Association of Mouse Mammary Tumor Virus With Human Breast Cancer: Histology, Immunohistochemistry and Polymerase Chain Reaction Analyses.

PURPOSE: The purpose of this study is to determine whether mouse mammary tumor virus (MMTV)-associated human breast cancer has the same or similar histology to MMTV-associated mouse mammary tumors. Such associations may indicate a role for MMTV in human breast cancer. METHODS: Immunohistochemical techniques (using antibodies directed against the signal peptide p14 of the envelope precursor protein of MMTV) and polymerase chain reaction (PCR) analyses were used to identify MMTV proteins and MMTV-like envelope gene sequences in a series of breast cancers from Australian women. The histological characteristics of these human breast cancer specimens were compared with MMTV positive mouse mammary tumors. The same methods were used to study benign breast tissues which 1-11 years later developed into breast cancer. RESULTS: MMTV p14 proteins were identified in 27 (54%) of 50 human breast cancers. MMTV env gene sequences were identified by PCR in 12 (27%) of 45 human breast cancers. There was a significant correlation between the presence of MMTV (identified by p14 immunohistochemistry) in human breast cancers and histological characteristics similar to MMTV positive mouse mammary tumors (p = 0.001). There was a non-significant correlation between the presence of MMTV env gene sequences (identified by PCR) in human breast cancers and histological characteristics similar to MMTV positive mouse mammary tumors (p = 0.290). MMTV p14 proteins were identified in 7 (54%) of 13 benign breast specimens that later developed into human breast cancers. MMTV by PCR was identified in two benign specimens one of whom later developed MMTV positive breast cancer. DISCUSSION: These observations offer evidence that MMTV may be associated with characteristic human breast cancer histology. p14-based immunohistochemistry appears to be a more reliable technique than PCR for the identification of MMTV in human breast cancer. Identification of MMTV-associated p14 proteins in benign breast tissues confirms prior PCR-based studies that MMTV infection occurs before the development of MMTV positive breast cancer. CONCLUSION: Many MMTV positive human breast cancers have similar histology to MMTV positive mouse mammary tumors. MMTV infection identified in benign breast tissues precedes development of MMTV positive human breast cancer. When considered in the context of prior studies, these observations indicate a likely role for MMTV in human breast cancer.

High risk human papilloma viruses (HPVs) are present in benign prostate tissues before development of HPV associated prostate cancer.

BACKGROUND: Although high risk HPVs are associated with an increased risk of prostate cancer it is not known if they have a causal role. The purpose of this study is to investigate the potential role of human papilloma viruses (HPVs) in prostate cancer. The aims are (i) to investigate the presence and confirm the identity of high risk HPVs in benign prostate tissues prior to the development of HPV positive prostate cancer in the same patients, and (ii) to determine if HPVs are biologically active. METHODS: We used polymerase chain reaction (PCR) to identify HPVs in specimens from 52 Australian men with benign prostate biopsies who 1 to 10 years later developed prostate cancer. Immunohistochemistry (IHC) was used to assess the expression of HPV E7 oncoproteins, cytokeratin and prostate specific antigen (PSA). We used RNASeq data from The Cancer Genome Atlas (TCGA) to identify possible HPV RNA sequences in prostate cancer. RESULTS: HPV screening using standard PCR was conducted on 28 of the 52 sets of benign and later prostate cancers. HPV L1 genes were identified in 13 (46%) benign and 8 (29%) of 28 later prostate cancers in the same patients. HPV E7 genes were identified in 23 (82%) benign and 19 (68%) of 28 subsequent prostate cancers in the same patients. The same HPV types were present in both the benign and subsequent prostate cancers in 9 sets of specimens. HPV type 16 was identified in 15% of benign and 3% of prostate cancers. HPV type 18 was identified in 26% of benign and 16% of prostate cancers. Small numbers of HPV types 45, 47, 76 and 115 were also identified. High confidence RNA-Seq evidence for high risk HPV types 16 and 18 was identified in 12 (2%) of the 502 TCGA prostate cancer transcriptomes. High risk HPV E7 oncoprotein was positively expressed in 23 (82%) of 28 benign prostate specimens but only in 8 (29%) of 28 of the later prostate cancer specimens. This difference is statistically significant (p = 0.001). Prostate specific antigen (PSA) was more highly expressed in 26 (50%) of 52 prostate cancer specimens as compared to prior benign prostate specimens in the same patients. CONCLUSIONS: High risk HPVs are present in benign prostate tissues prior to the development of HPV positive prostate cancer. There is a significantly higher expression of HPV E7 oncoproteins in benign prostate tissues as compared to late prostate cancer that subsequently developed in the same patients. This observation suggests that HPV oncogenic activity is an early phenomenon in a majority of prostate oncogenesis. TCGA RNA-Seq data suggests that HPV is biologically active in some prostate tumour samples.

Erratum to: Mouse mammary tumor-like virus (MMTV) is present in human breast tissue before development of virally associated breast cancer.

[This corrects the article DOI: 10.1186/s13027-016-0113-6.].

Mouse mammary tumor-like virus (MMTV) is present in human breast tissue before development of virally associated breast cancer.

BACKGROUND: There is substantial evidence that a virus homologous to mouse mammary tumor virus (MMTV) may have a role in human breast cancer. The present study indicates that those who developed breast cancer associated with an MMTV-like virus had this virus in their non-cancerous breast tissues years before the cancer developed. METHODS: Polymerase chain reaction (PCR) techniques and sequencing were used to identify MMTV-like envelope gene sequences (MMTV-like env sequences) in Australian benign breast biopsy specimens from women who several years later developed breast cancer. Murine contamination was excluded by stringent laboratory procedures, and the absence of intracisternal A particle sequences and mitochondrial cyclooxygenase sequences. RESULTS: MMTV-like env sequences (also called HMTV sequences to denote their source) were found in 9 of 25 breast cancer specimens (36%). Among 25 non-cancerous breast biopsies of these same patients taken 1 to 11 years earlier, six contained MMTV-like sequences (24%). Five of the six were among the nine virally-associated breast cancers. In two pairs of specimens, benign and malignant, env sequences were 97% identical. CONCLUSIONS: The identification of MMTV (MMTV-like) sequences in breast tissues prior to the development of MMTV positive breast cancer fulfills a key criterion for a possible causal role for the MMTV-like virus in human breast cancer.

Early Human Papilloma Virus (HPV) Oncogenic Influences in Breast Cancer.

BACKGROUND: Human papilloma viruses (HPVs) may act early in breast oncogenesis ("hit-and-run" phenomena). METHODS: The authors used immunohistochemistry for the identification of HPV E7 oncogenic protein expression in 32 sets of benign and subsequent breast cancer specimens from the same Australian patients. RESULTS: HPV E7 oncoprotein was clearly expressed in the nuclei of 23 (72%) of the 32 benign specimens and 20 (62.5%) of the subsequent 32 breast cancer specimens in the same patients. There was no HPV E7 protein expression in seven (30%) of the 23 breast cancer specimens that had prior HPV E7 protein-positive benign breast biopsies in the same patients. CONCLUSIONS: This observation suggests that HPV oncogenic influences occur early in some breast cancers. This finding confirms the previous observations. This early influence of HPVs may be the reason why there is no increase in the prevalence of HPV-associated breast cancer in immunocompromised patients as compared to HPV-associated cervical cancer.

Identification of Human Papilloma Viruses in Atheromatous Coronary Artery Disease.

OBJECTIVE: To identify human papilloma viruses (HPV) in atheromatous coronary arteries. BACKGROUND: Atheromatous arterial disease is primarily an initial inflammatory response to unknown stimuli. The crucial question is "what causes the initial inflammation in atheromatous disease?" HPV infections may be relevant as US women with vaginal, high risk for cancer, HPV infections, are at up to threefold increased risk of cardiovascular disease as compared with vaginal HPV-negative women. These studies did not include analyses of HPV in atheromatous coronary arteries. METHODS: Atheromatous coronary arteries were identified and collected from 20 deceased donors. Polymerase Chain Reaction techniques were used to identify HPV gene sequences. Immunohistochemistry methods were used to identify HPV E7 proteins. RESULTS: HPV types 16 and 18 were identified in 11 (55%) of 20 specimens. HPV E7 protein was identified in 10 (50%) of 20 specimens. Positive and negative HPV identification and HPV E7 expression in coronary smooth muscle cells were significantly correlated (cc = 0.503, p = 0.024). The HPV E7 proteins were expressed in smooth muscle cells and plasma cells, foam cells, and macrophages located in the atheromatous plaque. HPV E7 proteins were not expressed in infiltrating lymph cells. CONCLUSION: HPV gene sequences were identified in 55% of atheromatous coronary arteries and may have a role in coronary artery disease.

Human Papilloma Virus Identification in Breast Cancer Patients with Previous Cervical Neoplasia.

PURPOSE: Women with human papilloma virus (HPV)-associated cervical neoplasia have a higher risk of developing breast cancer than the general female population. The purpose of this study was to (i) identify high-risk HPVs in cervical neoplasia and subsequent HPV positive breast cancers which developed in the same patients and (ii) determine if these HPVs were biologically active. METHODS: A range of polymerase chain reaction and immunohistochemical techniques were used to conduct a retrospective cohort study of cervical precancers and subsequent breast cancers in the same patients. RESULTS: The same high-risk HPV types were identified in both the cervical and breast specimens in 13 (46%) of 28 patients. HPV type 18 was the most prevalent. HPVs appeared to be biologically active as demonstrated by the expression of HPV E7 proteins and the presence of HPV-associated koilocytes. The average age of these patients diagnosed with breast cancer following prior cervical precancer was 51 years, as compared to 60 years for all women with breast cancer (p for difference = 0.001). CONCLUSION: These findings indicate that high-risk HPVs can be associated with cervical neoplasia and subsequent young age breast cancer. However, these associations are unusual and are a very small proportion of breast cancers. These outcomes confirm and extend the observations of two similar previous studies and offer one explanation for the increased prevalence of serious invasive breast cancer among young women.

Human Papilloma Viruses and Breast Cancer.

PURPOSE: Human papillomaviruses (HPV) may have a role in some breast cancers. The purpose of this study is to fill important gaps in the evidence. These gaps are: (i) confirmation of the presence of high risk for cancer HPVs in breast cancers, (ii) evidence of HPV infections in benign breast tissues prior to the development of HPV-positive breast cancer in the same patients, (iii) evidence that HPVs are biologically active and not harmless passengers in breast cancer. METHODS: RNA-seq data from The Cancer Genome Atlas (TCGA) was used to identify HPV RNA sequences in breast cancers. We also conducted a retrospective cohort study based on polymerase chain reaction (PCR) analyses to identify HPVs in archival specimens from Australian women with benign breast biopsies who later developed breast cancer. To assess whether HPVs in breast cancer were biologically active, the expression of the oncogenic protein HPV E7 was assessed by immunohistochemistry (IHC). RESULTS: Thirty (3.5%) low-risk and 20 (2.3%) high-risk HPV types were identified in 855 breast cancers from the TCGA database. The high risk types were HPV 18 (48%), HPV 113 (24%), HPV 16 (10%), HPV 52 (10%). Data from the PCR cohort study indicated that HPV type 18 was the most common type identified in breast cancer specimens (55% of 40 breast cancer specimens) followed by HPV 16 (13%). The same HPV type was identified in both the benign and subsequent breast cancer in 15 patients. HPV E7 proteins were identified in 72% of benign breast specimens and 59% of invasive breast cancer specimens. CONCLUSION: There were four observations of particular interest: (i) confirmation by both NGS and PCR of the presence of high-risk HPV gene sequences in breast cancers, (ii) a correlation between high-risk HPV in benign breast specimens and subsequent HPV-positive breast cancer in the same patient, (iii) HPVs in breast cancer are likely to be biologically active (as shown by transcription of HPV DNA to RNA plus the expression of HPV E7 proteins), (iv) HPV oncogenic influences may occur early in the development of breast cancer.

Human papillomavirus and Epstein Barr virus in prostate cancer: Koilocytes indicate potential oncogenic influences of human papillomavirus in prostate cancer.

INTRODUCTION: The purpose of this study is to determine if high risk human papillomaviruses (HPV) and Epstein Barr virus (EBV) are both present in the same prostate cancer specimens. METHODS: We used a range of analytical techniques including in situ polymerase chain reaction (IS-PCR) and standard liquid PCR followed by sequencing of the product to seek to identify HPV and EBV in normal, benign, and malignant prostate tissues. RESULTS: Both HPV type 18 and EBV gene sequences were identified in a high and approximately equal proportion of normal, benign, and prostate cancer specimens. These sequences were located in the nuclei of prostate epithelial cells. HPV associated koilocytes were identified in 24% of prostate cancer specimens. CONCLUSIONS: The presence of both HPV and EBV gene sequences in most of the same normal, benign, and malignant prostate specimens is particularly noteworthy because of recent experimental evidence demonstrating that EBV and HPV can collaborate to increase proliferation of cultured cervical cells. Because the presence of EBV and HPV in normal, benign, and malignant prostate tissues appears to be ubiquitous, it is possible that they are harmless. On the other hand HPV type 18 in particular, has high oncogenic potential and may be associated with some prostate cancers. The identification of HPV associated koilocytes in prostate cancer specimens is an indication of HPV infection and potential oncogenic influences of human papillomavirus in prostate cancer.

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

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.

High risk human papillomavirus and Epstein Barr virus in human breast milk.

BACKGROUND: Multiple viruses, including human immunodeficiency virus, Epstein Barr virus (EBV) and mouse mammary tumour virus have been identified in human milk. High risk human papillomavirus (HPV) sequences have been identified in breast cancer. The aim of this study is to determine if viral sequences are present in human milk from normal lactating women. FINDINGS: Standard (liquid) and in situ polymerase chain reaction (PCR) techniques were used to identify HPV and EBV in human milk samples from normal lactating Australian women who had no history of breast cancer.High risk human papillomavirus was identified in milk samples of 6 of 40 (15%) from normal lactating women - sequencing on four samples showed three were HPV 16 and one was HPV 18. Epstein Barr virus was identified in fourteen samples (33%). CONCLUSION: The presence of high risk HPV and EBV in human milk suggests the possibility of milk transmission of these viruses. However, given the rarity of viral associated malignancies in young people, it is possible but unlikely, that such transmission is associated with breast or other cancers.

Mouse mammary tumor virus-like sequences in human breast cancer.

Mouse mammary tumor virus (MMTV) sequences have been reported to be present in some human breast cancers, but it is unclear whether they have any causal role. In mice, MMTV promotes tumor formation indirectly by insertional mutagenesis of Wnt oncogenes that lead to their activation. In this study, we investigated the status of Wnt-1 in human breast cancers harboring MMTV-like sequences encoding viral envelope (env) genes. We confirmed the detection of env sequences in the nucleus of human breast cancer specimens that are similar in appearance to mouse mammary tumors expressing MMTV env sequences. MMTV env sequences in human breast cancers were also nearly indistinguishable from env sequences in mouse MMTV isolates. Further, Wnt-1 expression was higher in specimens of env-positive ductal carcinoma in situ and invasive ductal carcinoma, relative to env-negative specimens. Our findings extend the evidence that MMTV sequences found in naturally occurring mouse mammary tumors can be found in some human breast cancers, prompting further evaluation of causal roles in these settings.

Cytotoxic effects of antipsychotic drugs implicate cholesterol homeostasis as a novel chemotherapeutic target.

The reported reduction in cancer risk in those suffering from schizophrenia may be because antipsychotic medications have antineoplastic effects. In this study, 6 antipsychotic agents with a range of structural and pharmacological properties (reserpine, chlorpromazine, haloperidol, pimozide, risperidone and olanzapine), were screened for their effect on the viability of cell lines derived from lymphoblastoma, neuroblastoma, non-small cell lung cancer and breast adenocarcinoma. We aimed to determine if antipsychotic drugs in general possess cancer-specific cytotoxic potential, and whether it can be attributed to a common mode of action. With the exception of risperidone, all drugs tested displayed selective inhibition of the viability of cancer cell lines compared with normal cells. Using Affymetrix expression microarrays and quantitative real-time polymerase chain reaction, we found that for the antipsychotic drugs, olanzapine and pimozide, cytotoxicity appeared to be mediated via effects on cholesterol homeostasis. The role of cholesterol metabolism in the selective cytotoxicity of these drugs was supported by demonstration of their increased lethality when coadministered with a cholesterol synthesis inhibitor, mevastatin. Also, pimozide and olanzapine showed accelerating cytotoxic effects from 12 to 48 hr in time course studies, mirroring the time-dependent onset of cytotoxicity induced by the amphiphile, U18666A. On the basis of these results, we concluded that the Class II cationic amphiphilic properties of antipsychotic drugs contribute to their cytotoxic effects by acting on cholesterol homeostasis and altering the biophysical properties of cellular membranes, and that drugs affecting membrane-related cholesterol pathways warrant further investigation as potential augmentors of standard cancer chemotherapy.

High prevalence of prostatic neoplasia in Australian men.

AIMS: The purpose of this study was to document the extremely high level of prostatic neoplasia among Australian men. METHODS: The study was based on histological assessment of prostate tissue sampled from an unselected series of 133 cadavers referred for coronial autopsy at the Department of Forensic Medicine, Sydney South West Area Health Service, New South Wales. RESULTS: Evidence of neoplastic growth was identified in prostate tissue sampled from 30% of the 70 men aged 50 years and over, with invasive carcinoma present in 25.7% of subjects, and suspected prostatic intraepithelial neoplasia (PIN) in a further 4.3% of subjects. These findings were in marked contrast to the 63 subjects under 50 years of age. In this age group one subject had invasive prostate carcinoma, and one other had suspected PIN. CONCLUSIONS: The prevalence of invasive prostate carcinoma is extraordinarily high in this sample of Australian men. Among those subjects who were 50 years of age or over, more than one in four had apparently undiagnosed invasive cancer of the prostate gland.

Mouse mammary tumor virus-like env sequences in human breast cancer.

Mouse mammary tumor virus (MMTV) has long been known as a causal agent of breast cancer in mice. To date, varied MMTV-like envelope gene (env) sequences have been identified in up to 74% of human breast cancers. However, the role and origin of these MMTV-like sequences in human breast cancer remain uncertain. Our study was initiated to study the integration of MMTV-like env sequences in human breast cancer. PCR screening has identified 28 (56%) Australian breast cancer specimens and 7 (87.5%) human breast cancer cell lines to be positive for MMTV-like env sequence. In the MCF-7 genome, a fragment containing an MMTV-like env sequence of approximately 1.9 kb plus a downstream rodent-like sequence of approximately 200 bp was found to be integrated into a bacterial-like beta-lactamase sequence by insertional mutagenesis. The identified MMTV-rodent fragment is present in some MCF-7 sublines but absent in the screened specimens and other cell lines. Sporadic mutations found in this fragment indicate it has multiple copies in the MCF-7 genome. Sequence analysis has identified a novel ORF of approximately 1.6 kb which is 94-99% identical to MMTV env genes. RT-PCR was performed on the MCF-7 cDNA but no MMTV-like env transcript was detected. This is the first report to reveal the locus of MMTV-like env sequence in human cells. The MMTV-like env sequence was shown to be distinct from the human endogenous retroviral sequences and is closely related to rodents.

Breastfeeding, breast milk and viruses.

BACKGROUND: There is seemingly consistent and compelling evidence that there is no association between breastfeeding and breast cancer. An assumption follows that milk borne viruses cannot be associated with human breast cancer. We challenge this evidence because past breastfeeding studies did not determine "exposure" of newborn infants to colostrum and breast milk. METHODS: We conducted a prospective review of 100 consecutive births of infants in the same centre to determine the proportion of newborn infants who were "exposed" to colostrum or breast milk, as distinct from being fully breast fed. We also report a review of the breastfeeding practices of mothers of over 87,000 newborn infants in the Australian State of New South Wales. This study was approved by the Human Research Ethics Committee of the University of New South Wales (Sydney, Australia). Approval 05063, 29 September 2005. RESULTS: Virtually all (97 of 100) newborn infants in this centre were "exposed" to colostrum or breast milk whether or not they were fully breast fed. Between 82.2% to 98.7% of 87,000 newborn infants were "exposed" to colostrum or breast milk. CONCLUSION: In some Western communities there is near universal exposure of new born infants to colostrum and breast milk. Accordingly it is possible for the transmission of human milk borne viruses. This is contrary to the widespread assumption that human milk borne viruses cannot be associated with breast cancer.

Viruses and human breast cancer.

There are well-established risk factors for breast cancer, most of which relate to estrogens and growth hormones in females. These include early-age menarche, late-age menopause, postmenopausal obesity and use of hormone therapy. However, these factors do not account for the sixfold difference in breast cancer incidence and mortality between countries and the fact that these differences dramatically lessen after migration; nor do they account for male breast cancer. Accordingly, hormone-responsive viruses have become major suspects as etiological agents for human breast cancer. Human papillomaviruses, mouse mammary tumor virus and Epstein-Barr virus are the prime candidate viruses as causes of human breast cancer. Human papillomaviruses and the mouse mammary tumor virus have hormone responsive elements that appear to be associated with enhanced replication of these viruses in the presence of corticosteroid and other hormones. This biological phenomenon is particularly relevant because of the hormone dependence of breast cancer. Viral genetic material for each of these candidate viruses has been identified by polymerase chain reaction in breast tumors but rarely in normal breast tissue controls. Pooled data from controlled studies show substantial odds ratios for the presence of viral genetic material in breast tumors compared with normal controls. These and additional data provide substantial, but not conclusive, evidence that human papillomavirus, the mouse mammary tumor virus and Epstein-Barr virus may have a role in the etiology of human breast cancer. If conclusive evidence for a role of these viruses in breast carcinogenesis can be developed, there is a practical possibility of primary prevention.