Edited by: Amit Manulal Oza, Princess Margaret Hospital and University of Toronto, Canada
Reviewed by: Connie Irene Diakos, University of Sydney, Australia; Brigitte Mlineritsch, Universitätsklinik für III. Medizin in Salzburg, Austria
Specialty section: This article was submitted to Women's Cancer, a section of the journal Frontiers in Oncology
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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.
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.
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 (
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.
Women with squamous or glandular precancer of the cervix have a significantly higher risk of subsequent breast cancer than the general female population – odds ratios 1.10 and 1.52, respectively (
Although these data indicate that HPVs may have a possible role in some breast cancers, it is unlikely that HPVs have a major causal role. There are two reasons for this view: (i) in immunocompromised patients (due to either human immunodeficiency viral infections or post organ transplantation therapy), there is no increased prevalence of breast cancer, which is in contrast to the twofold to sixfold increased prevalence of HPV-associated cervical cancer in these patients (
Data from The Cancer Genome Atlas (TCGA), published on line by the Larsson Lab (
There is an apparent conflict between (i) data which indicate an increased risk of breast cancer among women with prior HPV-associated cervical pathology (
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.
We used the following research approaches: (i) a retrospective cohort study of women with breast cancer who had prior precancer or neoplasia of the cervix, (ii) a study of HPV-associated biomarkers in these breast cancers to determine if the HPVs were biologically active, and (iii) identification of koilocytes by light microscopy. We also compared the outcomes of these studies with normal breast tissues from women who had not developed breast cancer after approximately 10 years.
All cervical and subsequent breast cancer specimens were from women residing in Australia. We reviewed approximately 4,000 pathology reports of breast cancer and identified 28 patients who had either HPV cervical infections or more commonly cervical intraepithelial neoplasia (CIN) 1–11 years prior to developing breast cancer. Archival formalin-fixed specimens from these patients were identified and collected from a large Australian pathology service (Douglass Hanly Moir – Pathology).
Eighteen unselected archival formalin-fixed “normal” breast specimens from women who had breast reduction surgery were used as a comparison group. Normal breast specimens from these women do not provide a strict control group as there is no information available about their cervical tissues, although cervical HPV infections are likely to be present in some of these women (
We sought to identify HPV DNA sequences in prior cervical neoplasia and later breast cancer specimens from the same patients to determine if the same HPV virus was present. If the HPV DNA was of the same type and sequences in both the prior cervical as in the later breast cancer tissues from the same subject, this provides potential evidence of prior HPV infection as a possible cause for malignancy in the breast.
For standard and quantitative polymerase chain reaction (PCR), genomic DNA (gDNA) was extracted from formalin-fixed paraffin-embedded (FFPE) breast and cervix tissues using a combination of heat treatment and a DNA extraction kit (DNeasy Blood and tissue extraction, QIAGEN). The method is described in Steinau et al. (
A blank extraction control (an extraction without FFPE tissues) was performed during the DNA extraction procedure. The gDNA extracts were quantified with a NanoDrop spectrophotometer (Thermo Scientific). The quality of gDNA was assessed by amplification of human β-actin. The primers used were β-actin5′_fwd (5′-CTTCTGCCGTTTTCCGTAGG-3′) and β-actin3′_rev (5′-TGGGATGGGGAGTCTGTTCA-3′) at the final concentration of 1 μM. Thermal cycles were 94°C for 15 min; 94°C for 30 s, 55°C for 30 s, and 72°C for 45 s for 30 cycles. HotStarTaq Master Mix Kit (QIAGEN) was used for PCR master mix. gDNA samples, which were β-actin positive, were selected for the detection of HPV genomes.
Both semi-nested PCR and real-time quantitative PCR were used for the detection of HPV. Archival DNA is fragmented and the original nested MY/Gp primers, in the L1 gene, did not always give sound results due to the length of the MY product. Therefore, a semi-nested PCR, MY11 to Gp6, followed by Gp5–Gp6 was used. Some of the primers have been altered slightly and are degenerate for HPV16 and 18. They are also capable of bringing up types 3, 11, 12, 45, 58, 73, and 75.
MY11 – (5′GCACAGGGYCAYAAYAATGG3′)
Gp5 – (5′TATTTGTTACTGTKGTWGATAC3′)
Gp6 – (5′GACATGKKGAGGAATATGATT3′)
Positive PCR products were routinely sequenced in addition to the negative controls, to ensure there was no contamination. The negative controls were no DNA (water) and a blank extraction (which tested the reagents), plus sequencing of the products of these controls in case the bands could not be seen on a gel. Negative outcomes of PCR analyses of selected breast cancer specimens provided negative controls based on breast tissues.
The HPV L1 gene in gDNA samples was amplified using a real-time PCR machine (Rotor Gene Q, QIAGEN). Thermal cycles used were 95°C for 5 min, 95°C for 10 s, and 60°C for 30 s for 60 cycles. The PCR reaction mix was the Quanti Fast SYBR Green kit (QIAGEN) and was used according to the manufacturer’s recommendation. The primers used for detection of HPV were
The quantity of gDNA used for PCR was determined by estimating theoretical gene copies of HPV 16 in breast and breast cancer tissues (
Polymerase chain reaction products were analyzed and selected for sequencing based on amplification profiles and the achievement of the known melting point (the specific temperature for each product). The analysis was performed using Rotor Gene Q software (QIAGEN).
DNA extracts from 11 selected (for positive HPV) cervical precancer and breast cancer specimens were independently analyzed for the presence of HPV DNA (Annika Antonnson, QIMR Berghofer Medical Research Institute), using methods as described in Ref. (
The HPV PCR products from GP5 to Gp6 were sequenced to determine the HPV type. The HPV genotypes were identified by BLAST via the US National Center for Biotechnology Information.
Archival tissues on slides were washed in xylene to remove the wax followed by washes in decreasing concentrations of alcohol. The tissues were subjected to pepsin digestion with varying times of digestion which were required for different tissues. These differences were probably due to fixation procedures, which could vary in duration. The digestion was stopped in 0.1M Tris buffer pH 8. Approximately 75 μl of PCR mix, containing inner nested PCR primers Gp5–Gp6. Digoxogenin (DIG) – dUTP (0.3 nM) (Roche) – was added to the tissue in a frame which was sealed. PCR cycling was the same as for standard PCR. Detection using Anti-DIG AP-Fab fragments (1 μl) (Roche) in buffer pH 7.5 followed by NBT/BCIP (2 μl) (Roche) in buffer pH 9.5 was stopped when a blue color was observed in the cells of the cancer specimen and not in the negative control. The tissues were counterstained with eosin. We used stringent negative controls that included omitting both DNA primers and Taq polymerase when conducting
The identification of HPV in normal and benign breast or breast cancer specimens was considered as positive if two or more of the following outcomes in the same specimen were observed: (i) HPV DNA sequences identified by standard PCR and/or real-time PCR and (ii) HPV positive
An assessment of HPV biological activity can be conducted in several ways: (i) the identification of HPV oncoprotein E7 and (ii) the identification of HPV-associated koilocytes.
Antibodies specific for HPV E7 proteins, have recently been developed and were used in this study. These antibodies were HPV E7 monoclonal “Cervimax” – Valdospan GmbH. Austria (
The identification of koilocytes by light microscopy is subjective. For this reason, two independent observers (James S. Lawson and Warick Delprado) reviewed all the specimens and assessed the presence of koilocytes.
The SPSS statistical package was used to (i) assess the significance of any differences in outcomes for the various investigations and (ii) assess for correlation of non-parametric data.
The same high-risk HPV types were identified in both the cervical and breast specimens from 13 (46%) of patients. High-risk HPV types 16, 18, 33, and 58 were identified in 19 (70%) invasive and 2 (100%) non-invasive breast cancer specimens (Table
Patient | Age | Diagnosis | HPV E7 | HPV |
---|---|---|---|---|
Immunohistochemistry | PCR consolidated | |||
1. Cervix | 46 | CIN1 | HPV 16/18 | |
Breast | 48 | IDC | Neg | HPV 18 |
2. Cervix | 60 | Normal | Neg | |
Breast | 66 | IDC Gr3 | Neg | HPV 18 |
3. Cervix | 62 | CIN1 | HPV 18 | |
Breast | 73 | DCIS | 2+ | HPV 18 |
4. Cervix | 53 | CIN3 | HPV 18 | |
Breast | 62 | IDC Gr2 | ||
5. Cervix | 44 | CIN2 | HPV 18 | |
Breast | 45 | IDC Gr2 | Neg | HPV 18 |
6. Cervix | 42 | HPV koilos | Neg | |
Breast | 47 | IDC Gr2 | 3+ | HPV 18 |
7. Cervix | 39 | CIN3 | ||
Breast | 45 | ILC Gr2 | 3+ | Neg |
8. Cervix | 40 | CIN3 | HPV 18 | |
Breast | 45 | IDC Gr3 | 3+ | HPV 18 |
9. Cervix | 33 | CIN2 | HPV 18 | |
Breast | 39 | IDC Gr2 | 3+ | HPV 18 |
10. Cervix | 44 | HPV koilos | HPV 18 | |
Breast | 44 | DCIS | Neg | HPV 18 |
11. Cervix | 57 | HPV koilos | HPV 18 | |
Breast | 68 | IDC Gr3 | Neg | HPV 18 |
12. Cervix | 43 | CIN3 | HPV 16/18 | |
Breast | 45 | IDC Gr2 | HPV 18 | |
13. Cervix | 29 | CIN 2 | HPV 16 | |
Breast | 32 | IDC | 3+ | HPV 18 |
14. Cervix | 46 | HPV koilos | HPV 18 | |
Breast | 47 | IDC Gr2 | 1+ | HPV 18 |
15. Cervix | 41 | CIN1 | ||
Breast | 46 | IDC Gr1 | 2+ | HPV 18 |
16. Cervix | 41 | CIN2 | HPV 16/18/58 | |
Breast | 50 | IDC Gr2 | 1+ | HPV 58 |
17. Cervix | 33 | CIN3 | HPV 18 | |
Breast | 34 | IDC Gr3 | Neg | HPV 18 |
18. Cervix | 29 | HPV koilos | Neg | |
Breast | 38 | I Muc C Gr2 | HPV 16/18 | |
19. Cervix | 55 | CIN1 | ||
Breast | 62 | IDC Gr1 | 2+ | HPV 18 |
20. Cervix | 57 | CIN1 | HPV 18 | |
Breast | 67 | IDC Gr1 | 1+ | HPV 16/18 |
21. Cervix | 36 | CIN1 | Neg | |
Breast | 40 | IDC Gr1 | 1+ | Neg |
22. Cervix | 54 | CIN1 | HPV | |
Breast | 64 | IDC Gr2 | 2+ | HPV 18 |
23. Cervix | 39 | CIN2 | HPV | |
Breast | 47 | ILC Gr2 | 1+ | Neg |
24. Cervix | 46 | CIN3 | HPV 16/18 | |
Breast | 52 | IDC Gr1 | Neg | HPV 18 |
25. Cervix | 53 | CIN1 | HPV 18 | |
Breast | 61 | IDC Gr2/DCIS | 3+ | Neg |
26. Cervix | 44 | CIN3 | Neg | |
Breast | 45 | IDC Gr3 | Neg | |
27. Cervix | 44 | Normal | HPV 18 | |
Breast | 48 | DCIS | 3+ | HPV 18 |
28. Cervix | 50 | HPV koilos | HPV 18 | |
Breast | 61 | IDC Gr1 | Neg | HPV 16/18 |
Normal breast | Cervical precancer | Breast DCIS | Breast IDC/ILC | |
---|---|---|---|---|
High-risk HPV sequences (types 16, 18, 33, 58) | 3/18 (17%) | 20/25 (80%) | 2/2 | 19/27 (70%) |
HPV E7 | 4/18 (22%) | 1/2 | 16/25 (64%) | |
Koilocytes | 0/18 (0%) | 27/28 (96%) | 1/2 | 8/25 (32%) |
DNA extracts from 11 cervical and breast cancer HPV positive specimens (as identified in the University of New South Wales laboratories) were independently analyzed (Annika Antonsson, QIMR Berghofer Medical Research Institute). HPV types 16, 18, and 33 were identified in 4 cervical and 2 breast specimens. The identification of different HPV types indicates that contamination is improbable.
The pathological characteristics of the prior cervical and later breast specimens from the same patient are shown in Table
The average age of patients at date of diagnosis of breast cancer following cervical pathology was 51 years (Table
The number of years between the age of cervical and subsequent breast cancer diagnosis was 3.7 years for women below the age of 50 and 9.0 years for women 50 years or over (
As shown in Tables
HPV E7 in an HPV positive invasive breast cancer specimen is shown in Figure
As indicated above, HPV E7 protein was expressed in 64% of the breast cancer specimens (Table
Koilocytes were identified in all of the cervical specimens and in 9 of 28 (32%) of the breast cancers, which had later developed in the same patients. Typical koilocytes are shown in squamous epithelial cells of cervical tissue and glandular epithelial cells of breast cancer in the same patient in Figure
Cohort studies are a valuable approach to the study of viruses and cancer because they offer insights into the natural history of the disease. Prospective cohort studies are usually preferred to case control or retrospective studies because the risk of bias is generally considered to be less. However, we have undertaken a retrospective cohort study, because it is a practical and low cost method of achieving sound outcomes.
We have shown that (i) high-risk HPVs are present in 70% of breast cancers among women who had prior HPV positive cervical neoplasia – HPV types were identical in both prior cervical neoplasia and later breast cancer in 46% of the patients, (ii) HPV E7 was expressed in 64% of invasive breast cancers, and (iii) HPV-associated koilocytes were identified in 32% of breast cancers which is an additional indicator of HPV biological activity. We have also demonstrated that HPV positive breast cancer following HPV-associated cervical cancer occurs significantly more commonly in younger age women.
Despite reviewing approximately 4,000 pathology reports of breast cancer specimens, only 28 (0.7%) women with breast cancer had prior cervical neoplasms. While this may be an under estimate because not all biopsy and surgical specimens would have been referred to the same pathology service, it suggests that prior HPV cervical neoplasia is associated with a very low prevalence of subsequent HPV positive breast cancer.
The use of a range of techniques, namely, standard and real-time PCR,
It is particularly noteworthy that virtually identical HPV type 18 and HPV type 58 sequences were identified in both CIN and 1–11 years later in breast cancer in 46% of the same subjects. This is compatible with the same virus infection having an oncogenic role in both the cervical and breast cancers in the same patients. HPV type 18 is the dominant HPV type in both breast and prostate cancers among Australian patients (
The identification of koilocytes in 32% of the breast cancer specimens is of interest. This is a high proportion of koilocytes as compared to the identification of <5% in other series of breast cancers without prior HPV-associated cervical neoplasia (
The observation that HPV E7 protein expression may be absent in HPV sequence positive breast cancer specimens is of interest. A possible explanation is that HPV infections may have an early role in breast oncogenesis. This view is supported by the recent findings by Ohba et al. (
Although it is accepted by research workers in this field that HPVs are present in many breast tumors, there is concern that the viral load is so low that HPVs may not be oncogenic in breast cancer (
The expression of HPV E7 proteins and HPV-associated koilocytes in these sets of specimens are an indication that HPVs in these breast specimens are biologically active and capable of being oncogenic. Accordingly, despite their extremely low viral load, HPVs appear to be biologically active and have oncogenic influences in some of the breast cancers in this study.
Of particular interest is the very young age of many of these patients. Approximately 39% of the patients with prior cervical neoplasia were 45 years of age or below at age of breast cancer diagnosis. Breast cancer in young women is often aggressive and appears to have distinct biological characteristics (
There has been a small but significant increase in the incidence among young (below age 40 years) US women of breast cancers with metastases during the past three decades (
These observations are in accord with the well-known bimodal peak frequencies of breast cancer at different ages of diagnosis (
Also of interest is the 3.7-year gap between age of cervical and breast cancer diagnosis among women aged below 50 years as compared with 9.0 years for women 50 years and over at age of breast cancer diagnosis. This is compatible with the greater influence of sex hormones among younger women on both breast cancer and HPV viral replication (
The mode of transmission of HPV to the breast is not known. However, it is possible that HPVs are transmitted to the genital tract during sexual activities and later transmitted by white blood cells throughout the body, including the breasts. This hypothesis is based on the young age of women with HPV positive breast cancer and who are sexually active and have a high incidence of HPV cervical infections plus the repeated identification of high-risk HPVs in white blood cells (
Other viruses, including mouse mammary tumor virus, Epstein–Barr virus, and bovine leukemia virus, may each have a role in breast cancer including collaborative roles with HPVs (
The findings in this study indicate that high risk for cancer 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.
The availability and widespread use of vaccines which can effectively control high-risk HPV infections may result in a decrease of HPV-associated breast cancer and, thereby, provide evidence whether HPVs have a causal role in breast cancer.
This project has formal ethics approval by the University of New South Wales Human Research Ethics Committee – number HREC HC11421. Participants gave written informed consent to participate in this study. Ethics approvals for the follow-up of women who donated normal breast tissues was given by the New South Wales Population and Health Services Research Ethics Committee – number 2009/12/203. This Ethics committee waived the need for consent. The reasons for waiving consent were (i) the specimens were archival having been collected in 1999, 2000, and 2001, (ii) all specimens were “de-identified” to the research group, and (iii) retrospective approaches to donors, all of whom had cosmetic surgery, may have caused unnecessary anxiety.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Financial support was provided by the Australian National Breast Cancer Foundation and the James and Margaret Lawson Research Fund. The contribution by Daria Salyanika was supported by the Center for Computational Science, University of Miami and partially by a grant (1R03CA171052-01A1) from the United States National Cancer Institute.