Gonser, Anna M.

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.

e17617 Background: Abnormal uterine bleeding (AUB) is a cardinal symptom of endometrial cancer (EC). However, 90-95% of those with AUB will undergo invasive endometrial sampling with the finding of benign etiology. Our group has identified EC-related methylated DNA markers (MDMs) detectable in vaginal fluid obtained via self-collected tampon with high sensitivity to detect EC. To provide alternative vaginal fluid collection options, we compared total DNA recovery and detection of EC MDMs between tampon and swab collection methods. Methods: Patients ≥45 yrs with AUB or undergoing routine gynecologic care, and patients ≥18 yrs with biopsy-confirmed EC or atypical endometrial hyperplasia (AEH) were prospectively enrolled for paired vaginal fluid biospecimens collection. Participants self-collected vaginal fluid via tampon before provider-collected vaginal fluid sampling via swab immediately prior to clinically indicated endometrial sampling or hysterectomy. Cases (EC/AEH) and benign controls were determined by expert review. DNA was extracted from vaginal fluid. Bisulfite converted DNA was then used in long-probe quantitative amplified signal (LQAS) assays for 10 MDMs and 2 reference markers (RMs). Total DNA recovery using the RMs and detection of EC-specific MDMs were compared across sampling techniques. MDMs were standardized using the log of the ratio of the MDM and RM1. Standardized MDMs were compared between swabs and tampons using median and interquartile range (IQR). Results: A total of 40 eligible participants were evaluated in this study, 20 cases and 20 controls. Median time between tampon and swab collection was 27 hours (range 1-53). DNA quantity as measured using both RM1 and RM2 were comparable between swab and tampon in both cases and controls; MDM detection was also comparable between swab and tampon in both cases and controls (Table 1; 8 representative MDMs shown). Though not powered for sensitivity and specificity estimation, EC-related MDMs appear higher in EC cases vs controls, using either sample type. Conclusions: Sampling vaginal fluid with provider-collected swabs or self-collected tampons are both effective for detection of validated EC MDMs. Studies are ongoing to optimize an MDM panel to detect EC in vaginal fluid. Control Tampon Control Swab Case Tampon Case Swab RM1 3.5 [3.3-3.8] 4.2 [3.8-4.2] 3.6 [3.2-3.9] 4.2 [3.8-4.5] MDM1 0.5 [0.4-0.7] 0.4 [0.2-0.6] 1.3 [0.8-1.8] 1.1 [0.6-1.7] MDM2 1.5 [1.2-1.9] 0.9 [0.7-1.2] 3.2 [2.2-4.7] 2.9 [1.7-4.5] MDM3 0.5 [0.4-0.6] 0.4 [0.3-0.5] 1.5 [0.9-2.3] 1.0 [0.8-2.3] MDM4 0.9 [0.6-1.3] 0.5 [0.4-0.7] 2.3 [1.3-3.7] 1.9 [1.0-3.6] MDM5 1.3 [0.6-1.4] 0.8 [0.6-1.0] 3.3 [2.5-4.1] 3.1 [2.4-3.9] MDM6 1.6 [1.3-2.1] 1.2 [0.8-1.7] 3.3 [2.6-4.3] 3.1 [2.3-4.1] MDM7 1.5 [1.1-1.7] 1.0 [0.4-1.4] 3.5 [2.6-4.1] 3.3 [2.1-3.8] MDM8 0.5 [0.3-0.6] 0.5 [0.4-0.7] 1.8 [1.0-2.7] 1.7 [1.0-2.5] Median (IQR) for RM1 and 8 MDMs by case/control and sampling technique.

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: Upper tract urothelial cancers (UTUC) can be difficult to characterize, and non-invasive surveillance tools are lacking. UTUC are often treated with nephroureterectomy (NU). To address these gaps, we sought to discover and validate methylated DNA markers (MDMs) for detection of UTUC. Methods: Archival formalin fixed paraffin embedded (FFPE) tissue samples from NU were pathologically reviewed and punched prior to DNA extraction. Reduced representation bisulfite sequencing (RRBS) was used to identify candidate MDMs that discriminated between UTUC and age-balanced control urothelial tissues (uninvolved ureter/renal pelvic of renal cell carcinoma at radical nephrectomy). Highest ranked (fold-change and p-value) candidate MDMs were biologically validated by quantitative methylation specific PCR in a 2nd independent set of UTUC and age-balanced controls. DNA from healthy donor urine and buffy coat samples was used to assess MDM background signal. Discrimination between UTUC and controls was assessed as the area under the receiver operator characteristic curve (AUC) with corresponding 95% confidence intervals. Results: RRBS assessed 33 UTUC and 26 ureter/renal pelvis controls. 3.01 x 106 CpGs mapped to the reference genome with at least 10X read depth. From 358 candidates, 20 MDMs were selected for biological validation in independent patient samples which included 20 renal pelvis & 16 ureter UTUC and 17 renal pelvis & 15 ureter control specimens. Of UTUC and control patients, 36% and 65% of were men, respectively. Median AUC for comparison of UTUC vs control tissue was 0.87 (IQR 0.81-0.88); AUCs with corresponding 95% CIs are shown for the 10 most accurate MDMs (Table) across all control types. Conclusion: We report discovery and validation of highly sensitive and specific MDMs with potential for accurate non-invasive screening supported by low background in urine and blood. AUCs (95% CI) for selected methylated DNA markers for UTUC vs control tissue, urine, and blood UTUC vs Marker Control Tissue Control Urine Control Buffy C2orf55 0.92 (0.84-1) 0.93 (0.87-1) 0.96 (0.91-1) ANKRD35 0.91 (0.84-0.99) 0.93 (0.86-1) 0.98 (0.94-1) DLX6 0.91 (0.84-0.98) 0.78 (0.66-0.9) 0.97 (0.93-1) Chr8.9998 0.90 (0.82-0.97) 0.91 (0.84-0.97) 0.98 (0.96-1) SP9 0.88 (0.8-0.97) 0.95 (0.9-1) 0.99 (0.98-1) Chr13.1127 0.88 (0.8-0.97) 0.94 (0.89-1) 0.92 (0.85-1) Septin9.r2 0.88 (0.79-0.97) 0.95 (0.88-1) 0.98 (0.94-1) LBX2 0.88 (0.79-0.97) 0.94 (0.88-1) 0.96 (0.91-1) SIM2.r1 0.88 (0.79-0.97) 0.91 (0.82-1) 0.94 (0.86-1) Chr14.1037 0.88 (0.78-0.97) 0.93 (0.85-1) 0.96 (0.9-1) Citation Format: Ojas Bhagra, William R. Taylor, Benjamin R. Gochanour, Patrick H. Foote, Calise K. Berger, Xiaoming Cao, Douglas W. Mahoney, Sara S. Then, Anna M. Gonser, Karen A. Doering, Kelli N. Burger, John C. Cheville, Matthew T. Gettman, John B. Kisiel. Discovery and validation of methylated DNA markers of upper tract urothelial tract cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3349.

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.

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.

e15567 Background: Radiographic surveillance of colorectal cancer (CRC) after therapy is costly and insensitive. We have reported high cross-sectional performance of methylated DNA markers (MDMs) for detection of primary CRC and metastatic recurrence. This study evaluated MDMs to detect recurrence and measure response to anti-cancer therapy in a longitudinal cohort. Methods: This was a nested case-control study drawn from a prospective cohort of adult patients with CRC who completed definitive treatment and followed forward from the start of surveillance by cross-sectional imaging (CT or MRI) and carcinoembryonic antigen (CEA), collected every 3-6 months under routine care. Blood was collected in LBgard® tubes (Biomatrica, San Diego CA). Cases had recurrent CRC (defined by RECIST v1.1 or radiographic findings that justify re-initiation of anti-cancer therapy as determined by treating clinicians). Controls remained NED (no radiographic recurrence and off therapy for ≥4 surveillance visits). From 6mL of plasma, MDMs ( CNNM1, ANKRD13B, FER1L4, ZNF568, CHST2, ZNF671, VAV3, QKI, GRIN2D, DTX1, PDGFD, SFMBT2, JAM3) were measured by target enrichment long-probe quantitative-amplified signal assays, normalized to B3GALT6, in blinded fashion. Previously published random forest models of MDMs +/- CEA, developed on independent patients with intact primary tumors, were used to develop a summary score. Results: With a median follow-up of 276 days (IQR 187-343) and 3 visits per patient (54 visits total), 18 patients had recurrence after start of surveillance. Another 18 patients remained NED after median 555 days (IRQ 469-662) and 4 surveillance visits per patient (76 visits total). Using a 90th %-ile cut-off summary score from 60 NED patients in a prior cross-sectional measurement, the MDM panel was positive in 15 of 18 patients who recurred and only 2 of the 18 NED patients (Table). Of the 76 patient visits for NED patients, 67 (88%) were below the threshold. The MDM panel score crossed the positivity threshold by a median of 127 days (IRQ 0-233) preceding clinical or radiographic documentation of recurrence. A random forest model with CEA was more sensitive but less specific (Table). In patients who resumed therapy, the MDM panel scores correlated closely with subsequent RECIST scores (not shown). Conclusions: Plasma MDMs rise in anticipation of recurrent CRC prior to radiographic detection and remain low in patients without radiographic disease over serial measurements, warranting further development of this tumor-agnostic liquid biopsy approach as a cost-effective tool to assist cancer surveillance.[Table: see text]