Gysbers, Brianna

9567 Background: Cutaneous melanoma surveillance is important to identify low-volume systemic disease, but imaging is costly and poorly accessible to patients; frequent skin checks lack sensitivity and specificity. We aimed to establish clinical feasibility of a liquid biopsy blood test, which quantifies validated, melanoma-specific, methylated DNA markers (MDMs), previously discovered, and reported by our team, using tissue extracted DNA. Methods: We prospectively collected blood from adult patients with histologically confirmed melanoma metastases and no other internal malignancies (within 5-years) who underwent surveillance by FDG-PET/CT (N = 88). Blood from age- and sex balanced cancer-free controls (N = 100) were compared. From PET/CT, we extracted the number of organs involved, SUV-max, and largest tumor diameter. Unequivocal metastasis was defined as SUV ≥ 4 and largest diameter > 5 mm. Because PET/CT is inadequate for the screening of brain metastases, we excluded the brain from the analysis. MDMs ( chr11.149, HOXA9, chr20.210, FLJ22536, CLIC5, SIX4, chr7.155, chr17.730, chr1.110) were assayed using target enrichment long-probe quantitative-amplified signal assays, normalized to B3GALT6, in blinded fashion. Using a logistic regression approach and nine candidate MDMs, we calculated the sensitivity for detecting patients with metastasis on PET/CT at 100% specificity. Results: 52/88 (59%) of melanoma patients showed evidence of metastasis on PET/CT at the time of blood draw. At 100% specificity, a panel of 4 MDMs ( HOXA9, chr20.210, chr17.730, chr1.110) yielded a sensitivity of 86.5% (45/52 cases) vs. 100 cancer-free controls. When applying this model to the 36 PET/CT-negative patients, specificity was as high as 97.2% (35/36 cases) while maintaining a sensitivity of 86.5% (one patient with a positive test result had a complete metabolic response to binimetinib / encorafenib prior to negative PET/CT). For patients with ≥ 2 organs involved by metastasis, sensitivity was 100% (29/29 cases). False-negative cases had metastasis in single organs and were characterized by minimal tumor burden and oral corticosteroid use. One false-negative patient had localized stage III disease without known primary melanoma. Conclusions: Plasma MDM levels appear highly concordant with FDG-PET/CT in patients with metastatic cutaneous melanoma. A liquid biopsy approach has potential to lower cost and improve patient access to surveillance. Additional prospective studies in larger intended use cohorts are needed to validate our results.

5091 Background: Controversy surrounding prostate-specific antigen (PSA) based screening for prostate cancer (PCa) highlights the need for non-invasive assays that better discriminate patients with and without clinically significant prostate cancer (csPCa). Such distinction avoids overtreatment in patients with absent or indolent disease while capturing pathology that would derive benefit from intervention. We analyzed the presence of specific methylated DNA markers (MDMs) within urine samples of patients with biopsy proven PCa and assessed the ability to discriminate these patients from healthy controls with no clinical suspicion for PCa. Methods: 24 healthy volunteers with no clinical suspicion for PCa were age-matched to 24 patients with biopsy-confirmed disease across all Gleason scores. Urine collected from subjects was centrifuged with the cell-free supernatant analyzed in a blinded fashion for methylation signal within specific DNA sequences across 14 genes ( HES5, ZNF655, ITPRIPL1, MAX.chr3.193, SLCO3A1, CHST11, SERPINB9, WNT3A, KCNB2, GAS6, AKR1B1, MAX.chr3.727, GRASP, ST6GALNAC2) by target enrichment long-probe quantitative-amplified signal assays. A patient was considered to have a positive MDM panel if any individual marker exceeded the corresponding100% specificity cut-off value (overall MDM panel specificity of 100%). MDM panel positivity was used to evaluate the sensitivity for distinguishing patients with PCa (any Gleason score) from controls as well as discriminate csPCa cancers from PCa Gleason 6. Results: Median age of healthy controls and PCa patients was 70 years (IQR 67-72) and 65 years (IQR 61-71), respectively. Median PSA was 6.4 (IQR 4.9-9.0) among PCa patients, including median PSA of 5 (IQR 4.0-6.1) for Gleason 6, and 7.8 (IQR 5.1-9.2) for Gleason ≥7 disease. Gleason 6, Gleason 7, and Gleason 8+ cancer was noted in 8, 10, and 6 patients, respectively. Utilizing an overall specificity cut-off of 100% for discriminating normal controls from PCa cases across the MDM panel revealed an overall sensitivity of 83% (95% CI: 63-95%) for detection of PCa (6 of 8 Gleason 6, 9 of 10 Gleason 7, 5 of 6 Gleason 8+) and 88% (95% CI: 62-98%) for csPCa (Gleason ≥ 7). When considering a 100% specificity threshold for controls and Gleason 6 patients, the sensitivity the MDM panel was 69% (95% CI: 41-89%) for csPCa (6 of 10 Gleason 7, 5 of 6 Gleason 8+). Conclusions: We describe a panel of 14 MDMs within urine that offer high specificity and sensitivity for detection of prostate cancers as well as selective identification of clinically significant disease states. Prospective comparison between urine MDMs and PSA blood testing is necessary to discern the differential clinical impact of each screening methodology.

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]