Insights into the role of MSLN-positive circulating tumor cell as an auxiliary diagnostic biomarker in epithelial ovarian cancer

Zhu Yang^1*Hang Xu¹Min Wang¹Shuting Wu²Qinke Li¹Jinlong Wang¹Siying Zhang¹Qiongming Liu³Mengting Wang³Ruifang Li⁴Zhiyuan Hu⁵Yi Liu¹

• ¹Second Affiliated Hospital, Chongqing Medical University, Chongqing, China
• ²Fujian Medical University, Fuzhou, Fujian Province, China
• ³Nanopep Biotech Corporation, Beijing, China
• ⁴Shanxi Medical University, Taiyuan, Shanxi Province, China
• ⁵Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Nanoscience Center - Chinese Academy of Sciences, Haidian District, Beijing, China

Background: Epithelial ovarian cancer (EOC) currently lacks highly specific biomarkers for clinical screening. This study aimed to identify and validate novel auxiliary diagnostic markers for EOC. Methods: Through integrated analysis of transcriptome sequencing data and single-cell RNA sequencing from public databases, we identified mesothelin (MSLN) as an EOC-specific target. MSLN expression was subsequently validated in EOC cell lines and clinical specimens by flow cytometry, immunofluorescence, and immunohistochemistry. The capture efficacy of Pep@MNPs (Magnetic nanoparticles functionalised with EpCAM peptides) on EOC cells was verified by scanning electron microscopy, Prussian blue staining and cell spiked-blood capture experiments. In a prospective cohort of 35 patients with undiagnosed ovarian masses, we employed immunofluorescence staining to detect MSLN-positive circulating tumor cells (MSLN(+)CTCs) and assessed their diagnostic performance using receiver operating characteristic (ROC) analysis. Results: MSLN was highly expressed in EOC cell line and tissues but lowly expressed in normal ovarian surface epithelial tissues. EOC cells can be captured by Pep@MNPs with high sensitivity and specificity. ROC curves analysis showed that MSLN(+)CTCs differentiated between benign and malignant lesions of the ovary with a sensitivity of 66.67% and a specificity of 95% (p = 0.0014), which was more specific than cancer antigen 125 (CA125) (sensitivity: 71.43%; specificity: 94.47%; p < 0.0001) and human epididymis protein 4 (HE4) (sensitivity: 84.62%; specificity: 89.47%; p = 0.0002). When MSLN(+)CTCs were combined with CA125, the sensitivity was 92.86% and the specificity was 94.74%, p < 0.0001, which greatly improved the diagnostic sensitivity while preserving high specificity. Conclusions: MSLN(+)CTCs represent a highly specific auxiliary biomarker for differentiating benign and malignant ovarian lesions. The combination of MSLN(+)CTCs with CA125 provides an optimal balance between sensitivity and specificity, offering promising clinical utility for EOC diagnosis.