Allawi, Hatim T.

6057 Background: The incidence rate of human papillomavirus associated oropharyngeal squamous cell carcinoma (HPV(+)OPSCC) has persistently increased over the past decade and is anticipated to rise further over the next decade. Cost effective, accurate, and less invasive screening options for HPV(+)OPSCC have remained stagnant leading to the interrogation of circulating tumor DNA in liquid biopsies such as plasma. We hypothesized that candidate methylated DNA markers (MDMs), previously described by our group, would be present in the plasma of patients with HPV(+)OPSCC and absent in controls. Methods: HPV(+)OPSCC cases were enrolled from a high-volume referral practice. Cancer-free control patients were identified from the surrounding 7-county catchment area and frequency matched to cases by age and alcohol use. cfDNA was extracted from 4 mL of plasma and bisulfite converted prior to Target Enrichment Long-probe Quantitative Amplified Signal assays for 15 MDMs. Strand counts for individual MDMs were normalized to a methylated genomic reference marker. An MDM score was derived via random forest modeling of the 15 MDMs. Area under the receiver operator characteristic curve (AUC) for discriminating cases from controls was evaluated for individual markers and for the MDM score. The sensitivity of the MDM score at 95% specificity was also evaluated. Results: The study consisted of 96 HPV(+)OPSCC and 100 cancer-free controls. The study population was predominately white (95%) with a median age of 61 yrs (IQR: 52-69 yrs). Eighty-nine percent of the cases were male with cancers affecting either the tonsil in 50 (52%) or base of the tongue in 46 (48%) patients; by design 50% of controls were male. Stage I:II:III:IV disease was present in 66%:22%:9%:3% of the cases. The median (IQR) AUC across the 15 MDMs was 0.83 (0.82-0.85) overall: 0.78 (0.77-0.83) for stage I disease and 0.92 (0.91-0.93) for stage II-IV disease. The MDM score for the combined panel of 15 MDMs achieved a cross-validated AUC of 0.93 (CI: 0.89-0.97) overall: 0.90 (CI: 0.84-0.96) for stage I disease and 0.98 (CI: 0.97-1.00) for stage II-IV disease. At 95% specificity, the cross-validated sensitivity of the MDM score for HPV(+)OPSCC was 80% (CI: 71-87%) overall: 73% (CI: 60-83%) for stage I disease and 94% (CI: 78-99%) for stage II-IV disease. Conclusions: We validated a set of previously discovered MDM markers in HPV(+)OPSCC in a robust case control cohort using plasma. The MDMs were detected in both early and late-stage disease. Additional prospective studies in larger intended use cohorts are needed to validate our results for clinical use.

ABSTRACT Lymphoma is one of the leading causes of cancer and cancer deaths and yet has not been amenable to population screening. The role of methylated DNA markers (MDMs) in the detection of lymphoma has not been extensively studied. We aimed to discover, validate, and test tissue–derived MDMs of lymphoma in archival plasma specimens. Reduced representation bisulfite sequencing (RRBS) was performed on a discovery set of frozen tissues. MDMs identified were converted to methylation–specific PCR assays and validated on independent formalin–fixed, paraffin‐embedded (FFPE) tissues. Target enrichment long–probe quantitative‐amplified signal (TELQAS) assays were developed and assayed in plasma–extracted, bisulfite‐converted DNA from independent treatment‐naïve lymphoma patients and healthy controls. Prediction of cancer status was modeled using random forest model with in silico cross‐validation. After discovery and validation in tissue, 16 TELQAS assays ( ZNF503 , VWA5B1 , HOXA9 , GABRG3 , ITGA5 , MAX.chr17.7190 , BNC1 , CDK20 , MAX.chr4.4069 , TPBG , DNAH14 , SYT6 , CACNG8 , FAM110B , ADRA1D , and NRN1 ) were selected for testing in plasma. These detected 78% (95% CI, 74%–82%) of lymphoma cases at 90% specificity. Excluding marginal zone and T‐cell lymphomas, sensitivity increased to 84% (80%–88%). MDMs in plasma show promise to detect lymphoma and are candidates for inclusion in multi‐cancer detection studies.

Introduction: Non-invasive assays are needed to better discriminate patients with prostate cancer (PCa) to avoid over-treatment of indolent disease. We analyzed 14 methylated DNA markers (MDMs) from urine samples of patients with biopsy-proven PCa relative to healthy controls and further studied discrimination of clinically significant PCa (csPCa) from healthy controls and Gleason 6 cancers. Methods: To evaluate the panel, urine from 24 healthy male volunteers with no clinical suspicion for PCa and 24 men with biopsy-confirmed disease across all Gleason scores was collected. Blinded to clinical status, DNA from the supernatant was analyzed for methylation signal within specific DNA sequences across 14 genes (HES5, ZNF655, ITPRIPL1, MAX.chr3.6187, SLCO3A1, CHST11, SERPINB9, WNT3A, KCNB2, GAS6, AKR1B1, MAX.chr3.8028, GRASP, ST6GALNAC2) by target enrichment long-probe quantitative-amplified signal assays. Results: Utilizing an overall specificity cut-off of 100% for discriminating normal controls from PCa cases across the MDM panel resulted in 71% sensitivity (95% CI: 49–87%) for PCa detection (4/7 Gleason 6, 8/12 Gleason 7, 5/5 Gleason 8+) and 76% (50–92%) for csPCa (Gleason ≥ 7). At 100% specificity for controls and Gleason 6 patients combined, MDM panel sensitivity was 59% (33–81%) for csPCa (5/12 Gleason 7, 5/5 Gleason 8+). Conclusions: MDMs assayed in urine offer high sensitivity and specificity for detection of clinically significant prostate cancer. Prospective evaluation is necessary to estimate discrimination of patients as first-line screening and as an adjunct to prostate-specific antigen (PSA) testing.

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-based MDMs may offer a cost-effective alternative to imaging for melanoma detection. Early results show 80% sensitivity and 100% specificity against cancer-free controls. Further study needed. #MelanomaResearch #EarlyDetection

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 Purpose: Many cancer deaths could be avoided, and survival could be improved with detection of cancer at an earlier stage. Screening programs for breast, colon, and cervical cancer as well as the NLST trial for lung cancer screening demonstrated significant improvement in survival rates with screening. However, for 2021, the American Cancer Society estimates these screened cancers represent less than ~40% of cancer incidence and deaths. The remainder of cancer deaths occur because of tumors in unscreened organs and, therefore, a multicancer test that detects cancer in these organs can increase survival rates. By focusing on detection of unscreened cancers, this type of multicancer test would be complementary to current screening procedures. Here we describe the validation of a combined panel of methylated DNA markers (MDMs) and proteins for multicancer detection through testing an independent set of case/control samples. Experimental Procedures: In this study, we further evaluate the performance of our previously identified panel of 15 MDMs and 5 proteins for multicancer detection (Allawi et al., 2021 AACR Annual Meeting) by testing 315 controls and 160 cases encompassing 6 cancer types (liver, esophageal, lung, ovarian, pancreatic, and stomach). All samples used in the study were case-control collections with smoking status, age, and gender matching between cases and asymptomatic controls. Testing was performed in blinded fashion using multiplex PCR followed by LQAS (Long probe Quantitative Amplified Signal) assay on bisulfite converted DNA extracted from 3 mL of plasma collected in LB Gard® blood tubes. Protein concentrations were determined from paired serum aliquots and combined with MDMs for a multi-analyte analysis. The subjects were divided into training and validation with equal representation of cancer type, staging, gender, and age. Two thirds of the cases and controls were used to train with a logistic prediction algorithm, and the remaining 1/3 were used to validate the model. Results: Using stepwise logistic regression, a training model of MDMs and protein markers resulted in an area under the receiver operating characteristics curve (AUC) of 0.97 and cancer sensitivity of 89% at 98% specificity. The same model predicted the validation set with an AUC of 0.96 and cancer sensitivity of 85% at a specificity of 95%. Applying the algorithm to the combined training and validation sets resulted in sensitivities and specificities of 88% and 97%, respectively. The sensitivities per cancer type ranged from 73% for pancreatic cancer to 97% for lung cancer. Conclusion and Next Steps: This study demonstrates the performance of our MDMs and protein markers and their importance as components in our multi-omics strategy for multicancer detection. The next steps would be to expand the testing to include additional cancer types and combine with NGS-based methods to improve performance and optimize workflow. Citation Format: Hatim T. Allawi, Slava Katerov, Abram Vaccaro, Harrison L. Fleming, Debra E. Rugowski, Brittany Otto, Justin Heilberger, Jillian Cassel, Jacquelyn Hennek, William Taylor, Graham Lidgard. Validation of a panel of methylated DNA and protein markers for multi-cancer detection in plasma [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 631.

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]

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.