MALDI-TOF/MS Serum Spectral Profiling for Breast Cancer Discrimination Using Direct and ACN-Assisted Workflows: A Retrospective Proof-of-Concept Study

Background/Objectives: Serum matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) profiling may complement breast cancer (BrC) assessment, but retrospective studies are vulnerable to demographic and batch confounding. We evaluated direct-serum (Workflow A) and acetonitrile (ACN)-assisted (Workflow B) profiling. Methods: Workflow A analyzed 60 archived sera (30 BrC, 30 controls) with sample-level nested 10-fold cross-validation and L2-regularized logistic regression. Workflow B included 40 sample groups (20 BrC, 20 negative/comparator; approximately four spots/sample); replicates were aggregated before repeated sample-level five-fold cross-validation and batch/source diagnostics. Results: Workflow A yielded an area under the receiver operating characteristic curve (AUC) of 0.932, but groups were not age matched (52 vs. 45 years; standardized mean difference approximately 1.03), precluding cancer-specific interpretation independent of age. Workflow B yielded a mean AUC of 0.978 ± 0.007 and a balanced accuracy of 0.968, but negative/comparator batch/date classification reached an AUC of 0.970, and cross-batch transfer was asymmetric (1.00 vs. 0.64); five comparator sera were commercially sourced. Conclusions: Workflow A provides preliminary group-discrimination evidence but remains age-confounded. Workflow B demonstrates strong apparent internal discrimination under substantial batch/source confounding and is best viewed as an analytical stress test, not independent validation. Candidate m/z signals remain unidentified; age-matched, batch-balanced external validation and orthogonal molecular identification are required before cancer-specific or biomarker claims.