Epstein-Barr virus, human papillomavirus and mouse mammary tumour virus as multiple viruses in breast cancer

Abstract

Background: The purpose of this investigation is to determine if Epstein Barr virus (EBV), high risk human papillomavirus (HPV), and mouse mammary tumour viruses (MMTV) co-exist in some breast cancers.

Materials and methods: All the specimens were from women residing in Australia. For investigations based on standard PCR, we used fresh frozen DNA extracts from 50 unselected invasive breast cancers. For normal breast specimens, we used DNA extracts from epithelial cells from milk donated by 40 lactating women. For investigations based on in situ PCR we used 27 unselected archival formalin fixed breast cancer specimens and 18 unselected archival formalin fixed normal breast specimens from women who had breast reduction surgery. Thirteen of these fixed breast cancer specimens were ductal carcinoma in situ (dcis) and 14 were predominantly invasive ductal carcinomas (idc).

Results: EBV sequences were identified in 68%, high risk HPV sequences in 50%, and MMTV sequences in 78% of DNA extracted from 50 invasive breast cancer specimens. These same viruses were identified in selected normal and breast cancer specimens by in situ PCR. Sequences from more than one viral type were identified in 72% of the same breast cancer specimens. Normal controls showed these viruses were also present in epithelial cells in human milk - EBV (35%), HPV, 20%) and MMTV (32%) of 40 milk samples from normal lactating women, with multiple viruses being identified in 13% of the same milk samples.

Conclusions: We conclude that (i) EBV, HPV and MMTV gene sequences are present and co-exist in many human breast cancers, (ii) the presence of these viruses in breast cancer is associated with young age of diagnosis and possibly an increased grade of breast cancer.

Figure 1. Typical nested PCR amplification for EBV, HPV and MMTV.

A. EBV PCR for the second round of amplification of a 209 bp fragment from DNA extracted from 9 breast cancer specimens, B is the no DNA control, Raji DNA as a positive control (+) and M is a size ladder from Puc Hinf1. (12). B. HPV PCR for the second round amplification of a 148 bp fragment from 10 breast DNA extractions. RB is the reagent control,(+ ) is the positive control from Hela DNA containing HPV 18, B1 is a first round no DNA control for the PCR, B2 is a second round no DNA control. C. MMTV PRC for the second round of amplification of a 643 bp fragment from DNA extracted from 8 patient samples. RB is the reagent blank. (+) is the positive control of DNA extracted from mouse tails. B1 is the first round no DNA control, subjected to a second round of PCR. D. Typical b-globin PCR (single amplification) for 6 breast archival specimens showing the integrity of the DNA. + is a positive control from Hela DNA, B is the no DNA control.

Figure 2. Variable sequence region of Epstein-Barr viral PCR products compared to the EBV genome (B95-8 strain).

EBV, EBV genome; Raji DNA used as a positive control; A1-4 sequences are based on DNA extracts from archival formalin fixed invasive ductal carcinoma (idc) breast cancer specimens; B1-6 sequences are based on DNA extracts from fresh frozen idc breast cancer specimens. M1-2 sequences are from normal breast epithelial cell (milk) DNA extracts. The alignment of sequences demonstrates the high level of nucleotide homology between the EBV genome, the Raji EBV positive control and EBV identified by standard PCR in fixed, fresh idc breast cancer specimens, and normal breast specimens.

Figure 3. EBV, HPV and MMTV identified in the same ductal carcinoma in situ specimen by in situ PCR.

A.EBV positive, B. HPV positive, C. MMTV positive, D. Negative control – no primer, E. Negative control -no Taq. HPV associated koilocytes are present in B.All photographs were taken with a 20X objective and have been cropped. Variations in colour detection can be seen when the in-situ PCR was done at a different time.

Figure 4. HPV and EBV identified by in situ PCR in the same breast cancer cell nuclei – Ductal carcinoma in situ.

A.HPV (inner nested primers X 200). B. HPV (outer nested primers X 400). C. HPV (inner nested primers X 400). D. EBV (inner nested primers X 200). E. EBV (outer nested primers X 400). F. EBV (inner nested primers X 400). G. MMTV negative (inner nested primers X 200). H. Negative control (no primers X 200).

Figure 5. Putative Reed Sternberg cells in ductal carcinoma in situ (by immunohistochemistry).

Panel A. Positive EBNA 1 expression in a putative Reed Sternberg cells. Panel B. Positive CD 15 expression in putative Reed Sternberg cells. It is not possible to determine whether these cells are Reed Sternberg or granuloma cells.

Figure 6. Positive EBNA, CD 15 and LMP1 expression in the same invasive ductal carcinoma breast cancer specimen.

Panel A. Positive EBNA1 expression. Panel B. Positive CD15 expression. Panel C. Positive LMP1 expression. It is not possible to determine whether these cells are Reed Sternberg or granuloma cells.