Kisiel, John B.

Abstract Background and Aims The FDA-approved multitarget stool-DNA [mt-sDNA] test is a successful colorectal cancer [CRC] screening tool in average-risk individuals but is not indicated for patients with inflammatory bowel disease [IBD]. We determined the performance of the mt-sDNA assay without the haemoglobin component [mt-sDNAHgb-] in patients with IBD, while measuring sensitivity for colorectal cancer and advanced colorectal neoplasia [ACRN]. Methods This was a multi-centre, proof-of-concept investigation in persons aged 18–84 years with a diagnosis of IBD, or primary sclerosing cholangitis [PSC] with IBD. Enrolment occurred between March 2013 and May 2016. Stool was tested with the mt-sDNA molecular markers only, minus the immunochemical haemoglobin component. Results The analysis set contained 355 samples. The median age was 52 [range 39–62] years, 45.6% were female and 93% were White. Two-thirds [63%] had ulcerative colitis [UC] and 10.1% had PSC/IBD. Colonoscopy revealed cancer in 8.5% [N = 30], advanced precancerous lesions [APLs] in 9.3% [N = 33] and non-advanced precancerous lesions in 7.6% [N = 27], and three-quarters [74.7%, N = 265] had negative findings. mt-sDNAHgb- sensitivity was 73.3% for any stage cancers, and 76.2% for ACRN. Sensitivity was highest for IBD-associated high-grade dysplasia at 100% and 84.6% for IBD-associated low-grade dysplasia ≥1 cm. The test showed higher sensitivity and lower specificity in UC than in Crohn’s disease. Increasing inflammation score was associated with a significant decrease in mt-sDNAHgb- test score [ = 0.028] amongst neoplasia-negative individuals, but not in patients with ACRN. Conclusions These data highlight the potential of multitarget stool-DNA marker testing as an important addition to colorectal cancer surveillance by complementing colonoscopic evaluations in IBD patients.

Abstract Objective: Aberrantly methylated DNA may be used to detect minimal residual disease or early recurrence in breast cancer. As a 1st step, we aimed to develop an MDM panel to delineate between healthy women, stage I-III breast cancer in remission with no evidence of disease (NED), and metastatic breast cancer (MBC). Methods: We prospectively enrolled patients into 3 groups: healthy controls (adult females with a normal mammogram in past 12 months with no history of breast cancer), NED [stage I-III breast cancer with no clinical evidence of recurrence 6 months - 3 years post-treatment (adjuvant endocrine therapy allowed)], and MBC (collected either at initial diagnosis of untreated metastatic disease or radiographic progression). DNA was extracted from 6 mL of plasma derived from blood collected in LBgard tubes (Exact Sciences Corp., San Diego CA), and was bisulfite-treated and assayed by target enrichment long-probe quantitative amplified signal (TELQASTM) method for a panel of 15 previously identified MDMs [Table], normalized by the control gene, B3GALT6. Areas under the receiver operating characteristic curve (AUC) were calculated for each MDM to assess discrimination of MBC from other groups. With 60 patients per group, we had greater than 80% power to detect a difference of 0.20 from the null AUC of 0.7. Results: We tested samples from 60 MBC (10 untreated, 50 recurrent), 60 NED, and 60 healthy women. The median age was 63 (IQR, 55-72) and was not different between groups. The NED group was all hormone-receptor (HR) positive, with 47 (78%) being human epidermal growth factor receptor 2 negative (HER2-) and 13 (22%) HER2+; 48 (80%) were stage I and 9 (15%) were N+. The MBC group had 35 (58%) patients with HR+/HER2- tumors, 11 (18%) HR+/HER2+, and 6 (10%) HR-/HER2-, 4 (7%) HR-/HER2+, with 4 (7%) unknown. Of the 41 MBC whose original stage and nodal status were known 9 (22%) were stage I and 22 (54%) were N+. The markers were able to discriminate between MBC and NED patient samples with AUCs from 0.97 to 0.72. AUCs for 10/15 MDMs (ITPRIPL1, EMX1, C17orf64, OSR2, TRIM67, CHST2, IFFO1, TRH, FAM59B, and MPZ) were >0.90. No difference in MDMs was observed between the healthy and NED groups. Conclusions: MDMs, assayed from plasma, appear highly discriminant for metastatic breast cancer cases in comparison to control patients with NED or healthy controls. Further clinical validation of this MDM panel in a larger cohort is warranted. Table 1. Plasma MDM discrimination of metastatic breast cancer (MBC) from healthy controls and previously treated breast cancer patients with no evidence of disease (NED) Citation Format: Karthik V. Giridhar, Fergus J. Couch, Jason P. Sinnwell, Seth W. Slettedahl, William R. Taylor, Douglas W. Mahoney, Patrick H. Foote, Maria C. O’Connell, Mariah J. Robran, Mary E. Devens, Anna M. Gonser, Nicole Larson, Karen A. Doering, Kelli N. Burger, Michael Kaiser, Hatim Allawi, Kathryn Ruddy, Janet Olson, John B. Kisiel. Plasma assay of methylated DNA markers (MDM) detects patients with metastatic breast cancer (MBC) compared to healthy controls and treated breast cancer patients with no evidence of disease [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-06.

Abstract Objectives: We hypothesize that a validated panel of methylated DNA markers (MDMs) for HPV(+)CSCC will be similar to but distinct from a cohort of HPV(+)OPSCC. Methods: Patients were excluded for recurrent tumor, prior pelvic or head or neck cancer/neoplasia, chemotherapy < 1 year, prior therapeutic radiation to the target region, prior transplant, insufficient target tissue (<5mm), or if <18 yrs. MDMs identified from sequenced differentially methylated regions were selected from a panel validated for HPV(+)CSCC and evaluated on DNA from independent FFPE HPV(+)OPSCC, HPV(+)CSCC, normal oropharynx tonsil, and normal cervix tissue using methylation-specific PCR. White blood cells (WBCs) were used as a background control. Results: 34 HPV(+)OPSCC, 36 HPV(+)CSCC, 26 normal tonsil tissue, and 24 normal cervical tissue patients met criteria. The HPV(+)OPSCC cohort was older (57 vs 44 yrs, p=0.027), had more frequent alcohol exposure (85% vs 64%, p=0.02), but similar ACE-27 comorbidity scores (p=0.078) and tobacco use (p=0.066). Tumor stages were stage I (47% and 83%), stage II (44% and 11%) and stage III (9% and 6%) for HPV(+)OPSCC and HPV(+)CSCC, respectively. Twenty-one MDMs were evaluated and areas under the receiver operator characteristic curve (AUC) are reported (Table 1). 95% confidence intervals excluded a non-diagnostic null hypothesis AUC of 0.5. 5/21 (24%) achieved an AUC ≥ 0.90, 15/21 (71%) achieved an AUC ≥ 0.8, and 19/21 (90%) exhibited better than chance classifications relative to control tonsil tissue (all p<0.001). Conclusion: MDMs have the potential to discriminate both HPV(+)OPSCC and HPV(+)CSCC from their normal tissue controls. 90% of the MDMs discovered for HPV(+)CSCC had better than chance discrimination in HPV(+)OPSCC suggesting a distinct signature between the two subtypes. Future discovery efforts specific to HPV(+)OPSCC is warranted. Methylated DNA Markers in HPV(+)OPSCC and HPV(+)CSCC CSCC AUC (95% CI) OPSCC AUC (95% CI) AUC CSCC vs AUC OPSCC Pvalue GRIN2D 0.72 (0.59-0.85) 0.91 (0.83-0.99) 0.019 EMX1 0.93 (0.86-0.99) 0.77 (0.65-0.89) 0.0191 VWC2 0.88 (0.79-0.97) 0.57 (0.42-0.72) 0.0001 ZNF610 0.99 (0.98-1) 0.89 (0.8-0.97) 0.0162 ZNF781.F 0.92 (0.86-0.99) 0.79 (0.67-0.91) 0.0585 TBX15 0.99 (0.96-1) 0.82 (0.72-0.93) 0.0025 TSPYL5 0.94 (0.87-1) 0.82 (0.72-0.93) 0.058 LOC645323 0.95 (0.89-1) 0.88 (0.8-0.97) 0.1952 ASCL1 0.98 (0.93-1) 0.86 (0.76-0.95) 0.0187 ABCB1 0.97 (0.93-1) 0.74 (0.61-0.86) 0.0001 ZNF69 0.89 (0.8-0.97) 0.95 (0.9-1) 0.2017 MAX.chr9 0.97 (0.92-1) 0.82 (0.72-0.93) 0.0113 ARHGAP12 0.91 (0.83-0.99) 0.9 (0.81-1) 0.9223 C1orf114 0.94 (0.86-1) 0.88 (0.8-0.97) 0.3366 MAX.chr6 0.99 (0.97-1) 0.86 (0.76-0.96) 0.0099 NEUROG3 0.95 (0.89-1) 0.7 (0.57-0.83) 0.0001 NID2 0.98 (0.95-1) 0.86 (0.77-0.95) 0.0207 TMEM200C 0.95 (0.89-1) 0.54 (0.39-0.69) 0.0001 TTYH1 0.99 (0.98-1) 0.82 (0.71-0.93) 0.0023 ZNF773 0.93 (0.86-1) 0.9 (0.82-0.98) 0.5696 ZNF781.R 1 (1-1) 0.92 (0.86-0.99) 0.0198 Citation Format: Kathryn M. Van Abel, David M. Routman, Daniel J. Ma, Karen A. Doering, Kelli N. Burger, Patrick H. Foote, William R. Taylor, Douglas W. Mahoney, Calise K. Berger, Xiaoming Cao, Sara S. Then, Alyssa M. Larish, Eric J. Moore, Joaquin G. Garcia, Rondell P. Graham, Jamie N. Bakkum-Gamez, John B. Kisiel. Comparison of methylated DNA markers between human papillomavirus mediated carcinoma in oropharynx and cervical cancer: A pilot study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3742.

10529 Background: The Centers for Medicare & Medicaid Services (CMS) recommends covering blood-based biomarker tests with proposed minimum performance thresholds for colorectal cancer (CRC) screening test every 3 years for average-risk, asymptomatic Medicare beneficiaries ages 50–85 years. A blood-based test is a non-invasive screening method to detect CRC but is limited by low sensitivity to detect adenomas. Using the CRC-AIM microsimulation model, predicted life-years gained (LYG) and CRC incidence and mortality reduction were compared between a blood-based test meeting the CMS minimum thresholds and stool-based screening tests (fecal immunochemical test [FIT], fecal occult blood test [FOBT], and multitarget stool DNA [mt-sDNA]). Methods: Outcomes of blood- and stool-based tests were simulated for average-risk individuals free of diagnosed CRC at age 40 and screened between ages 45–75 years per USPSTF recommendations. Per CMS proposed criteria, CRC sensitivity and specificity for a blood-based test were set at 74% and 90%, respectively, and adenoma sensitivity was set at 10%. Published adenoma and CRC sensitivity and specificity were used for each stool test. For the primary analysis, adherence was assumed to be 100%. For secondary analysis, adherence was set at 30–70%, in 10% increments. Outcomes were per 1000 individuals. Results: At 100% adherence, LYG was 229 for a blood test and ≥305 for stool tests, corresponding to at least 25% lower LYG for a blood test relative to all stool tests (Table). Absolute CRC incidence and mortality reductions were at least 19% and 18% lower, respectively, for a blood test vs all stool tests. Secondary analysis indicates that at identical adherence rates that are less than 100%, a blood test had lower LYG vs all stool tests (Table). When the adherence of any test was 50%–70%, a blood test resulted in at least 20% lower LYG vs any stool test, and absolute reductions in CRC incidence and mortality were at least 9% and 10% lower, respectively, vs any stool test. Conclusions: This model suggests that if blood-based CRC screening tests do not sufficiently detect advanced adenomas, clinical outcomes will be inferior to stool-based testing due to lack of cancer prevention. Further discovery efforts to identify blood-based markers associated with both invasive and preinvasive neoplasia are needed to address this deficiency. [Table: see text]

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.

7562 Background: Lymphoma is the 6th most common cancer and a top 10 cause of cancer deaths. To date, there are no blood test approaches to population screening, and accurate surveillance markers are limited. Methylated DNA markers (MDMs) are broadly informative for early detection of cancer but have not been extensively studied for lymphomas. We sought to discover and validate MDMs in DNA extracted from lymphoma tissue, then test these MDMs in archival blood plasma specimens. Methods: Reduced representation bisulfite sequencing (RRBS) was performed on DNA extracted from a discovery set of frozen tissues of classic Hodgkin lymphoma (CHL, n=24), non-Hodgkin lymphoma (NHL, n=78: T-cell-TCL (8); diffuse large B-cell-DLBCL (18); follicular-FL (12); mantle cell-MCL (20); marginal zone-MZL (15)), and lymphoma cell line samples (27); controls included benign lymph node (n=11) and healthy donor buffy coat (n=30). 30 MDMs were ranked by fold-change and AUC and used to design methylation-specific PCR assays for biological validation on independent DNA samples extracted from FFPE tissue from 13 normal lymph node and 63 lymphoma samples and 36 buffy coats from healthy patients. Target enrichment long-probe quantitative-amplified signal assays were developed for 16 MDMs and then assayed in plasma-extracted, bisulfite-converted DNA samples from 390 independent treatment-naïve lymphoma patients (100 CHL and 290 NHL:100 DLBCL, 73 FL, 41 MCL, 41 MZL, 35 TCL), and 210 controls without cancer. Lymphoma plasma samples and 159 controls were provided by the Lymphoma SPORE (CA97274); 51 controls came from a 7-county population archive. Classification of lymphoma cases vs controls was modeled with random forest method and cross-validated across 500 bootstrap samples of each dataset. Results: For MDMs tested in DNA from independent biological validation samples, cross-validated random forest models identified 60/63 cases (95% sensitivity) and 12/13 normal controls (92% specificity). In plasmas, a panel of 16 MDMs ( ZNF503, VWA5B1, HOXA9_5195, GABRG3, ITGA5, MAX_chr17_793, BNC1_2407, CDK20, MAX_chr4_184, TPBG, DNAH14, SYT2, CACNG8, FAM110B, and NRN1) detected 78% (95% CI, 74-82%) of lymphoma cases at 90% specificity. Excluding MZL and TCL, sensitivity increased to 84% (80-88%) including 26/49 (53% (38-67%)) of stage I and 59/71 (83% (71-90%)) of stage II cases. Conclusions: MDMs show promise to detect lymphoma. These markers could be evaluated as part of multicancer early detection testing and could also be evaluated as response markers to treatment and subsequent surveillance.[Table: see text]

e17610 Background: Methylated DNA markers (MDMs) associated with sporadic ovarian cancer (OC) are detected in plasma from patients with treatment naïve tumors, but not cancer-free controls. To assess potential benefits to patients at risk for hereditary OC, the aim of this study was to measure the performance of validated sporadic OC MDMs in OC and benign ovarian tissue (BOT), surgically obtained from germline BRCA1 or BRCA2 ( BRCA1/2) mutation carriers. Methods: BRCA1/2 carriers with OC and BRCA1/2 carriers who had undergone risk-reducing salpingo-oophorectomy (SO) were identified at a comprehensive cancer center. BRCA1/2 carriers in both OC and BOT groups were balanced on age and year of surgery with non- BRCA carriers with sporadic OC or benign indications for SO. OC and BOT formalin fixed paraffin embedded (FFPE) tissues were macrodissected following pathologist selection of representative sites; DNA was extracted, and bisulfite converted. 15 OC MDMs ( GDF6, IFFO1, MAX.chr1.147790358, MAX.chr6.10382190, MAX.chr11.14926602, C2CD4D, PDRM14, NCOR2, SKI, DSCR6, SIM2, PALLD, CDO1, GPRIN1, BCAT1) previously identified in sporadic OC were assayed by quantitative methylation specific PCR, normalized by β-actin, by blinded personnel. Areas under the receiver operating characteristic curve (AUC) were generated for each MDM to assess discrimination of OC from benign tissue and compared between BRCA1/2 and sporadic (non- BRCA) patients, using a z-test. Results: Among BRCA1/2 carriers, there were 48 OC ( BRCA-OC) and 48 BOT ( BRCA-BOT); among non- BRCA, we matched 48 OC (Sporadic-OC) and 48 BOT (non- BRCA-BOT). The median AUC for OC MDM discriminated between OC and BOT with AUCs in BRCA1/2 carriers was 0.87, (IQR, 0.83-0.93). Sporadic-OC vs non- BRCA-BOT median AUC was 0.88 (IQR, 0.83-0.92). AUCs were not significantly different for each MDM when stratified by BRCA or sporadic status. MDMs with an AUC of ≥0.9 in either group are shown (Table). Conclusions: OC MDMs identified and validated in sporadic OC are highly discriminant for OC from BOT in DNA extracted from tissues of people who carry a BRCA1/2 mutation. Testing the performance of these OC MDMs in plasma from BRCA1/2 carriers with and without OC may be of clinical value for high-risk patients and inform multi-cancer early detection testing strategies.[Table: see text]

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.