Krockenberger, Martin

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 The multi-target stool DNA (mt-sDNA) test screens for colorectal cancer by analyzing DNA methylation/mutation and hemoglobin markers to algorithmically derive a qualitative result. A new panel of highly discriminant candidate methylated DNA markers (MDM) was recently developed. Performance of the novel MDM panel, with hemoglobin, was evaluated in a simulated screening population using archived stool samples weighted to early-stage colorectal cancer and prospectively collected advanced precancerous lesions (APL). Marker selection study (MSS) and separate preliminary independent verification studies (VS) were conducted utilizing samples from multi-center, case–control studies. Sample processing included targeted MDM capture, bisulfite conversion, and MDM quantitation. Fecal hemoglobin was quantified using ELISA. Samples were stratified into 75%/25% training-testing sets; model outcomes were cross-validated 1,000 times. All laboratory operators were blinded. The MSS included 232 cases (120 colorectal cancer/112 APLs) and 490 controls. The VS featured 210 cases (112 colorectal cancer/98 APLs) and 567 controls; APLs were 86.7% adenomas and 13.3% sessile serrated lesions (SSL). Average age was 65.5 (cases) and 63.2 (controls) years. Mean sensitivity in the VS from cross-validation was 95.2% for colorectal cancer and 57.2% for APLs, with specificities of 89.8% (no CRC/APLs) and 92.4% (no neoplasia). Subgroup analyses showed colorectal cancer sensitivities of 93.4% (stage I) and 94.2% (stage II). APL sensitivity was 82.9% for high-grade dysplasia, 73.4% for villous lesions, 49.8% for tubular lesions, and 30.2% for SSLs. These data support high sensitivity and specificity for a next-generation mt-sDNA test panel. Further evaluation of assay performance will be characterized in a prospective, multi-center clinical validation study (NCT04144738). Prevention Relevance: This study highlights performance of the next-generation mt-sDNA test, which exhibits high sensitivity and specificity for detecting colorectal cancer and APLs. This noninvasive option has potential to increase screening participation and clinical outcomes. A multi-center, clinical validation trial is underway. See related commentary by Bresalier, p. 93

63 Background: Population-based colorectal cancer (CRC) screening can reduce mortality by detecting and removing advanced precancerous lesions (APL) and early-stage invasive disease. One guideline-included strategy is the multi-target stool DNA test (mt-sDNA), which combines detection of methylation DNA markers (MDMs), KRAS mutations, and fecal hemoglobin. Since the mt-sDNA pivotal study was conducted, novel biomarkers have been discovered. A panel of highly discriminant MDMs ( LASS4, LRRC4, PPP2R5C, and reference marker ZDHHC1) was identified through a blinded, case-control study of archival specimens. Here, we evaluated the performance of this novel mt-sDNA panel, combined with fecal hemoglobin, in archival stool samples weighted to early-stage CRC and prospectively collected APL, simulating a screening population. Methods: The verification study featured 777 samples—210 cases (112 CRC [49 stage I, 38 stage II, 17 stage III, and 8 stage IV] and 98 APL) and 567 controls (176 non-APL and 391 colonoscopy-negative)—from three trials (NCT01397747, NCT01260168, and NCT02503631). Median APL size was 12 mm (interquartile range: 10 mm to 15 mm), with 86.7% adenomas (n = 85) and 13.3% sessile serrated polyps (SSPs; n = 13). The average age was 65.5 years for cases (57% men) and 63.2 for controls (47% men). Samples were processed through homogenization, targeted MDM capture, bisulfite conversion, and MDM quantitation using Long‐probe Quantitative Amplified Signal (LQAS). Fecal hemoglobin was quantified using enzyme-linked immunosorbent assay (ELISA). Samples were split into stratified 75%/25% training-testing sets and model outcomes were cross-validated 1,000 times. Results: Mean performance from the cross-validation analysis is summarized in the table below. Overall sensitivity was 95.2% for CRC and 57.2% for APL, with specificities of 89.8% (no CRC/APL) and 92.4% (no neoplasia). Subgroup analyses showed high sensitivity for early-stage CRC, with 93.4% at stage I and 94.2% at stage II. By APL subtype, sensitivity was 82.9% for high-grade dysplasia, 73.4% for villous lesions, 49.8% for tubular lesions, and 30.2% for SSPs. Conclusions: These data support high sensitivity and specificity for a second-generation mt-sDNA panel. A multicenter pivotal trial evaluating the panel is underway (NCT04144738). [Table: see text]