Quantitative assessment of STAT3 and HPV16 E6 transcripts using Flow-FISH approach for early detection of progressive cervical lesions

BACKGROUND: hybridization (FISH) showed diagnostic clinical relevance in screening for pre-cancerous cervical lesions. However, use of FISH does not provide clear objective diagnostic threshold to establish accurate signal positivity. AIM: To assess the feasibility of FISH for quantitative detection of STAT3 and HPV E6/E7 transcripts using a flow cytometry-based approach. METHODS: HPV-negative (C33a), HPV16-positive (SiHa) and HPV18-positive (HeLa) cervical cancer cell lines were analyzed for STAT3 and HPV E6/E7 transcript expression using FISH approach. Signal specificity and distribution were assessed by fluorescence microscopy using target-specific and scrambled probes. The same probes were further evaluated using flow cytometry-based FISH to determine their suitability for quantitative transcript detection. RESULTS: Fluorescence microscopy revealed strong and discrete STAT3-associated signals in SiHa and HeLa cells, whereas C33a cells exhibited comparatively diffuse fluorescence. Scrambled control probes produced minimal background staining, supporting the specificity of STAT3 probe. HPV16 E6 probe also produced detectable signals but comparable fluorescence intensity was observed across all cell lines irrespective of HPV status and was similar to scrambled probe controls. Moreover, when assessed by flow cytometry-based FISH, the same probes displayed limited performance. STAT3-positive populations were low and accounted for approximately 10% of cells, while no clearly distinguishable HPV16 E6-positive population could be identified in any of the tested cell lines. These findings indicate that optimized microscopy-based FISH assay may not be directly compatible with flow-based transcript detection platforms. CONCLUSION: Therefore, re-optimization of assay is required for quantitative transcript detection for HPV-associated cervical cancer screening using flow cytometry-based FISH. The manuscript addresses the bottlenecks and potential strategies to mitigate the issues.