Kielar‐Grevstad, Elle

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.

Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death in the United States and worldwide. HCC screening among patients at elevated risk is associated with improved early detection and overall survival, but current ultrasound-based screening strategies are challenged by low adherence and suboptimal sensitivity. Blood-based screening tests have the potential to enhance adherence and improve early-stage HCC detection if they can demonstrate favorable performance compared with ultrasound-based screening. Here we present the design and rationale for ALTUS (A lternative to Ultrasound), a prospective, longitudinal, multicenter study in the United States to investigate the performance of the multi-target HCC blood test (mt-HBT) for the detection of HCC in a screening population. Methods: Adults with liver cirrhosis or chronic hepatitis B infection will be enrolled to undergo standard-of-care screening imaging and concurrent blood collection for the mt-HBT. All participants will undergo contrast-enhanced CT or MRI imaging with central radiology LI-RADS assessment as the reference method to determine HCC status. Participants without an identified malignancy will undergo a second screening visit and blood collection, and longitudinal clinical and imaging data will be collected up to 18 months from enrollment. The primary study objectives are to demonstrate that the mt-HBT is non-inferior to ultrasound for early-stage HCC sensitivity and to assess mt-HBT HCC specificity. The secondary objective is to assess mt-HBT overall sensitivity. Results: ALTUS is in progress with readout of the primary analysis expected in 2025. Conclusions: This prospective head-to-head comparison of the mt-HBT versus ultrasound will provide novel data regarding the performance and utility of the mt-HBT for HCC screening.

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.