Ahlquist, David A.

Early detection improves hepatocellular carcinoma (HCC) outcomes, but better noninvasive surveillance tools are needed. We aimed to identify and validate methylated DNA markers (MDMs) for HCC detection. Reduced representation bisulfite sequencing was performed on DNA extracted from 18 HCC and 35 control tissues. Candidate MDMs were confirmed by quantitative methylation‐specific PCR in DNA from independent tissues (74 HCC, 29 controls). A phase I plasma pilot incorporated quantitative allele‐specific real‐time target and signal amplification assays on independent plasma‐extracted DNA from 21 HCC cases and 30 controls with cirrhosis. A phase II plasma study was then performed in 95 HCC cases, 51 controls with cirrhosis, and 98 healthy controls using target enrichment long‐probe quantitative amplified signal (TELQAS) assays. Recursive partitioning identified best MDM combinations. The entire MDM panel was statistically cross‐validated by randomly splitting the data 2:1 for training and testing. Random forest (rForest) regression models performed on the training set predicted disease status in the testing set; median areas under the receiver operating characteristics curve (AUCs; and 95% confidence interval [CI]) were reported after 500 iterations. In phase II, a six‐marker MDM panel (homeobox A1 [HOXA1], empty spiracles homeobox 1 [EMX1], AK055957 , endothelin‐converting enzyme 1 [ECE1], phosphofructokinase [PFKP], and C‐type lectin domain containing 11A [CLEC11A]) normalized by beta‐1,3‐galactosyltransferase 6 (B3GALT6) level yielded a best‐fit AUC of 0.96 (95% CI, 0.93‐0.99) with HCC sensitivity of 95% (88%‐98%) at specificity of 92% (86%‐96%). The panel detected 3 of 4 (75%) stage 0, 39 of 42 (93%) stage A, 13 of 14 (93%) stage B, 28 of 28 (100%) stage C, and 7 of 7 (100%) stage D HCCs. The AUC value for alpha‐fetoprotein (AFP) was 0.80 (0.74‐0.87) compared to 0.94 (0.9‐0.97) for the cross‐validated MDM panel (P < 0.0001). Conclusion : MDMs identified in this study proved to accurately detect HCC by plasma testing. Further optimization and clinical testing of this promising approach are indicated.

Abstract Purpose: Gastric adenocarcinoma is the third most common cause of cancer mortality worldwide. Accurate and affordable noninvasive detection methods have potential value for screening and surveillance. Herein, we identify novel methylated DNA markers (MDM) for gastric adenocarcinoma, validate their discrimination for gastric adenocarcinoma in tissues from geographically separate cohorts, explore marker acquisition through the oncogenic cascade, and describe distributions of candidate MDMs in plasma from gastric adenocarcinoma cases and normal controls. Experimental Design: Following discovery by unbiased whole-methylome sequencing, candidate MDMs were validated by blinded methylation-specific PCR in archival case–control tissues from U.S. and South Korean patients. Top MDMs were then assayed by an analytically sensitive method (quantitative real-time allele-specific target and signal amplification) in a blinded pilot study on archival plasma from gastric adenocarcinoma cases and normal controls. Results: Whole-methylome discovery yielded novel and highly discriminant candidate MDMs. In tissue, a panel of candidate MDMs detected gastric adenocarcinoma in 92% to 100% of U.S. and South Korean cohorts at 100% specificity. Levels of most MDMs increased progressively from normal mucosa through metaplasia, adenoma, and gastric adenocarcinoma with variation in points of greatest marker acquisition. In plasma, a 3-marker panel (ELMO1, ZNF569, C13orf18) detected 86% (95% CI, 71–95) of gastric adenocarcinomas at 95% specificity. Conclusions: Novel MDMs appear to accurately discriminate gastric adenocarcinoma from normal controls in both tissue and plasma. The point of aberrant methylation during oncogenesis varies by MDM, which may have relevance to marker selection in clinical applications. Further exploration of these MDMs for gastric adenocarcinoma screening and surveillance is warranted. Clin Cancer Res; 24(22); 5724–34. ©2018 AACR.

Abstract Purpose: Lung cancer is the leading cause of cancer deaths worldwide. Most present symptomatically at late stage with high lethality. Early detection reduces mortality but accurate and readily accessible tools for population screening are limited. By whole methylome sequencing, we have identified novel methylated DNA markers (MDMs) for lung cancer in tissue (Giakoumopoulos et al. ASCO 2016). Using top candidate MDMs in the present study, we now explore their clinical accuracy for lung cancer detection when assayed from plasma. Experimental Procedures: Archival plasmas from two independent study groups were tested in blinded fashion. Lung cancer cases and controls (apparently healthy smokers) for each group were balanced on age and sex (Group 1: 64 cases, 231 controls; Group 2: 23 cases, 80 controls). Using multiplex PCR followed by QuARTS (Quantitative Allele-Specific Real-time Target and Signal amplification) assay, a post-bisulfite quantification of MDMs on DNA extracted from plasma was performed. We selected 31 MDM candidates for initial evaluation in Group 1 (1 ml plasma/patient); top individual MDMs were subsequently tested in Group 2 to identify optimal MDM panels for lung cancer detection (2 ml/patient). Results: From Group 1 analyses, 13 high performance MDMs were selected for further testing (CYP26C1, SOBP, SUCLG2, SHOX2, ZDHHC1, NFIX, FLJ45983, HOXA9, B3GALT6, ZNF781, SP9, BARX1, EMX1) with individual areas under the receiver operator curve (AUCs) ranging from 0.593 to 0.939. Discrimination by individual MDMs was corroborated in Group 2 in which data was analyzed using two methods: a logistic regression fit and a regression partition tree approach. The logistic fit model identified a 4-marker panel (ZNF781, BARX1, EMX1, and SOBP) with an AUC of 0.96 and an overall sensitivity of 91% and 90% specificity. Analysis of the data using a regression partition tree approach identified 4 markers (ZNF781, BARX1, EMX1, and HOXA9) with AUC of 0.93 and an overall sensitivity of 96% and specificity of 94%. For both approaches, B3GALT6 was used as a standardizing marker of total DNA input. Conclusion: A panel of MDMs assayed in plasma achieved high sensitivity and specificity for all types of lung cancer. Further clinical evaluation and validation of this promising panel in larger patient groups are clearly indicated. Citation Format: Hatim T. Allawi, Maria Giakoumopoulos, Evan Flietner, Austin Oliphant, Carla Volkmann, Brian Aizenstein, Tamara Sander, Drew Eckmayer, Ashley Poenitzsch Strong, Melissa Gray, Barry Berger, Tracy Yab, William Taylor, Douglas Mahoney, John B. Kisiel, David E. Midthun, David A. Ahlquist, Graham P. Lidgard. Detection of lung cancer by assay of novel methylated DNA markers in plasma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 712. doi:10.1158/1538-7445.AM2017-712

Abstract Stool DNA testing has emerged as a sensitive, noninvasive option for colorectal cancer (CRC) screening. A multi-target stool DNA test (MT-sDNA) has recently become available in the U.S. as the first FDA-approved molecular-based cancer screening test (“Cologuard” (Exact Sciences)). It is also approved by the Centers for Medicare & Medicaid Services and is incorporated into new screening guidelines by ACS, USPSTF, NCCN, and various other societies. Stool DNA tests target signature genetic and/or epigenetic markers that continuously exfoliate from CRC and its precursors. In early comparison studies (Clin Gastroenterol Hepatol. 2012;10:272), stool assay of exfoliated molecular markers proved to be more sensitive than DNA testing on paired plasma for detection of colorectal neoplasia, and differences were most striking with early stage CRC and precancers. Such findings suggest that the mechanism of luminal exfoliation into stool may provide a biological advantage in early detection as it appears to occur sooner in the oncologic cascade than does vascular invasion into the circulation. Average-Risk CRC Screening: The performance of the optimized MT-sDNA test (which assays a panel of 2 methylated genes, mutant KRAS, and hemoglobin) has been evaluated in multiple clinical studies. Case-control studies showed that CRC and precancers at greatest risk for progression could be detected with high sensitivity, that detection was unaffected by tumor stage or site within the colon, and that sensitivity for polyps increased in proportion to lesion size (Gastroenterology 2012;142:248, Clin Gastroenterol Hepatol 2013;11:1313). In subsequent cross-sectional validation studies from the screening setting (NEJM 2014; 370:1287, Mayo Clin Proc 2016;91:61), MT-sDNA achieved sensitivities for CRC of 92-100% at specificities (based on a denominator of patients with normal colonoscopy) of 90-93%; sensitivities for adenomatous or serrated polyps &gt;1cm, &gt;2cm, and &gt;3cm were respectively 42-43%, 60-66%, and 67-82%. Neoplasm detection by MT-sDNA was significantly higher than by fecal blood testing in these studies. Cumulative or “program” performance of MT-sDNA has been modeled. At the recommended testing frequency of every 3 years, MT-sDNA exhibits a higher benefit-to-risk ratio than the other endorsed CRC screening approaches and generates the fewest number of colonoscopies (i.e., highest program specificity) over a screening lifetime (JAMA 2016;315:2564). Furthermore, because polyps are slow-growing, it has been estimated that the program sensitivity of MT-sDNA for a cohort of advanced adenomas may exceed 90% by the 2nd or 3rd screening rounds (Dig Dis Sci 2015;60:623). Thus, over a lifetime of screening, regular use of MT-sDNA every 3 years has the potential to compare favorably with colonoscopy performed every 10 years in the detection of both CRC and those polyps at greatest risk for progression. In the screening setting, effective neoplasm detection is based not only on test sensitivity but on patient compliance and test access as well. The user-friendly features of MT-sDNA have potential to enhance screening participation rates, and these include noninvasiveness, operator-independence, and avoidance of bowel preparation and diet/medication restrictions. Furthermore, unlike with colonoscopy which requires an average of two missed work days, MT-sDNA can be done from home with no missed work and access by mail is virtually unlimited. Early outcomes with MT-sDNA indicate that &gt;40% of patients tested had not undergone CRC screening previously (based on questionnaire reports to Exact Sciences from first 100,000 test users). Further studies are needed to determine the uptake of this new test and its impact on CRC screening participation and long-term outcomes. Dysplasia Surveillance in Inflammatory Bowel Disease (IBD): Patients with IBD are at increased risk for CRC. Although IBD patients are currently advised to undergo surveillance colonoscopy annually or every 2 years, only half comply with this rigorous regime. Complementary approaches are needed. In early case-control studies (APT 2013;37:546, Gastroenterology 2016;150:S48), stool DNA testing targeting a panel of informative methylated gene markers detected 90-100% of CRC and high grade dysplasia in IBD patients using colonoscopy and surgery as reference standards. Modeling suggests that stool DNA testing every 1-2 years followed by chromo-endoscopy if positive is more cost-effective than either chromo-endoscopy or white-light colonoscopy alone (Kisiel et al. Clin Gastroenterol Hepatol (in press)). A multi-center study of stool DNA for IBD surveillance is underway. Extending Detection to Upper GI Neoplasms: Together, upper GI cancers account for roughly twice as many deaths as CRC does. Yet, they remain largely unscreened due, in part, to relatively lower single site prevalence and lack of cost-effective screening tools. Since all mucosal-based GI neoplasms exfoliate contents into a common excretory conduit, molecular stool testing allows the re-imagination of the GI tract as a single screen-able organ. Perceived in this way, it is the aggregate prevalence that counts and the value of a single screening intervention could theoretically be extended beyond the colon. While preliminary data suggest that tumors at all GI tract sites can be detected by stool DNA testing (Gastroenterology 2009; 136:2068), much technical optimization and substantial clinical testing are required to evaluate the feasibility and practicality of this intriguing approach. In summary, stool DNA testing has potential to favorably affect detection accuracy, patient compliance, and access with general CRC screening. Early data suggest that this approach may have value as a complement to colonoscopy in the surveillance of IBD. And, potential future applications of stool DNA testing could include universal GI cancer screening. Citation Format: David A. Ahlquist. Stool DNA detection of colorectal neoplasia. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr IA13.

341 Background: Precursors to 1/3 of colorectal cancer (CRC), serrated polyps have historically been underdetected by screening due to their inconspicuous, nonhemorrhagic, and proximal nature. New generation sDNA-MT show high sensitivity for both CRC and advanced adenomas (Gastroenterology 2012;142:248), but screen detection of serrated polyps by this approach requires further validation. Aim: To assess and compare noninvasive detection of sessile serrated polyps >1cm (SSP) by sDNA-MT and a commercial quantitative FIT. Methods: In a blinded prospective study, a single stool sample used for both tests was collected from 456 asymptomatic adults prior to screening or surveillance colonoscopy (criterion standard). All 29 patients with SSP were included as cases and all 232 with no neoplastic findings as controls. Buffered stool samples were processed and frozen on receipt; Exact Sciences performed sDNA-MT in batches using optimized analytical methods (Gastroenterology 2012;142:S770). The sDNA-MT panel targets methylated BMP3 (mBMP3) and NDRG4, mutant KRAS, β-actin, and hemoglobin. FIT (Polymedco OC-FIT Check) was performed in separate lab <2 days post defecation and evaluated at cutoffs of 50 (FIT-50) and 100 ng/ml (FIT-100). Results: Age/sex distributions were similar in cases and controls: 40% vs 45% > 65 years and 51% vs 59% women, p=NS respectively. SSP median size was 1.2cm (range 1-3cm), 93% were proximal, and 64% had synchronous diminutive polyps. Among sDNA-MT markers, mBMP3 proved highly discriminant for SSP detection (AUC=0.87, p<0.00001); other DNA markers were less informative and provided no incremental sensitivity. Hemoglobin alone showed no discrimination (AUC=0.50, p=NS). At matched specificities, SSP detection by stool mBMP3 was significantly greater than by either FIT-50 or FIT-100 (Table). Conclusions: From a screening and surveillance setting, SSP can be detected noninvasively by stool assay of exfoliated DNA markers, especially mBMP3. FIT appears to have no value in SSP detection. [Table: see text]

Abstract Background: Studies of colorectal cancer screening by multitarget stool DNA (MT-sDNA) show false-positive (FP) rates of 7% to 13%. It is unclear whether FP patients are at increased long-term risk of adverse outcomes. Methods: We compared subsequent clinical events among patients with apparent FP MT-sDNA with those in patients reported as true negative (TN). This was a retrospective cohort study of participants in pre-FDA approval MT-sDNA studies having nonadvanced or negative baseline colonoscopy findings from a single referral center. Per-protocol and calibrated cutoffs defined FP and TN groups. From the time of stool collection, we measured differences between FP and TN groups in time to death, subsequent cancer diagnosis, and onset of alarm symptoms. Results: Of 1,050 eligible patients, only 6 were lost to follow-up. Median age was 65.6 years [interquartile range (IQR), 56.8–72.3]; 54% were female. Median follow-up time was 4 years (IQR, 3.5–5.3). Eight aerodigestive (lung and gastrointestinal tract) cancers occurred. FP status by calibrated, but not per-protocol, cutoffs was associated with subsequent aerodigestive cancer; however, cumulative incidence did not exceed SEER expectations from the general population. By any cutoff method, FP status was not associated with mortality or alarm symptoms. Conclusions: Although FP status was associated with long-term aerodigestive cancers, new cases were not temporally related and did not exceed incidence estimates from general population. Impact: These observations do not justify aggressive follow-up evaluation for patients with FP MT-sDNA at this time. Larger studies are needed to confirm these early findings. Cancer Epidemiol Biomarkers Prev; 26(4); 614–21. ©2016 AACR.