BACKGROUND: There is increasing evidence that high-risk human papilloma virus (HPV) is involved in cancers in addition to cervical cancer. For example, it is generally accepted that HPV has a role in a significant proportion of head and neck tumours, and it has long been hypothesised that hormone dependent oncogenic viruses, such as HPV may have causal roles in some human breast cancers. A number of reports have identified HPV DNA in breast tissue and breast cancer specimens, but these rely on standard polymerase chain reaction (PCR), which is criticised for its propensity for contamination. METHODS: We have used two different technologies, in situ and standard PCR (with sequencing), and histology based on light microscopy. RESULTS: We unambiguously demonstrate the presence of high-risk HPV in the cells of breast cancer specimens and breast cancer cell lines. In addition, we also show that the oncogenic characteristics of HPV associated breast cancer are very similar to HPV-associated cervical cancer. Specifically, that putative koilocytes are present in some HPV associated breast cancers. INTERPRETATION: The above observations indicate a likely causal role for high-risk HPV in human breast cancer and offer the possibility of primary prevention of some breast cancers by vaccination against HPV.

BackgroundThe 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 MethodsAll 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).ResultsEBV 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.ConclusionsWe 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.

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Human papilloma viruses (HPVs) are accepted as being carcinogenic in human cervical and anogenital cancers. The suspicion that HPVs may also have a role in human breast cancer is based on the identification of HPVs in human breast tumours and the immortalisation of normal human breast cells by HPV types 16 and 18. For this investigation, DNA that had been previously extracted and fresh frozen at À701C from 50 unselected invasive ductal breast cancer specimens were screened by polymerase chain reaction (PCR) for HPV type 16, 18 and 33 gene sequences. We show that HPV 18 gene sequences are present in DNA extracted from breast tumours in Australian women. Overall, 24 (48%) of the 50 samples were HPV positive. Overall no correlations with tumour grade, patient survival, steroid receptor status, ERB-2, p53 expression and mutation were observed. Human papilloma viruses may have a role in human breast cancer. We speculate that HPVs may be transmitted by hand from the female perineum to the breast. It is accepted that human papillomavirus (HPV) types 16 and 18 are carcinogenic, and that probably HPV types 31 and 33 are also carcinogenic in human cervical and anogenital cancers (IARC, 1995). The suspicion that HPVs may also have a role in human breast cancer is based on the identification of HPVs in human breast tumours and the immortalisation of normal human breast cells by HPV 16 and 18 (Band et al, 1990;De Villiers et al, 2005).Human papilloma virus 16 has been identified in breast tumours in Italian women and breast tumours in Norwegian women who had previous cervical neoplasia (Hennig et al, 1999). Human papilloma virus 33 has been identified in breast cancer in Chinese and Japanese women (Yu et al, 1999) MATERIALS AND METHODSFor this investigation, DNA that had been previously extracted and fresh frozen at À70 O C from 50 unselected invasive ductal breast cancer specimens were screened by polymerase chain reaction (PCR) for HPV type 16, 18 and 33 gene sequences. The DNA samples were amplified twice per sample using GenomiPhit DNA Amplification Kit (Amersham Biosciences). The DNA quality was confirmed by PCR, amplifying 268 bp of the b-globin gene. These samples were then screened for the presence of HPV by PCR using primers that could detect 140 bp in the E6 region of HPV 16, 18 and 33 (Yu et al, 1999). DNA extracted from cervical cancer cell lines HeLa and SiHa cells were used as positive controls for HPV 18 and 16, respectively. Plasmid plink 322 HPV 33 was used as a positive control for HPV 33. DNA from leukaemia Raji cells was used a negative control. The PCR products were separated on 7.6% PAGE and visualised by SYBR Green I (Molecular Probes). The screening was repeated five times using different batches of DNA samples amplified by the GenomiPhit DNA Amplification Kit (Amersham Biosciences). Human papilloma virus-positive samples were sequenced (there was sufficient material to sequence 18 of 24 HPV-positive samples).Grade of tumour and survival of patients were known for each sample. Screening for exons 5...

The E6 oncoprotein of human papillomaviruses (HPVs) that are associated with cervical cancer utilizes the cellular ubiquitin–protein ligase E6-AP to target the tumor suppressor p53 for degradation. In normal cells (i.e., in the absence of E6), p53 is also a target of the ubiquitin–proteasome pathway. Under these conditions, however, p53 degradation is mediated by Mdm2 rather than by E6-AP. Here we show in a mutational analysis that, surprisingly, the structural requirements of p53 to serve as a proteolytic substrate differ between E6 proteins derived from different HPV types and, as expected, between Mdm2 and E6 proteins in vitro and in vivo . Stable expression of such mutants in HPV-negative and HPV-positive cell lines demonstrates that in HPV-positive cancer cells, the E6-dependent pathway of p53 degradation is not only active but, moreover, is required for degradation of p53, whereas the Mdm2-dependent pathway is inactive. Because the p53 pathway was reported to be functional in HPV-positive cancer cells, this finding indicates clearly that the ability of the E6 oncoprotein to target p53 for degradation is required for the growth of HPV-positive cancer cells.