-type samples at 0.55% and the lowest percent mutation of KRAS positive samples at 8.34%. Based on this data, this assay has the potential to be more sensitive analytically than standard sequencing. Conclusion: The QuARTS assay detects KRAS mutations in tissue with high sensitivity and specificity. This assay method could provide an accurate and high throughput approach to detection of KRAS mutations in biospecimens for multiple clinical applications. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A22.

Abstract BACKGROUND Aberrantly methylated genes represent important markers for cancer diagnosis. We describe a multiplex detection approach to efficiently quantify these markers for clinical applications such as colorectal cancer screening. METHODS Quantitative allele-specific real-time target and signal amplification (QuARTS) combines a polymerase-based target amplification with an invasive cleavage-based signal amplification. The fluorescence signal is detected in a fashion similar to real-time PCR. We measured the dynamic range and analytical sensitivity of multiplex QuARTS reactions with titrated plasmid DNA. We used the QuARTS technology to quantify methylated BMP3, NDRG4, VIM, and TFPI2 genes on 91 DNA samples extracted from colorectal tissues, including 37 cancers, 25 adenomas, and 29 healthy epithelia. The assays were designed in triplex format that incorporated ACTB as a reference gene. Percent methylation was calculated by dividing methylated strands over ACTB strands and multiplying by 100. RESULTS The QuARTS method linearly detected methylated or unmethylated VIM gene down to 10 copies. No cross-reactivity was observed when methylated assays were used to amplify 105 copies of unmethylated gene and vice versa. The multiplex assay detected methylated genes spiked in unmethylated genes at a 0.01% ratio and vice versa. At a diagnostic specificity cutoff of 95%, methylated BMP3, NDRG4, VIM, and TFPI2 detected 84%, 92%, 86%, and 92% of colorectal cancers and 68%, 76%, 76%, and 88% of adenomas, respectively. CONCLUSIONS The QuARTS technology provides a promising approach for quantifying methylated markers. The markers assayed highly discriminated colorectal neoplasia from healthy epithelia.

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 >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<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.