Abstract LB252: Novel performance quantification of MCED testing to aid clinical decisions: Analysis of a sequential reflex blood-based methylated ctDNA test

Abstract Background: Although ctDNA-based liquid biopsies show promise for multi-cancer early detection (MCED), current performance evaluations using aggregate metrics across cancers mask biological heterogeneity and cancer-specific test performance, limiting clinical validity and utility. To better elucidate the clinical validity of MCED testing, we assessed the detection performance of a novel sequential reflex ctDNA-based test in a case-control study, focusing on cancer-specific intrinsic accuracy and incidence-adjusted PPV of each tissue of origin (TOO). Methods: Peripheral blood samples (NCT05435066) from treatment-naïve cancer patients (N=1039) and individuals with no reported cancer (N=1,000) were analyzed using a sequential reflex test by Harbinger Health. The Primary Test profiles ctDNA methylation for cancer signal detection followed by a Reflex Test analyzing a broader set of biomarkers for cancer signal confirmation and TOO localization. Test performance was established using 10-fold cross-validation with patient-level splits. Cancer incidence for age>50 was estimated at 1.32% based on SEER 2022 data. Prospective PPV was calculated using cancer-specific intrinsic accuracy estimated from the case-control study in combination with SEER incidence values. Results: In this cohort (mean age 57.6 ± 14.3 years; 61.5% female; 71.6% White; 17.4% Black or African American), sequential reflex testing achieved 98.7% specificity. In a representative selection of common or high-mortality cancers lacking organized screening, TOO-specific prospective PPVs were 94% for upper gastrointestinal (UGI), 85% for colorectal, 80% for hepatobiliary (HB: liver, biliary duct), 39% for lung, 38% for pancreaticobiliary (PB: pancreas, gallbladder), and 34% for head & neck (H&N). Corresponding odds of correct case-type were 16:1 (UGI), 6:1 (colorectal), 4:1 (HB), 2:3 (lung), 3:5 (PB), and 1:2 (H&N). Early-stage (I-II) intrinsic accuracy (sensitivity) was 70% for H&N, 51% for HB, and 40% for PB, with lower sensitivities for lung and GI cancers. In a modeled cohort of 100,000 individuals receiving the test, 107 pancreaticobiliary cases are expected (38 early stage); the test identifies 67 of the 107 (15 early stage) with odds in favor of correct classification of 3:5. Conclusion: This analytical framework for MCED testing quantifies misclassification proportions corresponding to each TOO readout, informing diagnostic pathways in healthcare systems. Our approach can facilitate MCED test optimization, standardize cross-platform comparisons, and clarify risk-benefit implications of follow-up; potentially expediting diagnosis, reducing costs, and improving equity in access. The reported sequential reflex test demonstrates high performance for early cancer detection and localization, warranting prospective validation in diverse populations. Citation Format: Elie Massaad, A. Gregory DiRienzo, Johanna B. Withers, Dorna Kashef, Jocelyn Charlton, James X. Sun, Franziska Michor, Kieran Chacko, Hutan Ashrafian. Novel performance quantification of MCED testing to aid clinical decisions: Analysis of a sequential reflex blood-based methylated ctDNA test [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB252.