Doering, Karen A.

5526 Background: HR-HPV DNA testing, with or without cervical cytology, provides excellent sensitivity for detection of cervical cancer (CC) and its precursors; negative test results indicate that risk of disease is extremely low and enable women to undergo reduced screening with safety. However, management of women who screen positive remains challenging as many will prove to have self-limited HR-HPV infections. DNA methylation is an early event in carcinogenesis that could enhance CC screening specificity. Methods: For discovery, DNA from 70 FFPE CC (36 squamous, 34 adenocarcinoma) tissues that were reviewed microscopically, 18 fresh frozen benign cervicovaginal (BCV) tissues collected at the time of benign hysterectomy, and 18 buffy coats from cancer-free women underwent reduced representation bisulfite sequencing (RRBS) to identify MDMs associated with CC. Candidate MDM selection was based on area under the receiver operating characteristic curve (AUC) discrimination, methylation fold change, and low background methylation among benign controls. Candidate MDMs were re-tested using methylation-specific PCR (MSP) to confirm performance. Blinded biological validation was performed using MSP on DNA extracted from independent FFPE CC (38 squamous, 43 adenocarcinoma) and BCV (40) tissues. The performance of CC MDMs was also tested in DNA extracted from cervical dysplasia (36 adenocarcinoma in situ (AIS), 32 cervical intraepithelial neoplasia (CIN) 2/3, 11 CIN 1) FFPE tissues. Results: From RRBS discovery and technical validation via MSP, 30 candidate MDMs showed marked methylation fold changes (10 to >1000) across both CC histologies compared to BCV tissue from cancer-free women. Each of the 30 MDMs highly discriminated CC from BCV tissue with 9 MDMs having an AUC >0.90 (Table). CC MDMs also highly discriminated AIS from BCV but did not perform well in CIN 2/3 and CIN 1 (Table). Conclusions: Whole methylome sequencing, stringent filtering criteria, and biological validation have yielded outstanding candidate MDMs for CC that highly discriminate CC from BCV, notably with high specificity. Performance in cervical dysplasias varied with higher positivity rates in AIS than in CIN 2/3 and CIN 1. Translation to testing these novel MDMs in lower genital tract biospecimens and the addition of HR-HPV to the CC panel are warranted.[Table: see text]

e15511 Background: Aberrantly methylated DNA marker (MDM) candidates are strongly associated with primary colorectal cancer (CRC) before treatment and detect CRC recurrence with high sensitivity when assayed from plasma. The relationship of these MDMs in association to chemotherapy treatment response is unknown. Methods: In a prospective cohort of patients receiving systemic therapy for advanced CRC, peripheral blood was collected serially during restaging visits. 15 patients were retrospectively identified to have partial response (PR), stable disease (SD) and progressive disease (PD) to treatment (n=5 for each group) based on the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Using paired samples from each patient before and after response assessment, we analyzed 11 MDMs ( GRIN2D, ZNF671, ANKRD13B, QKI, VAV3, JAM3, SFMBT2, CHST2, ZNF568, FER1L4 and CNNM1) to assess correlation with treatment response. Cell-free DNA was extracted and bisulfite treated before MDMs were quantified by target enrichment long-probe quantitative-amplified signal assay and normalized to a methylated sequence of B3GALT6. Continuous variables are summarized as a median with corresponding interquartile ranges (IQR) and comparisons between subgroups were based on the Wilcox Rank Sums test. Results: The median interval between pre- and post-response assessment visits was 69 days (IQR: 63-83 days) and the level of tumor burden at pre-assessment was similar across all response types (Table 1). Patients with PD had higher levels of methylated GRIN2D, ZNF671 and ANKRD13B than those with PR or SD at baseline and may offer additional prognostic value over CEA which was similar in the PR and PD groups before treatment (Table 1). Elevation of pre-assessment MDMs preceded radiographic evidence of disease progression by 82 days (IQR 69-83 days). Conclusions: Three MDMs, GRIN2D, ZNF671 and ANKRD13B, were found to reflect treatment response (PD vs. PR + SD) as shown in the table. Although this pilot study was limited by a small sample size, it demonstrated the feasibility of using plasma-based MDMs in monitoring treatment response to systemic therapy for advanced CRC and should be compared to CEA in a larger study.[Table: see text]

Abstract 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.

Abstract 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.

Abstract 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.

e22024 Background: Disease surveillance in high risk melanoma patients using PET/CT is recommended every 6 months, an algorithm that is extremely expensive and often difficult to access. Biomarkers capable of detecting recurrence and low volume metastasis have application in tools to improve the cost effectiveness of surveillance. Towards this long term goal, our aim was to discover melanoma-specific methylated DNA markers (MDMs). Methods: Candidate MDMs were identified by reduced representation bisulfite sequencing of selected age- and sex-balanced snap frozen metastatic melanoma, benign control skin and white blood cells. Sequences were filtered for highest methylation difference, lowest control methylation background ( < 1%), and highest discrimination by variance inflated logistic regression. Using formalin-fixed tissues, the top 32 MDMs were further evaluated in metastatic melanoma from 35 patients, primary melanoma from 26 patients, and 73 control samples (47 benign precursor lesions or normal skin, and 26 healthy buffy coat samples to assess for background DNA in circulation). Candidate markers were blindly assayed by quantitative methylation specific PCR. After normalization by within sample ACTB level, random forest modeling with cross-validation identified marker combinations with greatest predictive accuracy. Results: On receiver operator characteristics analyses of individual marker candidates, areas under the curve (AUCs) ranged from 0.43 to 0.97. Median AUC was 0.835. At 100% specificity, a cross-validated panel of five MDMs (see Table), yielded a sensitivity of 33/35 cases (94.3%; 95% CI, 86.6-100%) for metastatic melanoma and 22/26 cases (84.6%; 95% CI, 70.7-98.5%) for primary melanoma. Furthermore, none of the 5 markers were detected in normal buffy coat samples. Conclusions: A panel of five novel MDMs assayed on tissue and undetectable in normal buffy coat achieves very high discrimination between melanoma and benign control tissues. This panel can be assessed in blood or other bodily fluids for application in melanoma surveillance. [Table: see text]

6072 Background: Effective screening tests for ovarian cancer (OC) are lacking; most cases present at advanced stage and portend poor prognosis. DNA methylation is an early event in carcinogenesis and can be detected in blood plasma samples from cancer patients. In DNA extracted from tissues, we first discovered, then validated discriminant methylated DNA marker (MDM) candidates for OC and subsequently tested independent plasma from women with and without OC. Methods: For discovery, DNA from 67 frozen tissues (18 high grade serous (HGS), 18 endometrioid, 15 clear cell (CC), 6 mucinous OCs; 10 benign fallopian tube epithelium (FT); and 19 buffy coats from cancer-free women underwent reduced representation bisulfite sequencing (RRBS) to identify MDMs associated with OC. 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 FFPE tissues from OCs (36 HGS, 22 endometrioid, 21 CC, and 14 mucinous) and 29 FT. Top performing MDMs in tissue were tested using long-probe quantitative amplified signal assays in independent pre-treatment plasma samples 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: After RRBS discovery and biological validation, 33 MDMs showed marked methylation fold changes (10 to > 1000) across all OC histologies vs FT. The top 11 MDMs ( GPRIN1, CDO1, SRC, SIM2, AGRN, FAIM2, CELF2, DSCR6, GYPC, CAPN2, BCAT1) were tested on plasma from 91 women with OC (76 (84%) 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 HGS, the panel correctly identified 83%, including 5/6 stage I/II, and the majority of other subtypes (Table). Conclusions: Whole methylome sequencing, stringent filtering criteria, and biological validation yielded outstanding candidate MDMs for OC that performed with promisingly high sensitivity and specificity in plasma. Larger plasma-based OC MDM testing studies, with larger numbers of non-HGS histologies are warranted. [Table: see text]

e16705 Background: The prevalence of pancreatic neuroendocrine tumors (PNETs) has increased in the last decade. Despite being clinically asymptomatic PNETs can be biologically aggressive. There is currently no reliable non-invasive diagnostic biomarker for PNETs. In this study we aimed to identify and validate methylated DNA marker (MDM) candidates that differentiate PNET from normal pancreas. Methods: For discovery, reduced representation bisulfite sequencing (RRBS) was performed on DNA extracted from frozen normal pancreas (n = 13) and PNET (n = 51) tissues. Area under the receiver operator characteristic curve (AUC), fold-change, and p-value criteria selected candidates MDMs for blinded validation in independent FFPE tissues from primary PNET (n = 67; solid = 50, cystic = 17), normal pancreas controls (n = 24), and normal buffy coat (n = 36) using methylation specific PCR. MDM distributions in primary PNETs were compared to primary lung (n = 36) and small bowel (n = 36) NETs and metastatic PNET tissue (n = 25). The discrimination accuracy of candidate markers was summarized as the AUC with corresponding 95% confidence intervals (CI). Results: From the RRBS discovery, 31 candidate MDMs were selected for validation. Four MDMs ( SRRM3, HCN2, SPTBN4 and TMC6) achieved individual AUCs ≥0.95 in the validation set (Table). These MDMs were similarly discriminant in metastatic PNET tissue and in primary lung and small bowel NETs. Three out of these 4 MDMs perfectly differentiated PNET tissue from buffy coat with AUC of 1 and may be ideally suited for further development of a blood-based assay. Conclusions: We identified and validated novel MDMs in tissue that discriminate PNETs from controls with normal pancreas and buffy coat with high accuracy. These MDMs also differentiated metastatic PNETs from normal pancreas tissue. Further exploration of these candidate tissue MDMs in plasma can potentially guide diagnosis and management of PNETs. Funding: P30DK084567. [Table: see text]

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]