Ahlquist, David A.

Abstract Background: Adenomatous polyps are the most common precursor to colorectal cancer, the second leading cause of cancer-related death in the United States. We sought to learn more about early events of carcinogenesis by investigating shifts in the gut microbiota of patients with adenomas. Methods: We analyzed 16S rRNA gene sequences from the fecal microbiota of patients with adenomas (n = 233) and without (n = 547). Results: Multiple taxa were significantly more abundant in patients with adenomas, including Bilophila, Desulfovibrio, proinflammatory bacteria in the genus Mogibacterium, and multiple Bacteroidetes species. Patients without adenomas had greater abundances of Veillonella, Firmicutes (Order Clostridia), and Actinobacteria (family Bifidobacteriales). Our findings were consistent with previously reported shifts in the gut microbiota of colorectal cancer patients. Importantly, the altered adenoma profile is predicted to increase primary and secondary bile acid production, as well as starch, sucrose, lipid, and phenylpropanoid metabolism. Conclusions: These data hint that increased sugar, protein, and lipid metabolism along with increased bile acid production could promote a colonic environment that supports the growth of bile-tolerant microbes such as Bilophilia and Desulfovibrio. In turn, these microbes may produce genotoxic or inflammatory metabolites such as H2S and secondary bile acids, which could play a role in catalyzing adenoma development and eventually colorectal cancer. Impact: This study suggests a plausible biological mechanism to explain the links between shifts in the microbiota and colorectal cancer. This represents a first step toward resolving the complex interactions that shape the adenoma–carcinoma sequence of colorectal cancer and may facilitate personalized therapeutics focused on the microbiota. Cancer Epidemiol Biomarkers Prev; 26(1); 85–94. ©2016 AACR.

SummaryBackgroundThe proportion of oesophageal adenocarcinoma that is detected concurrently with initial Barrett's oesophagus diagnosis is not well studied.AimTo compare the proportion of prevalent adenocarcinoma vs. incident adenocarcinoma found during surveillance of Barrett's.MethodsWe performed a systematic search of MEDLINE, EMBASE and Web of Science (from their inception to 31 May 2015) for cohort studies in adults with Barrett's (nondysplastic Barrett's ± Barrett's with low‐grade dysplasia) with minimum average follow‐up of 3 years, and providing numbers of prevalent adenocarcinoma detected (concurrently with Barrett's diagnosis and up to 1 year afterwards) vs. incident adenocarcinoma detected (greater than 1 year after Barrett's diagnosis). Pooled weighted proportions of prevalent and incident adenocarcinoma were calculated, using a random effects model.ResultsOn meta‐analysis of 13 studies reporting on 603 adenocarcinomas in 9657 Barrett's patients, 85.1% of adenocarcinomas were classified as prevalent [95% confidence interval (CI), 78.1–90.2%) and 14.9% as incident (95% CI, 9.8–21.9%), with substantial heterogeneity (I2 = 66%). Among nine studies reporting on 787 high‐grade dysplasia and oesophageal adenocarcinomas in 8098 Barrett's patients, the proportion of prevalent high‐grade dysplasia‐oesophageal adenocarcinoma was similar at 80.5% (95% CI, 68.1–88.8%, I2 = 87%). These results remained stable across multiple subgroup analyses including study quality, setting, duration of follow‐up and presence of baseline dysplasia.ConclusionsIn our meta‐analysis, four of five patients diagnosed with adenocarcinoma or high‐grade dysplasia at index endoscopy or within 1 year of Barrett's follow‐up were considered to be prevalent cases. Continued efforts are needed to identify patients with Barrett's before the development of adenocarcinoma.

Abstract Purpose: Methylated DNA markers can discriminate upper from lower GI neoplasms (Gastroenterology 2013;144(5):S84). It is unknown if organ-specific molecular prediction of GI tumor site can be achieved. Experimental design: An unbiased search for neoplasm markers at each major GI organ site was conducted by reduced representation bisulfite sequencing. Top marker candidates were then blindly validated by methylation specific PCR on independent tissue DNA samples from normal mucosa [17 esophagus (E), 13 stomach (S), 33 pancreas (P), 35 bile duct/liver (BD), 21 colorectal (CR)] and neoplasia [42 E, 43 S, 36 P, 48 BD, 97 CR]. Recursive partitioning (rPart) trees modeled all stepwise combinations of single markers to classify controls vs neoplasms and then neoplasms by site (CR, gastroesophageal or pancreaticobiliary). A 2nd model was designed from tissue data for blinded clinical piloting on 42 independent plasma DNA samples (14 normal and 28 cancers (14 P, 14 CR). Results: From the top 100 markers, 95 were validated and used for rPart modeling. A 3- marker panel (chr12.133484978-5047, BMP3, chr11.123301058-255) was selected for the universal detection of GI neoplasms, as it classified neoplasms and control tissues with 95% accuracy. Two markers (chr7.25896389-501, QKI) then assigned lower vs upper GI neoplasms with 94% accuracy. Finally, 3 markers (PDGFD, ELOVL2, PCBP3) called pancreaticobiliary vs gastroesophageal neoplasms with 94% accuracy. All 8 markers applied in a single model (Table) accurately predicted control vs tumor by site [88% (p <0.0001)]. In plasma, a 2-marker panel (BMP3, QKI) was 78% accurate in assigning patients to cancer or control groups (p<0.0009) and 83% accurate in assigning cancer origin to CR or P sites (p<0.0001). Predicted by ModelTissue SourceControlsColorectal neoplasiaPancreatico-biliary cancerGastro-esophageal neoplasiaControls1169102Colorectal neoplasia18617Pancreatico-biliary cancer10654Gastro-esophageal neoplasia12872 Conclusion: Panels of methylated DNA markers were identified for both universal detection of GI neoplasia and accurate prediction of tumor site. Pilot testing of these markers in plasma supports potential clinical feasibility. Citation Format: John B. Kisiel, William R. Taylor, Tracy C. Yab, Hassan M. Ghoz, Patrick H. Foote, Mary E. Devens, Douglas W. Mahoney, Thomas C. Smyrk, Lisa A. Boardman, Gloria M. Petersen, Navtej S. Buttar, Lewis R. Roberts, Graham P. Lidgard, David A. Ahlquist. Accurate site prediction of gastrointestinal cancer by novel methylated DNA markers: Discovery & validation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4252. doi:10.1158/1538-7445.AM2015-4252

Abstract Stool DNA testing has evolved into a highly accurate and well-validated test for the screen-detection of colorectal neoplasia. An optimized multi-target stool DNA test (MT-sDNA) achieves the same high point-sensitivities as reported with colonoscopy for early-stage colorectal cancer (CRC) and significantly higher sensitivity than by fecal immunochemical blood testing (FIT) for detection of both cancer and advanced precancer. Thus, MT-sDNA sets a new high criterion standard for the noninvasive screen-detection of colorectal neoplasia. With clinical application, MT-sDNA has potential to meaningfully address current gaps in our approach to CRC screening through benefits of its high accuracy on screening effectiveness, user-friendly features on patient compliance, and easy mail-out distribution on test access. After an unprecedented parallel review, the U.S. Food & Drug Administration (FDA) and Center for Medicaid and Medicare Services (CMS) simultaneously endorsed MT-sDNA for general CRC screening in August 2014, and the test is now available for clinical use. This overview briefly summarizes the clinical validation, addresses central clinical questions at implementation, and touches on future applications of stool DNA testing. Despite efforts to date, CRC remains the 2nd leading cause of cancer mortality in the US and screening rates are suboptimal. Conventional screening tools are variably hampered by invasiveness, unpleasant bowel preparation, access limitations, interruption of work routines, and a bias toward left-sided lesion detection which compromise efficacy and participation. There is an imperative to improve screening effectiveness through more accurate, patient-friendly, and accessible approaches. Multi-Target Stool DNA Test (MT-sDNA) Stool DNA testing is grounded in the rational biology of exfoliation, inherent discrimination afforded by tumor-associated genetic and epigenetic alterations, and exquisite analytical sensitivity of new assay methods (Pathology 2012;44:8-0). Key technical innovations leading to the current high test performance include use of a stool buffer to prevent DNA degradation, discriminant new marker panels, and advanced analytical platforms that substantially augment sensitivity. MT-sDNA, an optimized and automated new generation test and the product of an intensive research and development effort, has been described in detail (Clin Gastroenterol Hepatol 2013; 11:1313). It targets two highly discriminant methylated genes (BMP3 and NDRG4), the informative KRAS mutations, a marker for total human DNA (β-actin), and fecal hemoglobin. Quantitative outputs analyzed in a software system yield a positive or negative result based on an established cutoff score. With tightly reproducible results via automation, MT-sDNA avoids the operator variation that hinders quality with endoscopic and subjective in vitro screening methods. This noninvasive test requires no bowel preparation, diet or medication restrictions, or missed work time. Stools can be collected at home and mailed to a central laboratory for processing. Clinical Validation Case-Control Studies. In the first large multicenter study using next generation technology (Gastroenterology 2012;142:248), a prototype MT-sDNA showed high neoplasm detection unaffected by tumor stage or site. In the second study (Clin Gastroenterol Hepatol 2013;11:1313), an optimized MT-sDNA detected 98% of CRC at specificity of 91% and again was unaffected by stage or site. Adenoma detection increased with size and degree of dysplasia: sensitivity for adenomas >1cm was 57%, >2 cm was 73%, and >3 cm was 83%; sensitivity was 83% with high-grade dysplasia (HGD), 94% of which occurred in lesions >2 cm. In a third study (PloS One 2014;9:e85659), MT-sDNA detected 55% of sessile serrated polyps (SSP) >1cm compared to 10% by fecal immune-testing for blood (FIT) at matched specificities. Pivotal Screen-Setting Study. In a multicenter cross-sectional study comprising >10,000 average risk persons from across North America (NEJM 2014;370:1287), MT-sDNA detected 92% of CRC (94% of stage I-II), 69% of HGD, 66% of adenomas >2cm, and 42% of advanced adenomas (>1cm or with villous features) + SSP >1cm. Specificity was 90% based on normal colonoscopy. MT-sDNA superiority over FIT was statistically significant for all lesion categories. Program Performance MT-sDNA could compare favorably with conventional methods in a longitudinal screening program. MT-sDNA achieves high point sensitivity for CRC, similar to that of colonoscopy. However, because it is practical to perform more frequently, MT-sDNA (followed by colonoscopy if positive) could improve program sensitivity by catching aggressive metachronous lesions missed by colonoscopy alone done at typical 10 year intervals. Point sensitivities of MT-sDNA for precancers could translate into very high programmatic sensitivity with repeated screening rounds, as we have estimated (Pathology 2012;44:80), and, therefore, into effective CRC prevention. Finally, MT-sDNA's point specificity of 90%, while lower than the average 95% reported for FIT, bodes well within a screening program as cumulative false-positives are most relevant. For example, MT-sDNA applied every 3 years (frequency supported by early modeling and approved by CMS) would yield roughly 3-4% false-positives/year, fewer than the 5% rate by FIT applied annually. Implementation With the clinical entry of MT-sDNA come both opportunities to improve screening outcomes and a collective responsibility to apply rational principles and evidence-based decision-making to guide and refine its use. Clinical Questions. Multiple questions will be briefly addressed in the oral presentation, including: Can FIT achieve same sensitivity by lowering its specificity?, How does MT-sDNA compare to plasma-based DNA tests?, Is MT-sDNA cost-effective?, What does the clinical use algorithm look like?, and Who would benefit from its use? With respect to the last question, while the FDA approved MT-sDNA for average-risk screening, other indications may evolve. A sound case can be made for interval testing between screening colonoscopies. As currently practiced, screening colonoscopy has a much lower benefit on incidence and mortality reduction from right-sided than left-sided CRC, interval cancers are disproportionately right-sided, and quality metrics of this operator-dependent procedure vary widely. Because of its very high sensitivity for CRC that is unaffected by tumor site and its superior sensitivity over FIT for detection of sessile serrated polyps and those adenomas at greatest risk of progression, MT-sDNA seems well-suited for consideration as an interval test. In instances of inadequately prepared or incomplete colonoscopy, which occur in 15 - 25% of colonoscopies, MT-sDNA testing may offer a convenient follow-up alternative to repeat colonoscopy or other imaging methods, particularly among those reticent or unwilling to undergo repeat bowel preparation and invasive testing. In low CRC risk surveillance populations, such as patients with a single small adenoma, use of MT-sDNA might be explored as a resource-savings approach. Those at high CRC risk represent another potential target group for surveillance. A premise for the recommendation of colonoscopy as the choice for high risk surveillance has been its unparalleled high sensitivity. However, in light of the recent MT-sDNA validation studies showing similarly high point-sensitivity for early-stage CRC and estimates suggesting comparable program-sensitivity for advanced precancers, it is intriguing to re-consider noninvasive surveillance in this setting. Further studies are clearly needed. Meanwhile, in high risk individuals refusing or unable to access colonoscopy and in those with relative contraindications to colonoscopy, surveillance by MT-sDNA might be considered. The Future: Expanded Applications Inflammatory Bowel Disease (IBD). IBD patients are at increased CRC risk, and stool DNA testing may help to improve surveillance detection and compliance rates. In a feasibility study (Aliment Pharmacol Ther 2013;37:546), highly accurate detection of both CRC and HGD in IBD patients was observed by stool assay of methylated DNA markers alone. Supra-colonic Cancers: There remains an enormous unmet need for early detection of cancers above the colon, which collectively exact a death toll 2-times higher than that of CRC in the US and 4-times higher globally. Based on our early observations, stool DNA testing represents a potential strategy to fill this void. If combined with CRC screening, it would be ideal to differentiate upper from lower GI lesions; and preliminary data suggest that site-specific DNA tumor markers can be identified (Gastroenterology 2013;144:S-84). Clearly, more studies are needed and warranted. As stool DNA testing moves from historical promise into primetime, efficient implementation systems and essential linkages with endoscopy will need to be established to achieve highest impact. Logical extensions of this powerful molecular technology open intriguing opportunities to expand its value. Citation Format: David A. Ahlquist. Stool DNA detection of colorectal neoplasia: A new high bar for noninvasive screening. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr PL02-01. doi:10.1158/1538-7445.AM2015-PL02-01

ABSTRACT The ability to assemble genomes from metagenomic sequencing avoids the need for culture and any associated culture biases. We assembled 24 essentially complete draft genomes from metagenomic pair-end and size-selected mate pair sequencing from 4 stool samples, 2 from subjects diagnosed with colorectal cancer and 2 from healthy controls.

Abstract Purpose: Discriminant markers for pancreatic cancer detection are needed. We sought to identify and validate methylated DNA markers for pancreatic cancer using next-generation sequencing unbiased by known targets. Experimental Design: At a referral center, we conducted four sequential case–control studies: discovery, technical validation, biologic validation, and clinical piloting. Candidate markers were identified using variance-inflated logistic regression on reduced-representation bisulfite DNA sequencing results from matched pancreatic cancers, benign pancreas, and normal colon tissues. Markers were validated technically on replicate discovery study DNA and biologically on independent, matched, blinded tissues by methylation-specific PCR. Clinical testing of six methylation candidates and mutant KRAS was performed on secretin-stimulated pancreatic juice samples from 61 patients with pancreatic cancer, 22 with chronic pancreatitis, and 19 with normal pancreas on endoscopic ultrasound. Areas under receiver-operating characteristics curves (AUC) for markers were calculated. Results: Sequencing identified >500 differentially hyper-methylated regions. On independent tissues, AUC on 19 selected markers ranged between 0.73 and 0.97. Pancreatic juice AUC values for CD1D, KCNK12, CLEC11A, NDRG4, IKZF1, PKRCB, and KRAS were 0.92*, 0.88, 0.85, 0.85, 0.84, 0.83, and 0.75, respectively, for pancreatic cancer compared with normal pancreas and 0.92*, 0.73, 0.76, 0.85*, 0.73, 0.77, and 0.62 for pancreatic cancer compared with chronic pancreatitis (*, P = 0.001 vs. KRAS). Conclusions: We identified and validated novel DNA methylation markers strongly associated with pancreatic cancer. On pilot testing in pancreatic juice, best markers (especially CD1D) highly discriminated pancreatic cases from controls. Clin Cancer Res; 21(19); 4473–81. ©2015 AACR.

Colorectal cancer (CRC), although a significant cause of morbidity and mortality worldwide, has seen a declining incidence and mortality in countries with programmatic screening. Faecal occult blood testing and endoscopic approaches are the predominant screening methods currently. The discovery of the adenoma–carcinoma sequence and a greater understanding of the genetic and epigenetic changes that drive the formation of CRC have contributed to innovative research to identify molecular markers for highly accurate, non-invasive screening tests for CRC. DNA, proteins, messenger RNA and micro-RNA have all been evaluated. The observation of tumour cell exfoliation into the mucocellular layer of the colonic epithelium and proven stability of DNA in a harsh stool environment make stool DNA a particularly promising marker. The development of a clinically useful stool DNA test has required numerous technical advances, including optimisation in DNA stabilisation, the development of assays with high analytical sensitivity, and the identification of specific and broadly informative molecular markers. A multitarget stool DNA test, which combines mutant and methylated DNA markers and a faecal immunochemical test, recently performed favourably in a large cross-sectional validation study and has been approved by the US Food and Drug Administration for the screening of asymptomatic, average-risk individuals. The ultimate way in which molecular marker screening assays will be used in clinical practice will require additional studies to determine optimal screening intervals, factors affecting compliance, management of false-positive results, and the use of these assays in high-risk populations, as well as other considerations.

SummaryBackgroundChronic ulcerative colitis (CUC) and colonic Crohn's disease (CD) increase colorectal neoplasia (CRN) risk. While sessile serrated polyp (SSP) is a known cancer precursor, serrated epithelial changes (SEC) are of uncertain prevalence and neoplastic risk.AimTo assess the serrated lesion detection rates in CUC and CD and documented incidence of subsequent CRN in a retrospective, single‐centre cohort study.MethodsPatients were identified by a central diagnostic index and pathology review confirmed SEC, SSP, CUC and CD diagnoses from 2006–12. Matched controls were identified from among all CUC and CD patients having colonoscopy during the second half of the time period. All were followed for incident CRN, estimated by the Kaplan–Meier method.ResultsBetween 2006 and 2012, 79 SEC and 10 SSP cases were identified. Detection rates were estimated to be 10/1000 and 2/1000 patients, for SEC and SSP respectively, among 4208 unique CUC or CD patients having colonoscopy from 2010–12. With only 10 cases, SSP patients were not further analysed. Cumulative incidence of subsequent CRN at 1 and 3 years was 12% (95% CI, 0–30%) and 30% (3–57%), respectively, in SEC patients compared to 4% (0–12%) and 9% (0–23%), respectively, in CUC or CD controls (P = 0.047, log‐rank). However, this statistical difference was not significant after patients were stratified for history of prior or synchronous dysplasia (P = 0.09).ConclusionsSerrated epithelial changes and sessile serrated polyps are uncommonly detected by colonoscopy in chronic ulcerative colitis and Crohn's disease patients. Histology with changes of serrated epithelium may be associated with risk of subsequent colorectal neoplasia, however further studies are needed to explore this relationship.

Abstract We have demonstrated that colorectal cancer (CRC) and advanced pre-cancers can be detected non-invasively by a manual multi-target stool DNA-based test (sDNA-MT) comprising exfoliated DNA markers (methylated BMP3 and NDRG4, mutant KRAS (7 mutations, codons 12, 13), plus β-actin) and fecal hemoglobin (Hb) (Lidgard, Gastroenterology 2012;142(5);S-770). We now report the clinical performance of this sDNA-MT test using an optimized automated analytic platform and logistic algorithm. This platform could facilitate the routine performance of sDNA-MT for CRC screening by molecular diagnostics capable clinical laboratories. Method: Stool samples were collected from 1003 subjects at 36 study sites after informed consent and prior to colonoscopy bowel preparation from those presenting for average risk CRC screening (283) or surveillance (176) at 2 sites. From referred subjects with CRC, Advanced Adenoma (AA) or Sessile Serrate Adenoma ≥ 1 cm, (SSA) stool was collected at least 7 days post-colonoscopy and prior to surgery or chemo-radiation (135; 21 sites) and similarly for subjects with no neoplastic findings on colonoscopy (Neg) (409; 13 sites). The study population included: cases (207), 58% male, median age 65 yrs. (38-87), CRC (93), AA (84), SSA ≥ 1 cm (30) and controls (796), 42% male, median age 65 yrs. (50-84), Neg (641) and non-advanced adenomas (NA) (155). Stool sample collection and DNA isolation were previously described. Automated methylation, mutation and actin assays were performed with a Hamilton STARlet fluid handler (Hamilton Robotics, Reno NV), and QuARTS (Quantitative Allele-specific Real-time Target and Signal amplification) run on an ABI 7500 FastDx real time thermal cycler (Applied Biosystems, Foster City, CA). Fecal Hb (ng/ml buffer) analysis was performed by automated sandwich ELISA. A “Positive” or “Negative” result was determined with an algorithm that included the methylation and mutation results and a logistic regression score, which combines DNA marker results with Hb and actin results. Algorithm results exceeding a threshold were called “Positive”. The algorithm provided good discriminative ability, stability, sensitivity and specificity. Robustness was tested with computer simulations and statistical techniques (leave-one-out and 10-fold cross validation). Results: At a 90% nominal specificity, sDNA-MT sensitivity was 98% for CRC (91/93) [Stage: I, 95% (20/21), II, 100% (23/23), III 96% (26/27), IV 100% (7/7) and I-III combined 97% (69/71)], 57% (65/114) for precursors ≥1 cm (AA, SSA), and 86% (12/14) for precursors with high grade dysplasia. CRC patients were typically referred to colonoscopy for symptoms and test sensitivity may be elevated relative to that seen with screening. Conclusion: With this study using a novel automated sDNA-MT analytic platform with logistic algorithm, we corroborate our earlier findings using a manual process and demonstrate a platform that allows testing to be performed routinely by molecular diagnostic capable laboratories. The high sensitivity of sDNA-MT for CRC across all stages and for advanced precursors with high-grade dysplasia could lead to improved non-invasive CRC screening performance with wide accessibility to patients. A large multi-site pivotal CRC screening study (DeeP-C study clinicaltrials.gov, NCT01397747) to support such use is underway. Citation Format: Graham P. Lidgard, Michael J. Domanico, Janelle J. Bruinsma, James Light, Zubin D. Gagrat, Rebecca L. Oldham-Haltom, Keith D. Fourrier, Hatim Allawi, Tracy C. Yab, Julie A. Simonson, Mary Devens, Russell I. Heigh, David A. Ahlquist, Barry M. Berger. An optimized molecular stool test for colorectal cancer screening: Evaluation of an automated analytic platform and logistic algorithm. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B12.