Comparative Evaluation of E6/E7 mRNA Expression and Viral DNA Load in Cervical Lesions with Multiple High-Risk HPV Infection.

Abstract

Multiple high-risk human papillomavirus (hrHPV) infections are frequently detected in cervical screening, but their biological interactions and clinical significance remain unclear. Because HPV DNA detection indicates viral presence but not transcriptional activity, this exploratory study evaluated viral load, genotype-specific E6/E7 mRNA expression, localization, and follow-up. Among 12,000 cervical specimens tested for hrHPV, 598 were positive and 72 showed multiple genotypes. Viral load was assessed using the BioPerfectus Multiplex Real-Time assay. A tissue-available subset of 36 dual HPV16+52 or HPV16+58 cases was analyzed by duplex RNAscope for genotype-specific E6/E7 mRNA expression and localization. Diagnoses were categorized as benign, LSIL, or HSIL. Viral load and RNAscope analyses used incompletely matched subsets and were interpreted as complementary, not paired. Elevated viral load (≥4.00 log10 copies/10,000 cells) was associated with HSIL. In cohort-level analyses, diffuse (+++) E6/E7 mRNA expression was more frequent in HSIL than elevated viral load (95.2% vs 55.5%, P = 0.01). RNAscope-negative cases were mostly benign or LSIL and showed no progression. Genotype-specific signals were spatially distinct, with rare overlap. Dual-positive cells showed fewer RNA signals than adjacent single-positive cells (P < 0.001). Higher E6/E7 mRNA expression showed possible association with LSIL progression, but events were limited. In multiplex hrHPV infection, transcriptional activity may better reflect cervical lesion biology than viral DNA burden alone. Genotype-specific E6/E7 mRNA expression may complement HPV DNA load. Spatially distinct signals and reduced signal intensity in rare dual-positive cells suggest possible interactions between co-infecting hrHPV genotypes, but require prospective validation in fully matched cohorts.