Breast cancer and human papillomavirus infection: no evidence of HPV etiology of breast cancer in Indian women

Abstract

Background: Two clinically relevant high-risk HPV (HR-HPV) types 16 and 18 are etiologically associated with the development of cervical carcinoma and are also reported to be present in many other carcinomas in extra-genital organ sites. Presence of HPV has been reported in breast carcinoma which is the second most common cancer in India and is showing a fast rising trend in urban population. The two early genes E6 and E7 of HPV type 16 have been shown to immortalize breast epithelial cells in vitro, but the role of HPV infection in breast carcinogenesis is highly controversial. Present study has therefore been undertaken to analyze the prevalence of HPV infection in both breast cancer tissues and blood samples from a large number of Indian women with breast cancer from different geographic regions.

Methods: The presence of all mucosal HPVs and the most common high-risk HPV types 16 and 18 DNA was detected by two different PCR methods - (i) conventional PCR assays using consensus primers (MY09/11, or GP5+/GP6+) or HPV16 E6/E7 primers and (ii) highly sensitive Real-Time PCR. A total of 228 biopsies and corresponding 142 blood samples collected prospectively from 252 patients from four different regions of India with significant socio-cultural, ethnic and demographic variations were tested.

Results: All biopsies and blood samples of breast cancer patients tested by PCR methods did not show positivity for HPV DNA sequences in conventional PCRs either by MY09/11 or by GP5+/GP6+/HPV16 E6/E7 primers. Further testing of these samples by real time PCR also failed to detect HPV DNA sequences.

Conclusions: Lack of detection of HPV DNA either in the tumor or in the blood DNA of breast cancer patients by both conventional and real time PCR does not support a role of genital HPV in the pathogenesis of breast cancer in Indian women.

Figure 1

(A-D) Quantity and quality of genomic DNA from breast cancer biopsy and PCR amplification of different regions of HPV genome. A: Estimation of quantity and quality of phenol-chloroform extracted genomic DNA from breast cancer biopsies as visualized on an ethidium bromide-stained 1% agarose gel. Lane M: Hind III-digested λ-DNA molecular weight marker. Lane 1-10: Genomic DNA samples from breast cancer biopsies. B: PCR amplification of HPV L-1 consensus sequences showing the amplimer of 450 bp along with β-globin of 268 bp. Lane M: Hae III-digested φ × 174 DNA molecular weight marker. Lane 1: positive control (HPV16 plasmid DNA), Lane 2: HPV16 positive cervical cancer biopsy DNA, Lane 3: Blood DNA from breast cancer patient, Lane 4: HPV negative cell line (C33a) DNA, Lane 5: MCF-7 cell DNA, Lane 6: HeLa cell DNA, Lanes 7-18: breast cancer biopsy DNA. All were positive for β-globin but none of them positive for HPV. C: PCR amplification of HPV16 E6 showing the amplimer of 503 bp along with β-globin of 268 bp. Lane M: Hae III-digested φ × 174 DNA molecular weight marker. Lane 1: positive control (HPV16 plasmid DNA), Lane 2: Cervical cancer DNA, Lane 3: Blood DNA from breast cancer patient, Lane 4: HPV negative cell line (C33a) DNA, Lane 5: MCF-7 cell DNA, Lane 6: Human placental DNA, Lane 7:SiHa cell DNA, Lanes 8-18: breast cancer biopsy DNA showing positivity for β-globin but negative for HPV. D: PCR amplification of HPV16 E7 showing the amplimer of 468 bp along with β-globin of 268 bp. Lane M: Hae III-digested φ × 174 DNA molecular weight marker. Lane 1: positive control (HPV16 plasmid DNA), Lane 2: Cervical cancer DNA, Lane 3: Blood DNA from breast cancer patient, Lane 4: HPV negative cell line (C33a) DNA, Lane 5: MCF-7 cell DNA, Lane 6: Human placental DNA, Lane 7:SiHa cell line DNA, Lane 8-18: breast cancer biopsy DNA showing positivity for β-globin but negative for HPV.

Figure 2

(A-D) Quantitative Real time PCR of HPV. Quantitative Real time PCR of GP5+/GP6+ to detect presence of HPV infection in blood and tumour biopsy DNA from breast cancer patients. Genomic DNA isolated from blood and tumour tissue biopsies from breast cancer patients were amplified using GP5+/GP6+ L1 consensus PCR primers using Biorad iQ SYBR Green supermix as described in Methods. Ten fold dilution series of WHO HPV16 international standards in the 4 log dynamic range (5 × 104-100) in C33a DNA diluents were used as reference for generation of standard curves. (A) DNA amplification; (B) Standard curve analysis showing efficiency of reaction and calculation of viral copy number; (C) Melt curve analysis showing specificity of HPV16 amplicon and (D) Post real time PCR electrophoresis run of standards and analyzed sample amplicons.