Algorithm Training and Testing for a Nonendoscopic Barrett's Esophagus Detection Test in Prospective Multicenter Cohorts.

BACKGROUND & AIMS: Endoscopic Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) detection is invasive and expensive. Nonendoscopic BE/EAC detection tools are guideline-endorsed alternatives. We previously described a 5-methylated DNA marker (MDM) panel assayed on encapsulated sponge cell collection device (CCD) specimens. We aimed to train a new algorithm using a 3-MDM panel and test its performance in an independent cohort. METHODS: Algorithm training and test samples were from 2 prospective multicenter cohorts. All BE cases had esophageal intestinal metaplasia (with or without dysplasia/EAC); control subjects had no endoscopic evidence of BE. The CCD procedure was followed by endoscopy. From CCD cell lysates, DNA was extracted, bisulfite treated, and MDMs were blindly assayed. The algorithm was set and locked using cross-validated logistic regression (training set) and its performance was assessed in an independent test set. RESULTS: Training (N = 352) and test (N = 125) set clinical characteristics were comparable. The final panel included 3 MDMs (NDRG4, VAV3, ZNF682). Overall sensitivity was 82% (95% CI, 68%-94%) at 90% (79%-98%) specificity and 88% (78%-94%) sensitivity at 84% (70%-93%) specificity in training and test sets, respectively. Sensitivity was 90% and 68% for all long- and short-segment BE, respectively. Sensitivity for BE with high-grade dysplasia and EAC was 100% in training and test sets. Overall sensitivity for nondysplastic BE was 82%. Areas under the receiver operating characteristic curves for BE detection were 0.92 and 0.94 in the training and test sets, respectively. CONCLUSIONS: A locked 3-MDM panel algorithm for BE/EAC detection using a nonendoscopic CCD demonstrated excellent sensitivity for high-risk BE cases in independent validation samples. (Clinical trials.gov: NCT02560623, NCT03060642.).

Next-generation Multi-target Stool DNA Panel Accurately Detects Colorectal Cancer and Advanced Precancerous Lesions.

The multi-target stool DNA (mt-sDNA) test screens for colorectal cancer by analyzing DNA methylation/mutation and hemoglobin markers to algorithmically derive a qualitative result. A new panel of highly discriminant candidate methylated DNA markers (MDM) was recently developed. Performance of the novel MDM panel, with hemoglobin, was evaluated in a simulated screening population using archived stool samples weighted to early-stage colorectal cancer and prospectively collected advanced precancerous lesions (APL). Marker selection study (MSS) and separate preliminary independent verification studies (VS) were conducted utilizing samples from multi-center, case-control studies. Sample processing included targeted MDM capture, bisulfite conversion, and MDM quantitation. Fecal hemoglobin was quantified using ELISA. Samples were stratified into 75%/25% training-testing sets; model outcomes were cross-validated 1,000 times. All laboratory operators were blinded. The MSS included 232 cases (120 colorectal cancer/112 APLs) and 490 controls. The VS featured 210 cases (112 colorectal cancer/98 APLs) and 567 controls; APLs were 86.7% adenomas and 13.3% sessile serrated lesions (SSL). Average age was 65.5 (cases) and 63.2 (controls) years. Mean sensitivity in the VS from cross-validation was 95.2% for colorectal cancer and 57.2% for APLs, with specificities of 89.8% (no CRC/APLs) and 92.4% (no neoplasia). Subgroup analyses showed colorectal cancer sensitivities of 93.4% (stage I) and 94.2% (stage II). APL sensitivity was 82.9% for high-grade dysplasia, 73.4% for villous lesions, 49.8% for tubular lesions, and 30.2% for SSLs. These data support high sensitivity and specificity for a next-generation mt-sDNA test panel. Further evaluation of assay performance will be characterized in a prospective, multi-center clinical validation study (NCT04144738). PREVENTION RELEVANCE: This study highlights performance of the next-generation mt-sDNA test, which exhibits high sensitivity and specificity for detecting colorectal cancer and APLs. This noninvasive option has potential to increase screening participation and clinical outcomes. A multi-center, clinical validation trial is underway. See related commentary by Bresalier, p. 93.

Plasma Assay of Cell-Free Methylated DNA Markers of Colorectal Cancer: A Tumor-Agnostic Approach to Monitor Recurrence and Response to Anticancer Therapies.

BACKGROUND: Radiographic surveillance of colorectal cancer (CRC) after curative-intent therapy is costly and unreliable. Methylated DNA markers (MDMs) detected primary CRC and metastatic recurrence with high sensitivity and specificity in cross-sectional studies. This study evaluated using serial MDMs to detect recurrence and monitor the treatment response to anti-cancer therapies. METHODS: A nested case-control study was drawn from a prospective cohort of patients with CRC who completed curative-intent therapy for CRC of all stages. Plasma MDMs were assayed vis target enrichment long-probe quantitative-amplified signal assays, normalized to B3GALT6, and analyzed in combination with serum carcinoembryonic antigen to yield an MDM score. Clinical information, including treatment and radiographic measurements of the tumor burden, were longitudinally collected. RESULTS: Of the 35 patients, 18 had recurrence and 17 had no evidence of disease during the study period. The MDM score was positive in 16 out of 18 patients who recurred and only 2 of the 17 patients without recurrence. The MDM score detected recurrence in 12 patients preceding clinical or radiographic detection of recurrent CRC by a median of 106 days (range 90-232 days). CONCLUSIONS: Plasma MDMs can detect recurrent CRC prior to radiographic detection; this tumor-agnostic liquid biopsy approach may assist cancer surveillance and monitoring.

Positive Predictive Value for Multitarget Stool DNA After Bariatric and Metabolic Surgery.

BACKGROUND AND AIMS: Bariatric and metabolic surgery (BMS) may adversely affect noninvasive stool tests for colorectal cancer (CRC) screening through several mechanisms. Multitarget stool DNA (mt-sDNA) is approved for CRC screening; however, performance in post-BMS patients is unknown. As the rates of BMS are anticipated to increase with rising incidence of obesity, it is important to evaluate mt-sDNA test performance among these patients. METHODS: In a multisite academic and community-based practice, we obtained mt-sDNA results from 10/2014 to 12/2019 through electronic records and an institutional BMS registry. Average CRC risk patients with BMS prior to a positive mt-sDNA underwent a detailed chart review. Follow-up colonoscopy findings were compared to those among BMS patients screened with colonoscopy alone and a historical cohort of patients without BMS, screened by mt-sDNA. The primary study endpoint was the positive predictive value (PPV) for advanced colorectal neoplasia. RESULTS: Among 336 average-risk patients who had mt-sDNA after BMS, mt-sDNA was positive in 49 (14.6%), 47/49 (96%) underwent follow-up colonoscopy, and the PPV for advanced neoplasia was 12/47 (25.5%). This is similar to the PPV for advanced colorectal neoplasia (425/1542, 28%) in a historical cohort of persons without prior BMS, screened by mt-sDNA at our center (P = .86). Among those who had prior BMS, the rate of advanced neoplasia was higher after mt-sDNA compared to screening colonoscopy alone. CONCLUSION: Despite anatomic and physiologic mechanisms that could alter blood or DNA content in stool, BMS does not appear to adversely affect the PPV of mt-sDNA.

Plasma Methylated DNA Markers for Melanoma Surveillance.

PURPOSE: Surveillance after primary melanoma treatment aims to detect early signs of low-volume systemic disease. The current standard of care, surveillance imaging, is costly and difficult to access. We therefore sought to develop methylated DNA markers (MDMs) as promising alternatives for disease surveillance. METHODS: We used reduced representation bisulfite sequencing (RRBS) to identify MDMs in DNA samples obtained from metastatic melanoma, benign nevi, and normal skin tissues. The identified MDMs underwent validation in an independent cohort of tissue and buffy coat DNA samples. Subsequently, we tested the validated MDMs in the plasma DNA of patients with metastatic melanoma undergoing surveillance with total body imaging and compared them with cancer-free controls. To estimate the overall predictive accuracy of the MDMs, we used random forest modeling with bootstrap cross-validation. RESULTS: Forty MDMs demonstrated discrimination between melanoma cases and controls consisting of benign nevi and normal skin. Nine MDMs passing biological validation in tissue were run on 77 plasma samples from individuals with a history of metastatic melanoma, 49 of whom had evidence of disease detected by imaging at the time of blood draw, and 100 cancer-free controls. The cross-validated sensitivity of the panel for imaging-positive disease was 80% with a specificity of 100% in cancer-free controls, resulting in an overall AUC of 0.88 (95% CI, 0.81 to 0.96). The survival estimates for patients with melanoma who tested positive for the panel at 6 months and 1 year were 67% and 56%, respectively, while those who tested negative had survival rates of 100% and 92%. CONCLUSION: MDMs identified by RRBS demonstrate a high degree of concordance with imaging results in the plasma of patients with metastatic melanoma. Further prospective studies in larger intended use cohorts are needed to confirm these findings.

Assessing the capacity of methylated DNA markers of cervical squamous cell carcinoma to discriminate oropharyngeal squamous cell carcinoma in human papillomavirus mediated disease.

OBJECTIVE: Early identification of human papillomavirus associated oropharyngeal squamous cell carcinoma (HPV(+)OPSCC) is challenging and novel biomarkers are needed. We hypothesized that a panel of methylated DNA markers (MDMs) found in HPV(+) cervical squamous cell carcinoma (CSCC) will have similar discrimination in HPV(+)OPSCC tissues. MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded tissues were obtained from patients with primary HPV(+)OPSCC or HPV(+)CSCC; control tissues included normal oropharynx palatine tonsil (NOP) and cervix (NCS). Using a methylation-specific polymerase chain reaction, 21 previously validated cervical MDMs were evaluated on tissue-extracted DNA. Discrimination between case and control cervical and oropharynx tissue was assessed using area under the curve (AUC). RESULTS: 34 HPV(+)OPSCC, 36 HPV(+)CSCC, 26 NOP, and 24 NCS patients met inclusion criteria. Within HPV(+)CSCC, 18/21 (86%) of MDMs achieved an AUC ≥ 0.9 and all MDMs exhibited better than chance classifications relative to control cervical tissue (all p < 0.001). In contrast, within HPV(+)OPSCC only 5/21 (24%) MDMs achieved an AUC ≥ 0.90 but 19/21 (90%) exhibited better than chance classifications relative to control tonsil tissue (all p < 0.001). Overall, 13/21 MDMs had statistically significant lower AUCs in the oropharyngeal cohort compared to the cervical cohort, and only 1 MDM exhibited a statistically significant increase in AUC. CONCLUSIONS: Previously validated MDMs exhibited robust performance in independent HPV(+)CSCC patients. However, most of these MDMs exhibited higher discrimination for HPV(+)CSCC than for HPV(+)OPSCC. This suggests that each SCC subtype requires a unique set of MDMs for optimal discrimination. Future studies are necessary to establish an MDM panel for HPV(+)OPSCC.

Methylated DNA Markers for Sporadic Colorectal and Endometrial Cancer Are Strongly Associated with Lynch Syndrome Cancers.

Lynch syndrome (LS) markedly increases risks of colorectal and endometrial cancers. Early detection biomarkers for LS cancers could reduce the needs for invasive screening and surgical prophylaxis.To validate a panel of methylated DNA markers (MDM) previously identified in sporadic colorectal cancer and endometrial cancer for discrimination of these cancers in LS.In a case-control design, previously identified MDMs for the detection of colorectal cancer and endometrial cancer were assayed by qMSP on tissue-extracted DNA. Results were normalized to ACTB values within each sample. Least absolute shrinkage and selection operator models to classify colorectal cancer and endometrial cancer were trained on sporadic cases and controls and then applied to classify colorectal cancer and endometrial cancer, in those with LS, and cross-validated.We identified colorectal cancer cases (23 with LS, 48 sporadic), colorectal controls (32 LS, 48 sporadic), endometrial cancer cases (30 LS, 48 sporadic), and endometrial controls (29 LS, 37 sporadic). A 3-MDM panel (LASS4, LRRC4, and PPP2R5C) classified LS-CRC from LS controls with an AUC of 0.92 (0.84-0.99); results were similar for sporadic colorectal cancer. A 6-MDM panel (SFMBT2, MPZ, CYTH2, DIDO1, chr10.4479, and EMX2OS) discriminated LS-EC from LS controls with an AUC of 0.92 (0.83-1.0); the AUC for sporadic endometrial cancer versus sporadic controls was nominally higher, 0.99 (0.96-1.0).MDMs previously identified in sporadic endometrial cancer and colorectal cancer discriminate between endometrial cancer and benign endometrium and colorectal cancer and benign colorectum in LS. This supports the inclusion of patients with LS within future prospective clinical trials evaluating endometrial cancer and colorectal cancer MDMs and may provide a new avenue for cancer screening or surveillance in this high-risk population. PREVENTION RELEVANCE: Lynch syndrome (LS) markedly increases risks of colorectal and endometrial cancers. Early detection biomarkers for LS cancers could reduce the needs for invasive screening and surgery. Methylated DNA markers previously identified in sporadic endometrial cancer and colorectal cancer discriminate between benign and cancer tissue in LS.

Neoplasia Diagnosis After Multi-target Stool DNA Is Enhanced Among Lowest Baseline Detectors.

BACKGROUND AND AIMS: Variation in colorectal neoplasia detection limits the effectiveness of screening colonoscopy. By evaluating neoplasia detection rates of individual colonoscopists, we aimed to quantify the effects of pre-procedural knowledge of a positive (+) multi-target stool DNA (mt-sDNA) on colonoscopy quality metrics. METHODS: We retrospectively identified physicians who performed a high volume of + mt-sDNA colonoscopies; colorectal neoplasia at post-mt-sDNA colonoscopy was recorded. These colonoscopists were stratified into quartiles based on baseline adenoma detection rates. Baseline colonoscopy adenoma detection rates and sessile serrated lesion detection rates were compared to post-mt-sDNA colonoscopy neoplasia diagnosis rates among each quartile. Withdrawal times were measured from negative exams. RESULTS: During the study period (2014-17) the highest quartile of physicians by volume of post-mt-sDNA colonoscopies were evaluated. Among thirty-five gastroenterologists, their median screening colonoscopy adenoma detection rate was 32% (IQR, 28-39%) and serrated lesion detection rate was 13% (8-15%). After + mt-sDNA, adenoma diagnosis increased to 47% (36-56%) and serrated lesion diagnosis increased to 31% (17-42%) (both p < 0.0001). Median withdrawal time increased from 10 (7-13) to 12 (10-17) minutes (p < 0.0001) and was proportionate across quartiles. After + mt-sDNA, lower baseline detectors had disproportionately higher rates of adenoma diagnosis in female versus male patients (p = 0.048) and higher serrated neoplasia diagnosis rates among all patients (p = 0.0092). CONCLUSIONS: Knowledge of + mt-sDNA enriches neoplasia diagnosis compared to average risk screening exams. Adenomatous and serrated lesion diagnosis was magnified among those with lower adenoma detection rates. Awareness of the mt-sDNA result may increase physician attention during colonoscopy. Pre-procedure knowledge of a positive mt-sDNA test improves neoplasia diagnosis rates among colonoscopists with lower baseline adenoma detection rates, independent of withdrawal time.

Detection of endometrial cancer using tampon-based collection and methylated DNA markers.

OBJECTIVE: Alterations in DNA methylation are early events in endometrial cancer (EC) development and may have utility in EC detection via tampon-collected vaginal fluid. METHODS: For discovery, DNA from frozen EC, benign endometrium (BE), and benign cervicovaginal (BCV) tissues underwent reduced representation bisulfite sequencing (RRBS) to identify differentially methylated regions (DMRs). Candidate DMRs were selected based on receiver operating characteristic (ROC) discrimination, methylation level fold-change between cancers and controls, and absence of background CpG methylation. Methylated DNA marker (MDM) validation was performed using qMSP on DNA from independent EC and BE FFPE tissue sets. Women ≥45 years of age with abnormal uterine bleeding (AUB) or postmenopausal bleeding (PMB) or any age with biopsy-proven EC self-collected vaginal fluid using a tampon prior to clinically indicated endometrial sampling or hysterectomy. Vaginal fluid DNA was assayed by qMSP for EC-associated MDMs. Random forest modeling analysis was performed to generate predictive probability of underlying disease; results were 500-fold in-silico cross-validated. RESULTS: Thirty-three candidate MDMs met performance criteria in tissue. For the tampon pilot, 100 EC cases were frequency matched by menopausal status and tampon collection date to 92 BE controls. A 28-MDM panel highly discriminated between EC and BE (96% (95%CI 89-99%) specificity; 76% (66-84%) sensitivity (AUC 0.88). In PBS/EDTA tampon buffer, the panel yielded 96% (95% CI 87-99%) specificity and 82% (70-91%) sensitivity (AUC 0.91). CONCLUSION: Next generation methylome sequencing, stringent filtering criteria, and independent validation yielded excellent candidate MDMs for EC. EC-associated MDMs performed with promisingly high sensitivity and specificity in tampon-collected vaginal fluid; PBS-based tampon buffer with added EDTA improved sensitivity. Larger tampon-based EC MDM testing studies are warranted.

Multitarget Stool DNA Testing Has High Positive Predictive Value for Colorectal Neoplasia on the Second Round of Testing.

BACKGROUND & AIMS: Multitarget stool DNA (mt-sDNA) testing is a stool-based screening test for colorectal cancer (CRC). In a single instance of testing, the pivotal Food and Drug Administration-approval study (NCT01397747) found that 16% of mt-sDNA tests were positive, and the positive predictive value (PPV) for CRC or advanced precursor lesions (APL) was 27.3%. We aimed to examine real-world longitudinal performance by determining the test-positive rate and PPV of mt-sDNA on the second round of testing. METHODS: Colonoscopy and pathology reports were reviewed retrospectively for patients with a negative mt-sDNA on the first round of screening and a positive mt-sDNA on the second round. The test-positivity rate and PPV for CRC, APL, and any colorectal neoplasia were calculated for the second mt-sDNA and compared with baseline PPVs from a previously published cohort of patients from our institution who tested positive on the first round of screening. RESULTS: A total of 2758 patients completed a second test at a median of 3.2 years after the first test. Of these, 422 (15%) had a positive second mt-sDNA. The PPV was 0.25% for CRC, 24% for APL, and 67% for any colorectal neoplasia. There was no significant difference in PPV on the second mt-sDNA test compared with the first round (24% vs 28% for APL; P = .12). CONCLUSIONS: mt-sDNA test positive rate and PPV were similar between the first and second rounds of screening. These observations confirm the utility of a second round of mt-sDNA screening and may inform estimates of mt-sDNA effectiveness for CRC screening.

High Positive Predictive Value of Multitarget Stool DNA After Aerodigestive Tract Radiotherapy.

BACKGROUND AND AIMS: Multitarget stool DNA (mt-sDNA) is approved for average-risk colorectal cancer screening; test performance in persons with prior radiation therapy (RT) has not been studied. RT can induce gastrointestinal bleeding and alter DNA methylation, which may affect mt-sDNA accuracy. Among patients previously treated with RT, we aimed to measure the positive predictive value (PPV) of mt-sDNA and compare these results to historical estimates of mt-sDNA PPV among average-risk patients. METHODS: After institutional review board approval, we conducted a retrospective cohort study of a multisite academic and community-based practice. Patients with RT and subsequent mt-sDNA use during the study period (2014-2016) were identified. The findings at diagnostic colonoscopy were compared with published reports among average-risk patients. Nominal P values were generated by 2-tailed Fisher's exact testing in comparisons of colorectal neoplasia (CRN) rates between groups. RESULTS: There were 220 patients who had RT before mt-sDNA testing. RT was delivered along the aerodigestive tract in 108 patients. Mt-sDNA tests were positive in 45 of 220 patients (20%), and colonoscopy findings were available for 42; 31 of 42 patients (74%) had CRN. PPV by mt-sDNA was similar when stratified by site of prior RT (along vs outside the aerodigestive tract; P = 1.00). Detection of advanced CRN (36%) was nominally higher than previously published retrospective (27%) and prospective (20%) studies. The median time from the start of RT to mt-sDNA use was 7 (interquartile range, 3-14) years. CONCLUSION: With a test positivity rate and PPV for CRN similar to reports among average-risk patients, prior RT does not appear to adversely affect mt-sDNA performance.

Tissue methylated DNA markers for sporadic pancreatic cancer are strongly associated with familial and genetically predisposed pancreatic cancer.

High-risk individuals (HRIs) with familial and genetic predisposition to pancreatic ductal adenocarcinoma (PDAC) are eligible for screening. There is no accurate biomarker for detecting early-stage PDAC. We previously demonstrated that a panel of methylated DNA markers (MDMs) accurately detect sporadic PDAC. In this study we compared the distribution of MDMs in DNA extracted from tissue of PDAC cases who carry germline mutations and non-carriers with family history, with control tissue and demonstrate high discrimination like that seen in sporadic PDAC. These results provide scientific rationale for examining plasma MDMs in HRIs with the goal of developing a minimally-invasive early detection test.

Methylated DNA markers for plasma detection of ovarian cancer: Discovery, validation, and clinical feasibility.

OBJECTIVE: Aberrant DNA methylation is an early event in carcinogenesis which could be leveraged to detect ovarian cancer (OC) in plasma. METHODS: DNA from frozen OC tissues, benign fallopian tube epithelium (FTE), and buffy coats from cancer-free women underwent reduced representation bisulfite sequencing (RRBS) to identify OC MDMs. Candidate MDM selection was based on receiver operating characteristic (ROC) discrimination, methylation fold change, and low background methylation among controls. Blinded biological validation was performed using methylated specific PCR on DNA extracted from independent OC and FTE FFPE tissues. MDMs were tested using Target Enrichment Long-probe Quantitative Amplified Signal (TELQAS) assays in pre-treatment plasma from women newly diagnosed with OC and population-sampled healthy women. A random forest modeling analysis was performed to generate predictive probability of disease; results were 500-fold in silico cross-validated. RESULTS: Thirty-three MDMs showed marked methylation fold changes (10 to >1000) across all OC subtypes vs FTE. Eleven MDMs (GPRIN1, CDO1, SRC, SIM2, AGRN, FAIM2, CELF2, RIPPLY3, GYPC, CAPN2, BCAT1) were tested on plasma from 91 women with OC (73 (80%) high-grade serous (HGS)) and 91 without OC; the cross-validated 11-MDM panel highly discriminated OC from controls (96% (95% CI, 89-99%) specificity; 79% (69-87%) sensitivity, and AUC 0.91 (0.86-0.96)). Among the 5 stage I/II HGS OCs included, all were correctly identified. CONCLUSIONS: Whole methylome sequencing, stringent filtering criteria, and biological validation yielded candidate MDMs for OC that performed with high sensitivity and specificity in plasma. Larger plasma-based OC MDM studies, including testing of pre-diagnostic specimens, are warranted.

Integrating Genome and Methylome Data to Identify Candidate DNA Methylation Biomarkers for Pancreatic Cancer Risk.

BACKGROUND: The role of methylation in pancreatic cancer risk remains unclear. We integrated genome and methylome data to identify CpG sites (CpG) with the genetically predicted methylation to be associated with pancreatic cancer risk. We also studied gene expression to understand the identified associations. METHODS: Using genetic data and white blood cell methylation data from 1,595 subjects of European descent, we built genetic models to predict DNA methylation levels. After internal and external validation, we applied prediction models with satisfactory performance to the genetic data of 8,280 pancreatic cancer cases and 6,728 controls of European ancestry to investigate the associations of predicted methylation with pancreatic cancer risk. For associated CpGs, we compared their measured levels in pancreatic tumor versus benign tissue. RESULTS: We identified 45 CpGs at nine loci showing an association with pancreatic cancer risk, including 15 CpGs showing an association independent from identified risk variants. We observed significant correlations between predicted methylation of 16 of the 45 CpGs and predicted expression of eight adjacent genes, of which six genes showed associations with pancreatic cancer risk. Of the 45 CpGs, we were able to compare measured methylation of 16 in pancreatic tumor versus benign pancreatic tissue. Of them, six showed differentiated methylation. CONCLUSIONS: We identified methylation biomarker candidates associated with pancreatic cancer using genetic instruments and added additional insights into the role of methylation in regulating gene expression in pancreatic cancer development. IMPACT: A comprehensive study using genetic instruments identifies 45 CpG sites at nine genomic loci for pancreatic cancer risk.

DNA Methylation Markers for Detection of Cholangiocarcinoma: Discovery, Validation, and Clinical Testing in Biliary Brushings and Plasma.

Cholangiocarcinoma (CCA) has poor prognosis due to late-stage, symptomatic presentation. Altered DNA methylation markers may improve diagnosis of CCA. Reduced-representation bisulfite sequencing was performed on DNA extracted from frozen CCA tissues and matched to adjacent benign biliary epithelia or liver parenchyma. Methylated DNA markers (MDMs) identified from sequenced differentially methylated regions were selected for biological validation on DNA from independent formalin-fixed, paraffin-embedded CCA tumors and adjacent hepatobiliary control tissues using methylation-specific polymerase chain reaction. Selected MDMs were then blindly assayed on DNA extracted from independent archival biliary brushing specimens, including 12 perihilar cholangiocarcinoma, 4 distal cholangiocarcinoma cases, and 18 controls. Next, MDMs were blindly assayed on plasma DNA from patients with extrahepatic CCA (eCCA), including 54 perihilar CCA and 5 distal CCA cases and 95 healthy and 22 primary sclerosing cholangitis controls, balanced for age and sex. From more than 3,600 MDMs discovered in frozen tissues, 39 were tested in independent samples. In the clinical pilot of 16 MDMs on cytology brushings, methylated EMX1 (empty spiracles homeobox 1) had an area under the curve (AUC) of 0.98 (95% confidence interval [CI], 0.95-1.0). In the clinical pilot on plasma, a cross-validated recursive partitioning tree prediction model from nine MDMs was accurate for de novo eCCA (AUC, 0.88 [0.81-0.95]) but not for primary sclerosing cholangitis-associated eCCA (AUC, 0.54 [0.35-0.73]). Conclusion: Next-generation DNA sequencing yielded highly discriminant methylation markers for CCA. Confirmation of these findings in independent tissues, cytology brushings, and plasma supports further development of DNA methylation to augment diagnosis of CCA.

Detection of Postcolonoscopy Colorectal Neoplasia by Multi-target Stool DNA.

INTRODUCTION: Significant variability between colonoscopy operators contributes to postcolonoscopy colorectal cancers (CRCs). We aimed to estimate postcolonoscopy colorectal neoplasia (CRN) detection by multi-target stool DNA (mt-sDNA), which has not previously been studied for this purpose. METHODS: In a retrospective cohort of patients with +mt-sDNA and completed follow-up colonoscopy, positive predictive value (PPV) for endpoints of any CRN, advanced adenoma, right-sided neoplasia, sessile serrated polyps (SSP), and CRC were stratified by the time since previous colonoscopy (0-9, 10, and ≥11 years). mt-sDNA PPV at ≤9 years from previous average-risk screening colonoscopy was used to estimate CRN missed at previous screening colonoscopy. RESULTS: Among the 850 studied patients with +mt-sDNA after a previous negative screening colonoscopy, any CRN was found in 535 (PPV 63%). Among 107 average-risk patients having +mt-sDNA ≤9 years after last negative colonoscopy, any CRN was found in 67 (PPV 63%), advanced neoplasia in 16 (PPV 15%), right-sided CRN in 48 (PPV 46%), and SSP in 20 (PPV 19%). These rates were similar to those in 47 additional average risk persons with previous incomplete colonoscopy and in an additional 68 persons at increased CRC risk. One CRC (stage I) was found in an average risk patient who was mt-sDNA positive 6 years after negative screening colonoscopy. DISCUSSION: The high PPV of mt-sDNA 0-9 years after a negative screening colonoscopy suggests that lesions were likely missed on previous examination or may have arisen de novo. mt-sDNA as an interval test after negative screening colonoscopy warrants further study.

Validation of a methylated DNA marker panel for the nonendoscopic detection of Barrett's esophagus in a multisite case-control study.

BACKGROUND AND AIMS: We previously identified a 5 methylated DNA marker (MDM) panel for the detection of nonendoscopic Barrett's esophagus (BE). In this study, we aimed to recalibrate the performance of the 5 MDM panel using a simplified assay in a training cohort, validate the panel in an independent test cohort, and explore the accuracy of an MDM panel with only 3 markers. METHODS: Participants were recruited from 3 medical centers. The sponge on a string device (EsophaCap; CapNostics, Concord, NC, USA) was swallowed and withdrawn, followed by endoscopy, in BE cases and control subjects. A 5 MDM panel was blindly assayed using a simplified assay. Random forest modeling analysis was performed, in silico cross-validated in the training set, and then locked down, before test set analysis. RESULTS: The training set had 199 patients: 110 BE cases and 89 control subjects, and the test set had 89 patients: 60 BE cases and 29 control subjects. Sensitivity of the 5 MDM panel for BE diagnosis was 93% at 90% specificity in the training set and 93% at 93% specificity in the test set. Areas under the receiver operating characteristic curves were .96 and .97 in the training and test sets, respectively. Model accuracy was not influenced by age, sex, or smoking history. Multiple 3 MDM panels achieved similar accuracy. CONCLUSIONS: A 5 MDM panel for BE is highly accurate in training and test sets in a blinded multisite case-control analysis using a simplified assay. This panel may be reduced to only 3 MDMs in the future. (Clinical trial registration number: NCT02560623.).

High Detection Rates of Pancreatic Cancer Across Stages by Plasma Assay of Novel Methylated DNA Markers and CA19-9.

PURPOSE: We have previously identified tissue methylated DNA markers (MDMs) associated with pancreatic ductal adenocarcinoma (PDAC). In this case-control study, we aimed to assess the diagnostic performance of plasma MDMs for PDAC. EXPERIMENTAL DESIGN: Thirteen MDMs (GRIN2D, CD1D, ZNF781, FER1L4, RYR2, CLEC11A, AK055957, LRRC4, GH05J042948, HOXA1, PRKCB, SHISA9, and NTRK3) were identified on the basis of selection criteria applied to results of prior tissue experiments and assays were optimized in plasma. Next, 340 plasma samples (170 PDAC cases and 170 controls) were assayed using target enrichment long-probe quantitative amplified signal method. Initially, 120 advanced-stage PDAC cases and 120 healthy controls were used to train a prediction algorithm at 97.5% specificity using random forest modeling. Subsequently, the locked algorithm derived from the training set was applied to an independent blinded test set of 50 early-stage PDAC cases and 50 controls. Finally, data from all 340 patients were combined, and cross-validated. RESULTS: The cross-validated area under the receiver operating characteristic curve (AUC) for the training set was 0.93 (0.89-0.96) for the MDM panel alone, 0.91 (95% confidence interval, 0.87-0.96) for carbohydrate antigen 19-9 (CA19-9) alone, and 0.99 (0.98-1) for the combined MDM-CA19-9 panel. In the test set of early-stage PDAC, the AUC for MDMs alone was 0.84 (0.76-0.92), CA19-9 alone was 0.87 (0.79-0.94), and combined MDM-CA19-9 panel was 0.90 (0.84-0.97) significantly better compared with either MDMs alone or CA19-9 alone (P = 0.0382 and 0.0490, respectively). At a preset specificity of 97.5%, the sensitivity for the combined panel in the test set was 80% (28%-99%) for stage I disease and 82% (68%-92%) for stage II disease. Using the combined datasets, the cross-validated AUC was 0.9 (0.86-0.94) for the MDM panel alone and 0.89 for CA19-9 alone (0.84-0.93) versus 0.97 (0.94-0.99) for the combined MDM-CA19-9 panel (P ≤ 0.0001). Overall, cross-validated sensitivity of MDM-CA19-9 panel was 92% (83%-98%), with an observed specificity of 92% at the preset specificity of 97.5%. CONCLUSIONS: Plasma MDMs in combination with CA19-9 detect PDAC with significantly higher accuracy compared with either biomarker individually.

Novel Methylated DNA Markers in the Surveillance of Colorectal Cancer Recurrence.

PURPOSE: We aimed to assess the concordance of colorectal cancer-associated methylated DNA markers (MDM) in primary and metastatic colorectal cancer for feasibility in detection of distantly recurrent/metastatic colorectal cancer in plasma. EXPERIMENTAL DESIGN: A panel of previously discovered colorectal cancer-associated MDMs was selected. MDMs from primary and paired metastatic colorectal cancer tissue were assayed with quantitative methylation-specific PCR. Plasma MDMs were measured blindly by target enrichment long-probe quantitative-amplified signal assays. Random forest modeling was used to derive a prediction algorithm of MDMs in archival plasma samples from primary colorectal cancer cases. This algorithm was validated in prospectively collected plasma samples from recurrent colorectal cancer cases. The accuracy of the algorithm was summarized as sensitivity, specificity, and area under the curve (AUC). RESULTS: Of the 14 selected MDMs, the concordance between primary and metastatic tissue was considered moderate or higher for 12 MDMs (86%). At a preset specificity of 95% (91%-98%), a panel of 13 MDMs, in plasma from 97 colorectal cancer cases and 200 controls, detected stage IV colorectal cancer with 100% (80%-100%) sensitivity and all stages of colorectal cancer with an AUC of 0.91 (0.87-0.95), significantly higher than carcinoembryonic antigen [AUC, 0.72 (0.65-0.79)]. This panel, in plasma from 40 cases and 60 healthy controls, detected recurrent/metastatic colorectal cancer with 90% (76%-97%) sensitivity, 90% (79%-96%) specificity, and an AUC of 0.96 (0.92-1.00). The panel was positive in 0.30 (0.19-0.43) of 60 patients with no evidence of disease in post-operative patients with colorectal cancer. CONCLUSIONS: Plasma assay of novel colorectal cancer-associated MDMs can reliably detect both primary colorectal cancer and distantly recurrent colorectal cancer with promising accuracy.

Novel methylated DNA markers accurately discriminate Lynch syndrome associated colorectal neoplasia.

Aim: Acquired molecular changes in Lynch syndrome (LS) colorectal tumors have been largely unstudied. We identified methylated DNA markers (MDMs) for discrimination of colorectal neoplasia in LS and determined if these MDMs were comparably discriminant in sporadic patients. Patients & methods: For LS discovery, we evaluated DNA from 53 colorectal case and control tissues using next generation sequencing. For validation, blinded methylation-specific PCR assays to the selected MDMs were performed on 197 cases and controls. Results:OPLAH was the most discriminant MDM with areas under the receiver operating characteristic curve ≥0.97 for colorectal neoplasia in LS and sporadic tissues. ALKBH5, was uniquely hypermethylated in LS neoplasms. Conclusion: Highly discriminant MDMs for colorectal neoplasia in LS were identified with potential use in screening and surveillance.