The complementary contributions of methylation and protein biomarker classes in a multi-cancer early detection (MCED) test

Background: Cancer is a complex disease driven by a multitude of molecular alterations. Multi-cancer early detection (MCED) tests rely on highly sensitive technologies to measure these diverse tumor-associated biomarker classes in blood. A multi-biomarker MCED testing approach may have the potential to detect more cancers than a single biomarker approach, as release of biomarkers into the circulation is driven by different mechanisms. We previously developed a multi-biomarker class (methylation and protein; MP V1) MCED classifier (Gainullin, medRxiv, 2025.08.24.25334244). Herein, we describe the contributions attributable to protein (P) and methylation (M) using a newly developed (MP V2) classifier. Methods: A prospectively collected case-control study (N=3,163; 729 cancers, 2,434 non-cancers) was used to develop a refined MP V2 classifier. MP V2 was trained to achieve robust early-stage sensitivity at a target specificity of ≥97.0%. M and P classifiers were trained individually; results were combined using a logical OR overarching classifier to generate the final call. MP V2 calls were deemed positive when either the M or P assay provided a positive call. We assessed the percentage of MP V2 positive calls due to P-only, M-only, and M+P (logical AND) positive calls. Results: Of the 378/590 positive MP V2 calls in the intended use (breast and prostate cancers excluded) cohort, M+P-positive calls were observed in 45.5%, P-only in 7.4%, and M-only in 47.1%. A P-only positive call was observed in 13.9%, 9.5%, 11.1%,6.5%, 5.7%, and 6.3% of stage I (N=36), II (N=63), stages I and II combined (N=99), III (N=123), IV (N=140), and unknown stage (N=16) MP V2 positive calls, respectively. An M-only positive call was observed in 63.9%, 65.1%, 64.6%, 52.0%, 28.6%, and 62.5% of stage I, II, stages I and II combined, III, IV, and unknown stage MP V2 positive calls, respectively. An M+P positive call was observed in 22.2%, 25.4%, 24.2%, 41.5%, 65.7%, and 31.1% of stage I, II, stages I and II combined, III, IV, and unknown stage MP V2 positive calls, respectively. Of the 64/2,434 (2.6%) false positive calls, none were positive for M+P; all false positives were a result of P-only or M-only positive calls. Conclusions: M and P biomarker classes provide independent contributions for MP V2 classifier positive calls, especially in early-stage (I and II) cases, demonstrating the potential value of adding proteins to methylation analysis in MCED testing. Prospective, real-world evaluation of the MP V2 classifier is currently underway.