Slettedahl, Seth W.

5624 Background: Endometrial cancer (EC) is the most common gynecologic cancer and abnormal uterine bleeding (AUB) is the predominant presenting symptom. While <10% of AUB represents underlying EC, endometrial sampling is currently required to rule out EC. To develop an alternative, non-invasive rule-out strategy, we aimed to refine a previously reported EC-associated methylated DNA marker (MDM) panel in vaginal swabs. Methods: Patients ≥18 years old with AUB who met ACOG recommendations for endometrial sampling and those undergoing a hysterectomy for biopsy-proven EC or atypical hyperplasia (AH) were prospectively enrolled in a multicenter cohort study. A vaginal fluid specimen was collected via swab by a provider during a speculum exam. DNA was extracted from samples and bisulfite treated, followed by a target and signal amplification method to detect 8 previously identified MDMs. The cross-validated EC classification model used the mean of standardized MDMs. Clinical classification of benign endometrium (BE), EC, and AH was based on central pathology review. EC diagnoses were based on hysterectomy specimens. Endometrioid and mucinous constituted type 1 EC; serous, carcinosarcoma, clear cell, and mixed histology ECs were collectively analyzed as type 2. Results: Of 693 participants enrolled, 670 were evaluable with valid results: 524 in the AUB cohort and 146 in the suspected EC cohort. Within the AUB cohort, 16 had EC or AH. The final analysis cohort included 508 BE, 156 EC, and 6 AH. Of the evaluable participants 20% identified as Black, 3% Asian or Pacific Islander, 75% White, and 3% Other; 7% reported Hispanic ethnicity. Median age was 66 and 50 years for EC and BE AUB, respectively; 80% of those with EC and 43% with benign AUB reported being postmenopausal. Among participants with EC, 102 (65%), 17 (11%), 30 (19%), 6 (4%) were stage I, II, III, and IV, respectively; one EC was unstaged. Type 2 EC accounted for 42 (27%) of all EC cases. A 4-MDM panel yielded an AUC of 0.97 (0.96-0.99, 95% CI) for EC; models with 5-8 MDMs gave identical performance. All-stage EC sensitivity was 97, 97, 96, 95, and 92% at specificities ranging from 75, 80, 85, 90, and 95%, respectively. At the 85% (82-88%) specificity threshold, sensitivities across EC stage, type, and age were consistently high (Table). Sensitivity for AH was 33% (10-70%). Conclusions: An MDM panel demonstrated high sensitivity for EC across stage and EC type, supporting the potential for a non-invasive vaginal swab-based test to rule out EC. Future studies are necessary to finalize the panel and algorithm, prior to validating the test. Clinical trial information: NCT06294886 . EC Sensitivity (%, 95% CI) at 85% Specificity All Stages (n=156)* 96 (92-98) Stage I (n=102) 94 (88 - 97) II (n=17) 100 (82 - 100) III (n=30) 100 (89 - 100) IV (n=6) 100 (61 - 100) Type 1 (n=114) 95 (89 - 98) 2 (n=42) 100 (92 -100) Age ≤ 66 yrs (n=86) 95 (89 - 98) > 66 (n=70) 97 (90 - 99) *n=1 EC was un-staged.

Abstract Esophageal squamous cell carcinoma (ESCC) remains a leading cause of cancer-related mortality worldwide, and there are limited molecular screening options. Following a prior discovery and validation studies in subjects from the United States, China, and Iran, we aimed to validate methylated DNA markers (MDM) for ESCC detection in Malawi, a country with one of the highest ESCC incidence. Fourteen MDMs were tested on 177 tissue samples. Ten MDMs showed excellent performance (AUCs ≥ 0.85); a random forest model achieved an AUC of 0.97. The consistent cross-population performance suggests potential for universal application of these MDMs and further justifies exploring nonendoscopic sampling methods, such as swallowed cell collection devices in combination with MDMs, for efficient and cost-effective screening in high-incidence populations. Prevention Relevance: This study validates MDMs for early detection of ESCC in Malawi, supporting their potential role in noninvasive, population-based screening strategies in Africa to enable early detection and timely intervention for cancer interception, potentially reducing cancer burden in high-incidence, resource-limited regions.

Abstract Aberrant DNA methylation and copy-number alterations (CNA) drive Barrett’s esophagus progression to esophageal adenocarcinoma; however, their combined utility for early detection is unclear. We aimed to identify and validate methylated DNA markers (MDM) and CNAs to distinguish esophageal adenocarcinoma/high-grade dysplasia (HGD) from nondysplastic Barrett’s esophagus (NDBE). In this multiphase, multicenter study, we discovered and validated MDMs and quantified CNAs utilizing whole-genome methylation sequencing of esophageal brushings. DNA biomarkers identified from discovery were further validated in independent patients with paired esophageal brushing and swallowed capsule sponge samples. MDMs were filtered against a reduced representation bisulfite sequencing dataset obtained from independent tissue samples to advance only concordant candidates. CNA burden was quantified using ichorCNA-derived aneuploidy scores (AS). Two hundred MDMs discovered in HGD (N = 18) and esophageal adenocarcinoma (N = 18) versus NDBE brushing samples (N = 18) were tested in independent samples (N = 146). A 52-MDM panel achieved a cross-validated AUC of 0.88 [95% confidence interval (CI), 0.82–0.95]; the addition of AS improved discrimination of HGD/esophageal adenocarcinoma from NDBE to 0.91 (95% CI, 0.86–0.97) AUC. At 80% specificity, the combined model detected 93% of esophageal adenocarcinoma and 88% of HGD cases. In paired capsule sponge samples, a 58-MDM panel achieved a cross-validated AUC of 0.77 (95% CI, 0.66–0.88); a combined 58-MDM and AS model achieved AUC 0.80 (95% CI, 0.7–0.9). MDMs and AS discerned HGD/esophageal adenocarcinoma from normal esophagus/NDBE in endoscopic brushing and capsule sponge samples. This approach may improve Barrett’s esophagus surveillance. Prevention Relevance: This study demonstrates that combining epigenetic and genomic biomarkers across minimally invasive sampling methods can accurately distinguish HGD/esophageal adenocarcinoma from nondysplastic Barrett’s esophagus, offering promising, less invasive strategies to improve BE surveillance and enable endoscopic therapy for esophageal adenocarcinoma prevention and treatment.

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.

Abstract Introduction: Barrett’s esophagus (BE) confers increased cancer risk. Endoscopic surveillance is recommended to distinguish non-dysplastic BE (NDBE), low grade dysplasia (LGD), high grade dysplasia (HGD), or adenocarcinoma (EAC) for endoscopic intervention. However, this paradigm is limited by sampling error, leading to missed/interval EAC. Herein, we aimed to classify these samples more accurately by performing whole genome methylation sequencing (WGMS) and utilizing multiple genomic features on endoscopic brush samples. Materials and methods: Endoscopic brush samples from patients with NDBE (n=18), HGD (18), EAC (18), and normal controls (18) were collected and underwent WGMS (discovery dataset). Sequence reads were processed through internal pipeline to extract CpG methylation and read level methylation ratio (alpha value). Copy number aberration (CNA) detection was performed using both CNVkit and ichorCNA. Differential methylated regions (DMRs) were identified through comparing EAC/HGD with NDBE and normal samples. Targeted methylation sequencing for selected DMRs and shallow WGMS were performed on EAC (41), HGD (26), LGD (23), NDBE (42), and normal controls (37) as validation. Ratio of the reads with alpha value&gt;0.6 for each DMR was used for further analysis. Detected CNA segments were summarized into chromosome arm loss or gain (aneuploidy score or AS representing the number of chromosome arms gained or lost in a sample) and trimmed median absolute deviation (tMAD). Classification models were developed using the discovery set and the best performing one was applied to the validation dataset. Results: Genome-wide DNA methylation largely segregated different groups of samples as diagnosed; however, some unexpected group crossings were observed. Top 200 DMRs were identified between EAC/HGD and NDBE and normal samples. CNA changes were widespread in EAC, very common in HGD but rare in NDBE (17/18 EACs,14/18 HGDs, 5/18 NDBEs with AS&gt;0). With tMAD &gt; 0.015, 16/18 EACs, 8/18 HGDs but none of 18 NDBEs passed the threshold. EAC or HGD samples without detectable CNAs had very low “tumor” fraction as determined by methylation-based cell type deconvolution or ichorCNA. Similar results were found in the validation dataset. A multi-omics (AS, tMAD, estimated tumor fraction, and DMR methylation) model could predict EAC, HGD, or NDBE with 0.81 accuracy through cross validation (10X) and 0.73 in the independent dataset. This increased to 0.98 and 0.90 in separating high risk EAC/HGD from NDBE, respectively. Conclusion: Multi-omics data obtained from single WGMS allow for a comprehensive analysis of BE samples with varying dysplasia levels. Using classification modeling, these characteristics can be effectively used to accurately identify and distinguish between EAC and HGD from NDBE, suggesting their utility as adjuncts to endoscopic surveillance biopsies. Citation Format: Zhifu Sun, Caroline L. Matchett, Seth W. Slettedahl, William R. Taylor, Panwen Wang, Calise Berger, Caryn E. Anderson, Melissa A. Passe, Ramona Lansing, Collin E. Chalmers, Patrick H. Foote, Jeanette E. Eckel Passow, Doug W. Mahoney, John B. Kisiel, Prasad G. Iyer. Multi-omics cancer risk assessment of Barrett’s esophagus with DNA methylation sequencing from endoscopic brush samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB249.

Abstract Lynch syndrome (LS) markedly increases risks of colorectal and endometrial cancers. Early detection biomarkers for LS cancers could reduce the needs for invasive screening and surgical prophylaxis. To validate a panel of methylated DNA markers (MDM) previously identified in sporadic colorectal cancer and endometrial cancer for discrimination of these cancers in LS. In a case–control design, previously identified MDMs for the detection of colorectal cancer and endometrial cancer were assayed by qMSP on tissue-extracted DNA. Results were normalized to ACTB values within each sample. Least absolute shrinkage and selection operator models to classify colorectal cancer and endometrial cancer were trained on sporadic cases and controls and then applied to classify colorectal cancer and endometrial cancer, in those with LS, and cross-validated. We identified colorectal cancer cases (23 with LS, 48 sporadic), colorectal controls (32 LS, 48 sporadic), endometrial cancer cases (30 LS, 48 sporadic), and endometrial controls (29 LS, 37 sporadic). A 3-MDM panel (LASS4, LRRC4, and PPP2R5C) classified LS-CRC from LS controls with an AUC of 0.92 (0.84–0.99); results were similar for sporadic colorectal cancer. A 6-MDM panel (SFMBT2, MPZ, CYTH2, DIDO1, chr10.4479, and EMX2OS) discriminated LS-EC from LS controls with an AUC of 0.92 (0.83–1.0); the AUC for sporadic endometrial cancer versus sporadic controls was nominally higher, 0.99 (0.96–1.0). MDMs previously identified in sporadic endometrial cancer and colorectal cancer discriminate between endometrial cancer and benign endometrium and colorectal cancer and benign colorectum in LS. This supports the inclusion of patients with LS within future prospective clinical trials evaluating endometrial cancer and colorectal cancer MDMs and may provide a new avenue for cancer screening or surveillance in this high-risk population. Prevention Relevance: Lynch syndrome (LS) markedly increases risks of colorectal and endometrial cancers. Early detection biomarkers for LS cancers could reduce the needs for invasive screening and surgery. Methylated DNA markers previously identified in sporadic endometrial cancer and colorectal cancer discriminate between benign and cancer tissue in LS.

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 &gt;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.

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]

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]