MALDI-MSI Profiling of Effusion Cytology Cell Blocks Distinguishes High-Grade Serous Ovarian Carcinoma from Benign Effusions.

Background/Objectives: Cytological analysis of pleural and peritoneal effusions is minimally invasive but may show limited sensitivity for malignancy detection because tumor cells can be scarce or morphologically difficult to identify. Proteomics-based mass spectrometry imaging (MSI) enables direct molecular profiling of cytological specimens and may improve the distinction between malignant and benign samples. This study investigated whether MALDI-based MSI profiling of formalin-fixed paraffin-embedded (FFPE) cytology cell blocks could discriminate high-grade serous ovarian carcinoma (HGSOC) from benign effusions and identify discriminatory peptide signatures. Methods: Forty-one FFPE cytological specimens derived from pleural and peritoneal effusions were analyzed, including 18 malignant samples and 23 benign control specimens with reactive or inflammatory cytological backgrounds. MALDI-MSI analyses were performed using proteomic profiling. In a preliminary comparative experiment, two section thicknesses (3 µm and 5 µm) were evaluated to optimize ion peak intensity yield. Classification analyses using linear discriminant analysis (LDA) and support vector machine (SVM) models were performed to identify discriminatory peptide signatures. Results: Comparative analysis of average mass spectra identified multiple differentially expressed ions with significant discriminatory performance (AUROC ≥ 0.7 or ≤0.3; Wilcoxon/Kruskal-Wallis, p < 0.001). Classification analyses achieved accuracy ranged from 91% to 94% for the discrimination of malignant and benign samples. Differential proteomic profiling identified Complement C3, Perilipin-3, arachidonate 5-lipoxygenase, Leukotriene A-4 hydrolase, fibrinogen beta and gamma chains, and serotransferrin as discriminatory proteins associated with immune modulation, lipid metabolism, cytoskeletal and extracellular matrix organization, and metabolic regulation. Notably, Complement C3 was found to be overexpressed in malignant tumor cells, supporting its potential role as a marker of tumor presence and progression. Conclusions: Proteomics-based mass spectrometry imaging enabled reliable discrimination of HGSOC from benign cytological specimens and revealed cancer-associated proteins linked to relevant biological processes. These findings support MALDI-MSI as a complementary molecular approach for the classification of challenging cytological specimens in routine diagnostic pathology.