Validation of protein biomarkers for early detection of cervical cancer and squamous intraepithelial lesions.

e17506 Background: Human papillomavirus infections often resolve on their own; however, ongoing high risk HPV infections can progress to cervical cancer, a significant global health issue responsible for approximately 660,000 new cases and 350,000 deaths in 2022. Current screening methods emphasize cytology or HPV DNA detection, with a limited focus on protein biomarkers. Thus, identifying sensitive and specific cervical cancer protein biomarkers is essential, as this analysis provides opportunities for developing affordable point-of-care screening tests. Methods: This study optimized a unified protocol for cell lysis and protein extraction to validate biomarkers using cervical cell lines and cervical swab samples. Four proteins—Topoisomerase II Alpha (TOP2A), Minichromosome Maintenance Complex Component 2 (MCM2), Valosin Containing Protein (VCP), and Cyclin-Dependent Kinase Inhibitor 2A (p16INK4a)—were quantified using enzyme-linked immunosorbent assays. Furthermore, the expression of target biomarkers in cervical tumors (G1-G3) and squamous intraepithelial lesions (SIL) was evaluated using immunohistochemistry. Cytological evaluation of swab samples categorized them as normal, reactive cellular changes (RCC), atypical squamous cells of undetermined significance (ASC-US), low grade SIL (LSIL), or high grade SIL (HSIL). The study utilized cervical cancer cell lines HeLa, Ca Ski, HT-3, C-33A, and PCS. Statistical analysis was performed using a gamma-generalized linear mixed model, and imaging tools (Aperio and Visiopharm) were employed for tissue analysis. Results: RIPA buffer and acetone precipitation achieved optimal cell isolation and high-yield protein recovery. Biomarker protein concentrations were normalized to Beta-actin. Compared to normal samples, HSIL, LSIL, and ASC-US samples showed overexpression of p16INK4a, while HSIL samples exhibited higher levels of VCP. VCP and p16INK4a were overexpressed across all cancer cell lines compared to primary cells. MCM2 was overexpressed in HT-3 and HeLa, while TOP2A was underexpressed in Ca Ski. Further tissue analysis confirmed strong staining of all target biomarkers in precancerous and cancerous tissues, with significant differences in VCP, TOP2A, and p16INK4a expression observed across tumor grades and SIL. Conclusions: Through a simplified sample preparation and protein extraction protocol, four protein biomarkers were effectively identified and validated for cervical cancer screening. This research emphasizes the importance of detecting a panel of proteins expressed in both HPV-positive and HPV-negative cervical cancers. The results highlight the promise of these target proteins in recognizing precancerous lesions and the potential benefits of incorporating protein biomarker-based screening for improving early detection and cervical cancer outcomes, especially in resource limited settings.