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.

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.

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.

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.

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.

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.

Methylated DNA Markers of Esophageal Squamous Cancer and Dysplasia: An International Study.

BACKGROUND: Discovery of methylated DNA markers (MDM) of esophageal squamous cell carcinoma (ESCC) has sparked interest in assessing these markers in tissue. We evaluated MDMs in ESCC from three geographically and ethnically distinct populations, and explored the feasibility of assaying MDMs from DNA obtained by swallowed balloon devices. METHODS: MDMs were assayed in ESCC and normal tissues obtained from the populations of United States, Iran, and China, and from exfoliative cytology specimens obtained by balloons in a Chinese population. Areas under the receiver operating curve (AUC) of MDMs discriminating ESCC from normal tissues were calculated. Random forest prediction models were built, trained on U.S. cases and controls, and calibrated to U.S.-only controls (model 1) and three-country controls (model 2). Statistical tests were used to assess the relationship between dysplasia and MDM levels in balloons. RESULTS: Extracted DNA from 333 ESCC and 322 normal tissues was analyzed, in addition to archival DNA from 98 balloons. For ESCC, model 1 validated in Iranian and Chinese tissues with AUCs of 0.90 and 0.87, and model 2 yielded AUCs of 0.99, 0.96, and 0.94 in tissues from the United States, Iran, and China, respectively. In Chinese balloons, MDMs showed a statistically significant trend of increasing levels with increasing grades of dysplasia (P < 0.004). CONCLUSIONS: MDMs accurately discriminate ESCC from normal esophagus in tissues obtained from high- and low-incidence countries. Preliminary data suggest that levels of MDMs assayed in DNA from swallowed balloon devices increase with dysplasia grade. Larger studies are needed to validate these results. IMPACT: MDMs coupled with minimally invasive collection methods have the potential for worldwide application in ESCC screening.

Accurate Nonendoscopic Detection of Barrett's Esophagus by Methylated DNA Markers: A Multisite Case Control Study.

INTRODUCTION: Nonendoscopic Barrett's esophagus (BE) screening may help improve esophageal adenocarcinoma outcomes. We previously demonstrated promising accuracy of methylated DNA markers (MDMs) for the nonendoscopic diagnosis of BE using samples obtained from a capsule sponge-on-string (SOS) device. We aimed to assess the accuracy of these MDMs in an independent cohort using a commercial grade assay. METHODS: BE cases had ≥ 1 cm of circumferential BE with intestinal metaplasia; controls had no endoscopic evidence of BE. The SOS device was withdrawn 8 minutes after swallowing, followed by endoscopy (the criterion standard). Highest performing MDMs from a previous study were blindly assessed on extracted bisulfite-converted DNA by target enrichment long-probe quantitative amplified signal (TELQAS) assays. Optimal MDM combinations were selected and analyzed using random forest modeling with in silico cross-validation. RESULTS: Of 295 patients consented, 268 (91%) swallowed the SOS device; 112 cases and 89 controls met the pre-established inclusion criteria. The median BE length was 6 cm (interquartile range 4-9), and 50% had no dysplasia. The cross-validated sensitivity and specificity of a 5 MDM random forest model were 92% (95% confidence interval 85%-96%) and 94% (95% confidence interval 87%-98%), respectively. Model performance was not affected by age, gender, or smoking history but was influenced by the BE segment length. SOS administration was well tolerated (median [interquartile range] tolerability 2 [0, 4] on 10 scale grading), and 95% preferred SOS over endoscopy. DISCUSSION: Using a minimally invasive molecular approach, MDMs assayed from SOS samples show promise as a safe and accurate nonendoscopic test for BE prediction.

Comparison of Tissue-Based Molecular Markers in Younger versus Older Patients with Colorectal Neoplasia.

BACKGROUND: Emerging colorectal cancer trends demonstrate increased incidence and mortality in younger populations, prompting consideration of average-risk colorectal cancer screening initiation at age 45 versus 50 years. However, screening test performance characteristics in adults 45-49 years have been minimally described. To inform the biologic rationale for multi-target stool DNA (mt-sDNA) screening in younger patients, we analyzed and compared tissue levels of methylation (BMP3, NDRG4) and mutation (KRAS) markers included in the FDA-approved, mt-sDNA assay (Cologuard; Exact Sciences Corporation). METHODS: Within 40-44, 45-49, and 50-64 year age groups, archived colorectal tissue specimens were identified for 211 sporadic colorectal cancer cases, 123 advanced precancerous lesions (APLs; adenomas >1 cm, high-grade dysplasia, ≥25% villous morphology, or sessile serrated polyp; 45-49 and 50-64 age groups only), and 204 histologically normal controls. Following DNA extraction, KRAS, BMP3, and NDRG4 were quantified using QuARTS assays, relative to ACTB (reference gene). RESULTS: None of the molecular marker concentrations were significantly associated with age (P > 0.05 for all comparisons), with the exception of NDRG4 concentration in APL samples (higher in older vs. younger cases; P = 0.008). However, NDRG4 levels were also statistically higher in APL case versus normal control samples in both the 45-49 (P < 0.0001) and 50-64 (P < 0.0001) year age groups. CONCLUSIONS: Overall, these findings support the potential for earlier onset of average-risk colorectal cancer screening with the mt-sDNA assay. IMPACT: These novel data address an identified knowledge gap and strengthen the biologic basis for earlier-onset, average-risk screening with the mt-sDNA assay.

Methylated DNA in Pancreatic Juice Distinguishes Patients With Pancreatic Cancer From Controls.

BACKGROUND & AIMS: Precursors of pancreatic cancer arise in the ductal epithelium; markers exfoliated into pancreatic juice might be used to detect high-grade dysplasia (HGD) and cancer. Specific methylated DNA sequences in pancreatic tissue have been associated with adenocarcinoma. We analyzed these methylated DNA markers (MDMs) in pancreatic juice samples from patients with pancreatic ductal adenocarcinomas (PDACs) or intraductal papillary mucinous neoplasms (IPMNs) with HGD (cases), and assessed their ability to discriminate these patients from individuals without dysplasia or with IPMNs with low-grade dysplasia (controls). METHODS: We obtained pancreatic juice samples from 38 patients (35 with biopsy-proven PDAC or pancreatic cystic lesions with invasive cancer and 3 with HGD) and 73 controls (32 with normal pancreas and 41 with benign disease), collected endoscopically from the duodenum after secretin administration from February 2015 through November 2016 at 3 medical centers. Samples were analyzed for the presence of 14 MDMs (in the genes NDRG4, BMP3, TBX15, C13orf18, PRKCB, CLEC11A, CD1D, ELMO1, IGF2BP1, RYR2, ADCY1, FER1L4, EMX1, and LRRC4), by quantitative allele-specific real-time target and signal amplification. We performed area under the receiver operating characteristic curve analyses to determine the ability of each marker, and panels of markers, to distinguish patients with HGD and cancer from controls. MDMs were combined to form a panel for detection using recursive partition trees. RESULTS: We identified a group of 3 MDMs (at C13orf18, FER1L4, and BMP3) in pancreatic juice that distinguished cases from controls with an area under the receiver operating characteristic value of 0.90 (95% CI, 0.83-0.97). Using a specificity cut-off value of 86%, this group of MDMs distinguished patients with any stage of pancreatic cancer from controls with 83% sensitivity (95% CI, 66%-93%) and identified patients with stage I or II PDAC or IPMN with HGD with 80% sensitivity (95% CI, 56%-95%). CONCLUSIONS: We identified a group of 3 MDMs in pancreatic juice that identify patients with pancreatic cancer with an area under the receiver operating characteristic value of 0.90, including patients with early stage disease or advanced precancer. These DNA methylation patterns might be included in algorithms for early detection of pancreatic cancer, especially in high-risk cohorts. Further optimization and clinical studies are needed.

Novel Methylated DNA Markers Discriminate Advanced Neoplasia in Pancreatic Cysts: Marker Discovery, Tissue Validation, and Cyst Fluid Testing.

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.

Hepatocellular Carcinoma Detection by Plasma Methylated DNA: Discovery, Phase I Pilot, and Phase II Clinical Validation.

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.

Highly Discriminant Methylated DNA Markers for the Non-endoscopic Detection of Barrett's Esophagus.

BACKGROUND: Minimally invasive methods have been described to detect Barrett's esophagus (BE), but are limited by subjectivity and suboptimal accuracy. We identified methylated DNA markers (MDMs) for BE in tissue and assessed their accuracy on whole esophagus brushings and capsule sponge samples. METHODS: Step 1: Unbiased whole methylome sequencing was performed on DNA from BE and normal squamous esophagus (SE) tissue. Discriminant MDM candidates were validated on an independent patient cohort (62 BE cases, 30 controls) by quantitative methylation specific PCR (qMSP). Step 2: Selected MDMs were further evaluated on whole esophageal brushings (49 BE cases, 36 controls). 35 previously sequenced esophageal adenocarcinoma (EAC) MDMs were also evaluated. Step 3: 20 BE cases and 20 controls were randomized to swallow capsules sponges (25 mm, 10 pores or 20 pores per inch (ppi)) followed endoscopy. DNA yield, tolerability, and mucosal injury were compared. Best MDM assays were performed on this cohort. RESULTS: Step 1: 19 MDMs with areas under the ROC curve (AUCs) >0.85 were carried forward. Step 2: On whole esophageal brushings, 80% of individual MDM candidates showed high accuracy for BE (AUCs 0.84-0.94). Step 3: The capsule sponge was swallowed and withdrawn in 98% of subjects. Tolerability was superior with the 10 ppi sponge with minimal mucosal injury and abundant DNA yield. A 2-marker panel (VAV3 + ZNF682) yielded excellent BE discrimination (AUC = 1). CONCLUSIONS: Identified MDMs discriminate BE with high accuracy. BE detection appears safe and feasible with a capsule sponge. Corroboration in larger studies is warranted. ClinicalTrials.gov number NCT02560623.

Detection of Gastric Cancer with Novel Methylated DNA Markers: Discovery, Tissue Validation, and Pilot Testing in Plasma.

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.

Novel Approach to Fecal Occult Blood Testing by Assay of Erythrocyte-Specific microRNA Markers.

BACKGROUND: Fecal occult blood testing (FOBT) has historically relied on methods to detect hemoglobin with no fundamental innovations in decades. AIM: To examine microRNA (miRNA) as a new marker class for FOBT. METHODS: Candidate miRNA markers were identified by small RNA sequencing of human whole blood compared to colorectal epithelia. Markers were tested in human blood cell subsets and blood from non-human species. We assessed assay linearity in blood spiking and marker stability in stool over incubation experiments. Levels of candidate erythrocyte markers were explored in stools from colorectal cancer (CRC) cases and controls. RESULTS: Based on small RNA sequencing and validation RT-qPCR, expression level of each of the top blood-enriched markers (hsa-miR-144-3p, 144-5p, 451a, 486-5p, 363-3p, 20b-5p) could perfectly discriminate blood from colorectal epithelia. All six markers arose from and showed specificity to human erythrocytes. Marker levels increased linearly with erythrocyte concentration in saline or stool and demonstrated a broader dynamic range than did immunochemical test for hemoglobin. Degradation of markers occurred in stool but was reduced with preservative buffers. Erythrocyte marker candidates for stool testing were selected in an exploratory set of stools (20 CRC, 40 normal). Candidates were then further tested in a feasibility set (29 CRC, 31 advanced adenoma, and 115 normal); a miRNA panel (hsa-miR-451a, 144-5p, and 200b-3p as normalizer) yielded an AUC of 0.89 (95% CI 0.82-0.95, P < .0001) for CRC. CONCLUSIONS: A novel miRNA-based approach accurately quantifies fecal blood levels over a broad, clinically relevant range.

DNA Methylation and Mutation of Small Colonic Neoplasms in Ulcerative Colitis and Crohn's Colitis: Implications for Surveillance.

BACKGROUND: Stool DNA testing in patients with inflammatory bowel disease (IBD) may detect colorectal cancer and advanced precancers with high sensitivity; less is known about the presence of DNA markers in small IBD lesions, their association with metachronous neoplasia, or contribution to stool test positivity. METHODS: At a single center in 2 blinded phases, we assayed methylated bone morphogenic protein 3, methylated N-Myc downstream-regulated gene 4, and mutant KRAS in DNA extracted from paraffin-embedded benign lesions, and matched control tissues of patients with IBD, who were followed for subsequent colorectal dysplasia. Stool samples from independent cases and controls with lesions <1 cm or advanced neoplasms were assayed for the same markers. RESULTS: Among IBD lesions (29 low-grade dysplasia, 19 serrated epithelial change, and 10 sessile serrated adenoma/polyps), the prevalence of methylation was significantly higher than in mucosae from 44 matched IBD controls (P < 0.0001 for methylated bone morphogenic protein 3 or methylated N-Myc downstream-regulated gene 4). KRAS mutations were more abundant in serrated epithelial change than all other groups (P < 0.001). Subsequent dysplasia was not associated with DNA marker levels. In stools, the sensitivity of methylated bone morphogenic protein 3 as a single marker was 60% for all lesions <1 cm, 63% for low-grade dysplasia ≥1 cm and 81% for high-grade dysplasia/colorectal cancer, all at 91% specificity (P < 0.0001). CONCLUSIONS: Selected DNA markers known to be present in advanced IBD neoplasia can also be detected in both tissues and stools from IBD patients with small adenomas and serrated lesions. Mutant KRAS exfoliated from serrated epithelial change lesions might raise false-positive rates. These findings have relevance to potential future applications of stool DNA testing for IBD surveillance.