HPV Viral Load and In Situ Hybridization Signal Patterns Indicate Diverse Patterns of Dysregulation in Cervical Carcinoma Pathogenesis
Mark F Evans, Kavita Munjal, Vanitha Rajendran, Christine S Adamson, Zhihua Peng, Kumarasen Cooper. University of Vermont, Burlington, VT; Sri Aurobindu Institute of Medical Sciences, Indore, Madhya Pradesh, India
Background: The invasive cervical carcinoma (ICC) phenotype is understood to arise from genomic aberrations occurring consequent to chronic high-risk HPV E6 and E7 gene expression. There have been few large-scale studies examining how HPV status is impacted by tumorigenesis. Using quantitative real-time PCR (qPCR), chromogenic in situ hybridization (CISH), and immunohistochemistry (IHC) we have assessed HPV-16 heterogeneity and its implications for understanding the pathways that result in ICC.
Design: Archival ICC specimens (n=192) from Madhya Pradesh, India, HPV-16 positive by GP5+/6+ PCR, were assessed by qPCR, biotinyl-tyramide-based CISH and p16INK4a IHC. QPCR data were recorded as HPV-16 copies per cell equivalent; CISH signals as 'diffuse' (episomal HPV) and 'punctate' (integrated HPV), number of punctate signals per cell nucleus, and percent tumor cells stained; and, IHC staining was scored with reference to nuclear and/or cytoplasmic staining and intensity, and percent tumor cells stained.
Results: Age: 25.0-90.0; mean 48.7; SD 12.5. Histopathology: 174 squamous cell carcinomas (SCC), 12 adenosquamous (AS), 6 other. VLs ranged from 0.006 to 1780.0 HPV16 copies per cell (mean 69.0; SD: 214.7). VL was significantly lower in AS than in SCC (P=0.01) and significantly higher in women ≥46 compared to ≤45 years. CISH positive status (67.9% specimens) correlated with VL (P<0.0001) as did p16INK4a diffuse staining (78.6% ICC, P=0.046). There was also a significant relationship between CISH and p16INK4a staining (P=0.0002). One CISH positive sample showed only diffuse (episomal HPV) signals; all others showed punctate staining, with 12 showing punctate and diffuse patterns. A wide variation was observed with respect to CISH signal intensity, number of punctate signals per cell, and percentage of tumor cells showing staining. A minority (21.4%) of tumors were negative or showed sporadic p16INK4a staining, whereas others showed homogenous or heterogeneous, and/or intense, medium or weak staining that was nuclear and cytoplasmic, or cytoplasmic alone.
Conclusions: ICCs display considerable inter- and intra-tumoral heterogeneity with respect to HPV viral load and CISH staining; p16INK4a staining heterogeneity was also observed. These data suggest alternative forms of dysregulation in the pathogenesis of ICCs. Some ICCs (low viral load, CISH and p16INK4a negative) may be positive for passenger rather than driver HPV or have lost HPV expression in the course of tumor progression.
Wednesday, March 21, 2012 9:30 AM
Poster Session V # 267, Wednesday Morning