Development of a novel multi-locus Flow-FISH based assay for detection of progressive cervical precancer lesions

Aberrant STAT3 activation and persistent expression of HPV16 E6E7 transcripts are pivotal drivers of cervical cancer (CaCx) progression. The present study was aimed to develop a Flow cytometry- based Florescence In situ hybridization (Flow-FISH) assay for simultaneous detection of STAT3 and HPV16 E6E7 transcripts at single-cell level. A set of 48 STAT3 multi locus probes and 18 HPV16 E6E7 probes were designed using Stellaris Probe Designer. Fluorescence microscopy using these probe sets generated discrete punctate signals for both individual and simultaneous hybridizations, enabling accurate transcript identification. Flow-cytometry analysis showed quantifiable STAT3 expression across CaCx cell lines, namely HeLa, SiHa and C33a. However, HPV16 E6E7 probes showed non-specific binding, which was addressed by redesigning the probes with increased stringency. The specificity of both probe sets was then evaluated through extensive sequence alignment against all known STAT3 transcript variants (n = 27) and 98 HPV16 isolates. The redesigned phase 2 HPV16 E6E7 probes were subsequently tested in cell lines, demonstrating robust detection in HPV16-positive SiHa and CaSki cells and complete absence of signal in HPV-negative controls (C33a, MSB1, SF21) or HPV18-positive HeLa cells. Dual-color flow cytometry enabled simultaneous quantification of STAT3 and HPV16 E6E7 transcripts in both cell lines and patient's exfoliated samples. Increased dual-positive fractions across LSIL, HSIL, and SCC samples were detected that corresponded with progressive viral oncogene activity and STAT3 co-activation. Overall, the optimized probe-based Flow-FISH assay provided a sensitive, specific, and high-throughput method for transcript-level diagnostics, with potential utility for stratifying cervical lesions.