Physical state & copy number of high risk human papillomavirus type 16 DNA in progression of cervical cancer

Abstract

Background & objectives: High-risk human papilloma virus (HR-HPV) infection and its integration in host genome is a key event in malignant transformation of cervical cells. HPV16 being a dominant HR-HPV type, we undertook this study to analyze if viral load and physical state of the virus correlated with each other in the absence of other confounding variables and examined their potential as predictors of progressive cervical lesions.

Methods: Both, viral load and integration status of HPV16 were determined by real time URR PCR and estimation of E2:E6 ratio in a total of 130 PGMY-RLB -confirmed, monotypic HPV16-infected cervical DNA samples from biopsies of cytology-confirmed low grade (LSIL, 30) and high grade (HSIL, 30), and invasive carcinoma, (squamous cell carcinoma SCC, 70) cases.

Results: Investigation of DNA samples revealed a gradual increase in HPV16 viral load over several magnitudes and increased frequency of integration from LSIL to HSIL and HSIL to invasive cancer in relation to the severity of lesions in monotypic HPV16-infected cervical tissues. In a substantial number of precancer (11/60) and cancer cases (29/70), HPV16 was detected in concomitant mixed form. The concomitant form of HPV16 genome carried significantly higher viral load.

Interpretation & conclusions: Overall, viral load and integration increased with disease severity and could be useful biomarkers in disease progression, at least, in HPV16-infected cervical pre-cancer and cancer lesions.

Fig. 1

Quantitative real-time PCR of HPV16 URR to determine HPV16 viral load in DNA isolated from cervical precancer and cancer tissue biopsies. Genomic DNA of HPV16 positive cervical precancer and cancer samples was amplified by type-specific HPV16 URR primers using Biorad iQ SYBR Green supermix (as described in Methods). Two-fold serial dilution of WHO HPV16 international standards starting from 5×10⁴ copies/reaction in C33a DNA diluents were used as reference (upper panels). Amplification of p53 exon5 which was used as normalization control for genomic DNA input (lower panels). DNA amplification threshold cycle analysis (left panels); standard curve analysis for determining the efficiency of reaction and calculation of viral copy number and quantitation of host genome equivalents (middle panel); Melt curve analysis showing specificity of HPV16 and p53 exon 5 amplicons were performed in each run (right panel).

Fig. 2

Determination of physical state HPV16 genome in cervical precancer and cancer tissue biopsies by HPV16 E2 and HPV16 E6 amplification. HPV16 E2 and E6 open reading frame (ORFs) were amplified using specific primers, and E2:E6 densitometric ratio was determined as described in ‘Methods’. Vector-free HPV16 full length plasmid harbouring intact E2 and E6 region was used as positive control and as a reference for normalization of E2:E6 ratio in clinical samples. Upper panels showing gel photograph of HPV16 E2 amplification (1139bp) and lower panels show HPV16 E6 amplification (506bp) in DNA of vector control and clinical samples. Normalized E2:E6 ratio of the samples is indicated at the bottom of the respective lane. Normalized E2:E6 ratio = 0 represents completely integrated HPV16 genome, value = 1 represent episomal viral genome, and values between 0 and 1 indicate mixed form of HPV16 genome. M, φX174 HaeIII-digested molecular weight marker; P, HPV16 plasmid; N, PCR negative control (C33a genomic DNA); L1-L3, LSIL cases; H1- H3, HSIL cases; C1- C4, invasive cancer cases.

Fig. 3

Association of HPV16 physical state with the viral load in different precancer and cancer cases. Distribution of normalized HPV16 E2:E6 ratio and HPV 16 viral load in cervical precancer (LSIL and HSIL) and cancer cases. Each circle indicates individual case. Normalized E2:E6 ratios and viral load were calculated as described in Methods. Normalized E2:E6 = 0 represents completely integrated HPV16; E2:E6 = 1 represents episomal viral genome, E2:E6 between 0 and 1 indicates concomitant HPV16 genome. Vertical red line and horizontal blue line represent median values of E2:E6 ratio and HPV16 viral load in each disease group, respectively.