Ahlquist, David A.

4088 Background: Methylated DNA markers (MDMs) are broadly informative for early detection of colorectal cancer (CRC) but have not been extensively studied for post-treatment surveillance and disease monitoring. We aimed to assess the feasibility of novel CRC-associated MDMs for detection of distant recurrent CRC (rCRC) in plasma. Methods: A panel of 13 MDMs previously identified to be discriminant for primary CRC was selected. In a cross-sectional analysis of plasma samples, MDMs were assayed blindly (by target enrichment long-probe quantitative amplified signal assay) from 160 age/sex-balanced patients (60 healthy controls, 60 with resected CRC and no evidence of disease (NED), and 40 rCRC after primary tumor resection). Plasma-derived carcinoembryonic antigen (CEA) was measured on all patients. Random forest modeling was used to derive a prediction algorithm of MDMs (with and without CEA) for the endpoint of rCRC relative to healthy controls. The accuracy of the algorithm was summarized as sensitivity, specificity, and area under the receiver operating characteristic curve (AUC) with 95% confidence intervals (CI) in the test set. Results: Median patient age was 55 (interquartile range: 49-64) years. As shown in the Table below, a single MDM with the highest AUC was significantly better than CEA ( p= 0.02). On cross validation, CEA provided no additional improvement to the performance of the panel of 13 MDMs ( p= 0.2). The cross-validated panel of MDMs detected rCRC liver metastases with 96% (79-100%) sensitivity, lung metastases with 78% (40-97%) sensitivity, and peritoneal/nodal metastases with 57% (18-90%) sensitivity. Lesions with Response Evaluation Criteria in Solid Tumors sum > 4 cm were detected with 94% (73-100%) sensitivity and ≤4 cm with 78% (52-94%) sensitivity. Conclusions: Novel MDMs in plasma detect rCRC with promising accuracy. The clinical utility of MDMs for non-invasive post-treatment surveillance and treatment monitoring in CRC warrants further evaluation in longitudinal studies with sufficient follow-up to exclude sub-clinical recurrence in those with NED. [Table: see text]

Colorectal adenomas are precursors of CRC. Recently, the gut microbiota, i.e., the collection of microbes residing in our gut, has been recognized as a key player in CRC development. There have been a number of gut microbiota profiling studies for colorectal adenoma and CRC; however, fewer studies have considered the gut metabolome, which serves as the chemical interface between the host and gut microbiota. Here, we conducted a gut metabolome profiling study of colorectal adenoma and CRC and analyzed the metabolomic profiles together with paired microbiota composition profiles. We found several chemical signatures of colorectal adenoma that were associated with some gut microbes and potentially indicative of future CRC. This study highlights potential early-driver metabolites in CRC pathogenesis and guides further targeted experiments and thus provides an important stepping stone toward developing better CRC prevention strategies.

OBJECTIVES: Multitarget stool DNA (MT-sDNA) testing has grown as a noninvasive screening modality for colorectal cancer (CRC), but real-world clinical data are limited in the post-FDA approval setting. The effect of previous colonoscopy on MT-sDNA performance is not known. We aimed to evaluate findings of colorectal neoplasia (CRN) at diagnostic colonoscopy in patients with positive MT-sDNA testing, stratified by patient exposure to previous colonoscopy. METHODS: We identified consecutive patients completing MT-sDNA testing over a 39-month period and reviewed the records of those with positive tests for neoplastic findings at diagnostic colonoscopy. MT-sDNA test positivity rate, adherence to diagnostic colonoscopy, and the positive predictive value (PPV) of MT-sDNA for any CRN and neoplastic subtypes were calculated. RESULTS: Of 16,469 MT-sDNA tests completed, testing returned positive in 2,326 (14.1%) patients. After exclusion of patients at increased risk for CRC, 1,801 patients remained, 1,558 (87%) of whom underwent diagnostic colonoscopy; 918 of 1,558 (59%) of these patients had undergone previous colonoscopy, whereas 640 (41%) had not. Any CRN was found in 1,046 of 1,558 patients (PPV = 67%). More neoplastic lesions were found in patients without previous colonoscopy (73%); however, the rates remained high among those who had undergone previous colonoscopy (63%, P < 0.0001). The large majority (79%) of patients had right-sided neoplasia. DISCUSSION: MT-sDNA has a high PPV for any CRN regardless of exposure to previous colonoscopy. Right-sided CRN was found at colonoscopy in most patients with positive MT-sDNA testing, representing a potential advantage over other currently available screening modalities for CRC.

Objective: Alaska Native (AN) people have among the world’s highest rate of colorectal cancer (CRC). We assessed perceptions of AN people and their health care providers of a new take-home multitarget stool DNA test (MT-sDNA; Cologuard) relative to colonoscopy. Methods: Cross-sectional surveys of AN people aged 40 to 75 years (mailed) and providers (online). Results: Participants included 1616 AN patients (19% response rate) and 87 providers (26% response rate; 57% AN people). Over half (58%) of patients preferred colonoscopy for CRC screening, while 36% preferred MT-sDNA. Unscreened patients were significantly more likely to state a preference for MT-sDNA than previously screened patients (42% vs 31%, P < .05) as were younger patients (<60 years old) compared with older patients (40% vs 30%, P < .05). Most providers thought that MT-sDNA would improve screening rates (69%), would recommend if available (79%), and be implementable (79%). Perceived barriers differed substantially between patients and providers in both type and magnitude. Leading colonoscopy barriers reported by patients were travel (44%) and bowel preparation (40%), while providers thought that fear of pain (92%) and invasiveness of the test (87%) were the primary barriers. For MT-sDNA, patients’ belief that colonoscopy was better (56%) and not knowing how to do the test (40%) were primary barriers, while providers thought stool collection (67%) and having a stool sample in their home (63%) were leading barriers. Conclusions: This study found that MT-sDNA has potential acceptability among AN people and their health care providers. Both groups reported a willingness to use MT-sDNA and did not perceive major barriers to its use. This preference was especially true of unscreened and younger patients. The majority of providers indicated they would use MT-sDNA if available and that it would improve CRC screening rates. In this population, where colonoscopy access is limited, MT-sDNA has the potential to improve CRC screening adherence.

INTRODUCTION: Despite the demonstrated effectiveness of average-risk screening, colorectal cancer (CRC) remains the 2 nd leading cause of cancer death in the US. For optimal prevention, CRC screening tools should be highly accurate, user-friendly, and broadly accessible. Patient and provider adoption of the FDA-approved, guideline-endorsed multi-target stool DNA (mt-sDNA) test has grown exponentially since its launch in 2014. Toward ongoing improvement in CRC screening effectiveness, we conducted a blinded case-control study to assess the detection accuracy of a mt-sDNA panel of novel, highly discriminant methylated DNA markers (MDMs). METHODS: We selected 12 candidate MDMs from prior whole methylome discovery and validation in tissue. Stool was obtained as part of previous large, multicenter studies and collected prior to or 7 days post-colonoscopy (prior to any clinically-indicated treatment). CRC, advanced adenoma (AA; size ≥1 cm, high-grade dysplasia, or ≥25% villous morphology), non-AA, or control (no neoplasia) status was defined by colonoscopy and histopathology. Samples were blinded to operators and randomized by characterization to balance the test sets prior to assessment. MDMs were quantified by target-specific DNA capture and Long-probe Quantitative Amplified Signal assay on bisulfite-converted DNA; fecal hemoglobin was quantified by immunoassay. Bootstrapping methods were used to select a panel of 3 MDMs, which was then 10-fold cross-validated; hemoglobin was also included, as was a colon epithelium-specific MDM to normalize stool MDM levels. RESULTS: Participant samples included 117 CRC, 120 AA, 161 non-AA, and 327 controls. Median age was 67 (IQR 61, 75) years; 51% were men. The AUC with best-fit was 0.97 for CRC and 0.84 for AA, and these high AUCs held up tightly on cross-validation (Figure 1). At 92% (95% CI 88-94%) specificity, sensitivity of the panel was 92% (95% CI 86-96%) for CRC. Neither CRC stage (Figure 2) nor CRC site significantly affected performance. At the same 92% specificity, sensitivity was 65% (95% CI 56-73%) for AAs overall, 67% (38-88%) for sessile serrated polyps ≥1 cm, and 71% (57-82%) for adenomas with villous features (Figure 3). CONCLUSION: These data support highly accurate performance of a novel panel of mt-sDNA markers for detecting both CRC (including early stage lesions) and AAs at greatest risk of progression. Prospective comparative studies are warranted to further establish the clinical potential of this novel screening tool.

INTRODUCTION: Colorectal cancer (CRC) screening is broadly endorsed for individuals 50–75 years of age. Given recent increases in CRC incidence and mortality for patients <50 years old, the American Cancer Society now recommends initiation of average-risk CRC screening at age 45 (Wolf AMD CA Cancer J Clin 2018). The performance of recommended tests, including the multi-target stool DNA (mt-sDNA; marketed as Cologuard™) test, has been minimally described in populations under 50 years old. To address this knowledge gap, we conducted a retrospective study of DNA markers (KRAS, BMP3, NDRG4) included in the mt-sDNA assay to quantify and compare tissue marker levels in sporadic CRC cases and normal colon controls from patients in 45–49 and 50–64 year-old age groups. Our primary hypothesis was that mt-sDNA marker levels would be statistically similar between cases in both age groups. METHODS: Patient samples were identified from a pathology tissue archive at Mayo Clinic, excluding those with inflammatory bowel disease or familial cancer syndromes. Formalin-fixed, paraffin-embedded tissues were reviewed by a GI pathologist for 45–49 year-old (n = 90) and 50–64 year-old (n = 96) CRC cases; normal colon samples (45–49, n = 76; 50–64, n = 92) were also included for comparison. DNA extracted from core punches was blinded and processed in a randomized order. Quantitative Allele specific Realtime Target and Signal amplification (QuARTS) assays were used to quantify KRAS mutations, and BMP3 and NDRG4 methylation. Marker level in each sample was reported as the QuARTS product of mutant KRAS , methylated BMP3 or NDRG4 relative to the ACTB product in each sample, reported as a percentage. At least 60 patients per group provided sufficient power to demonstrate differences in marker levels of ±25% between case groups and between control groups using the Wilcoxon Rank Sum test. RESULTS: CRC cases in the 45–49 vs 50–64 year-old groups did not differ significantly with respect to sex ( P = 0.77) or race/ethnicity ( P = 0.06); left-sided CRC was more common in the 45–49 vs 50–64 year-old group (87% vs. 72%; P = 0.02). None of the marker levels were statistically different between cases of the younger vs older groups or between controls of these groups (Figure 1). CONCLUSION: These data support the expectation of no age-related differences in CRC detection by mt-sDNA, as the relevant molecular markers, when they occur, are present at similar levels across age groups 45–49 and 50–64 years.

Abstract Purpose: The burden of esophageal cancer continues to rise, and noninvasive screening tools are needed. Methylated DNA markers (MDM) assayed from plasma show promise in detection of other cancers. For esophageal cancer detection, we aimed to discover and validate MDMs in tissue, and determine their feasibility when assayed from plasma. Experimental Design: Whole-methylome sequencing was performed on DNA extracted from 37 tissues (28 EC; 9 normal esophagus) and 8 buffy coat samples. Top MDMs were validated by methylation specific PCR on tissue from 76 EC (41 adeno, 35 squamous cell) and 17 normal esophagus. Quantitative allele-specific real-time target and signal amplification was used to assay MDMs in plasma from 183 patients (85 EC, 98 controls). Recursive partitioning (rPART) identified MDM combinations predictive of esophageal cancer. Validation was performed in silico by bootstrapping. Results: From discovery, 23 candidate MDMs were selected for independent tissue validation; median area under the receiver operating curve (AUC) for individual MDMs was 0.93. Among 12 MDMs advanced to plasma testing, rPART modeling selected a 5 MDM panel (FER1L4, ZNF671, ST8SIA1, TBX15, ARHGEF4) which achieved an AUC of 0.93 (95% CI, 0.89–0.96) on best-fit and 0.81 (95% CI, 0.75–0.88) on cross-validation. At 91% specificity, the panel detected 74% of esophageal cancer overall, and 43%, 64%, 77%, and 92% of stages I, II, III, and IV, respectively. Discrimination was not affected by age, sex, smoking, or body mass index. Conclusions: Novel MDMs assayed from plasma detect esophageal cancer with moderate accuracy. Further optimization and clinical testing are warranted.

OBJECTIVES: Pancreatic cystic lesions (PCLs) may be precancerous. Those likely to harbor high-grade dysplasia (HGD) or pancreatic cancer (PC) are targets for surgical resection. Current algorithms to predict advanced neoplasia (HGD/PC) in PCLs lack diagnostic accuracy. In pancreatic tissue and cyst fluid (CF) from PCLs, we sought to identify and validate novel methylated DNA markers (MDMs) that discriminate HGD/PC from low-grade dysplasia (LGD) or no dysplasia (ND). METHODS: From an unbiased whole-methylome discovery approach using predefined selection criteria followed by multistep validation on case (HGD or PC) and control (ND or LGD) tissues, we identified discriminant MDMs. Top candidate MDMs were then assayed by quantitative methylation-specific polymerase chain reaction on archival CF from surgically resected PCLs. RESULTS: Of 25 discriminant MDMs identified in tissue, 13 were selected for validation in 134 CF samples (21 cases [8 HGD, 13 PC], 113 controls [45 ND, 68 LGD]). A tree-based algorithm using 2 CF-MDMs ( TBX15 , BMP3 ) achieved sensitivity and specificity above 90%. Discrimination was significantly better by this CF-MDM panel than by mutant KRAS or carcinoembryonic antigen, with areas under the receiver operating characteristic curve of 0.93 (95% confidence interval: 0.86–0.99), 0.71 (0.57–0.85), and 0.72 (0.60–0.84), respectively. Cutoffs for the MDM panel applied to an independent CF validation set (31 cases, 56 controls) yielded similarly high discrimination, areas under the receiver operating characteristic curve = 0.86 (95% confidence interval: 0.77–0.94, P = 0.2). DISCUSSION: Novel MDMs discovered and validated in tissue accurately identify PCLs harboring HGD/PC. A panel of 2 MDMs assayed in CF yielded results with potential to enhance current risk prediction algorithms. Prospective studies are indicated to optimize and further evaluate CF-MDMs for clinical use.

3599 Background: Existing tools for post-treatment CRC surveillance and monitoring are insensitive and expensive. MDMs are broadly informative for early detection of CRC but have not been extensively studied for disease monitoring. As a first step, we sought to assess the concordance of novel CRC-associated MDMs in pCRC and mCRC. Methods: A panel of 14 MDMs previously identified to be highly discriminant for pCRC was selected on the basis of high median fold-change of MDM levels relative to buffy coat (682 (IQR: 132-19347)). Surgically resected pCRC and paired mCRC were identified from institutional pathology databases. Quantitative methylation-specific PCR was used to assay MDMs. 30 paired samples per metastatic subtype were calculated to be sufficient. MDM levels were compared using two-sample and paired Wilcoxon rank sum tests. Results: 87 patients with paired pCRC and mCRC including 57 synchronous and 30 metachronous metastases were included. 41/87 (47%) had neoadjuvant and 59/87 (68%) had adjuvant chemotherapy. All synchronous metastases were to liver. Metachronous metastases were to liver in 19/30 (63%) and to lung in 11/30 (37%). The levels of 14 selected MDMs were remarkably similar between paired pCRC and mCRC (Table). Individual MDM levels and the average level of all MDMs combined were not significantly different ( p> 0.0018 by Bonferroni correction). Conclusions: MDM levels are highly concordant in pCRC and mCRC. Thus, MDMs discovered from pCRC should be further studied for non-invasive surveillance after surgical resection and monitoring of treatment response in mCRC. [Table: see text]