The presence of HPV in breast tissue and the potential causal association between human papillomavirus (HPV) and breast cancer (BC) remains controversial. The aim of the present study was to compare the HPV prevalence in BC tissues, adjacent normal breast tissues and breast benign disease tissues and to investigate the possible association between HPV and breast tumor development in Chinese women. Paraffin-embedded specimens from 187 pairs of BCs including tumor and normal breast tissue adjacent to tumors and 92 breast benign lesions between June 2009 and July 2014 were investigated by nested polymerase chain reaction (PCR) and type-specific PCR, respectively. With strictly quality control, HPV positive infection was detected in three BC tissues. No HPV positive infection was detected in all normal breast tissue adjacent to tumors and benign breast tissues. Through our detailed analysis, rare HPV infection in this study suggests that HPV might not be associated with BC progression.
Citation: Li J, Ding J, Zhai K (2015) Detection of Human Papillomavirus DNA in Patients with Breast Tumor in China. PLoS ONE 10(8): e0136050. https://doi.org/10.1371/journal.pone.0136050
Editor: Ming Yang, Beijing University of Chemical Technology, CHINA
Received: May 8, 2015; Accepted: July 29, 2015; Published: August 21, 2015
Copyright: © 2015 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper.
Funding: The authors received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Breast cancer (BC) is one of the most prevalent malignancies in women, in both the developed and the developing world . It is well recognized as a biological heterogeneous disease in regard to its clinical, histological and molecular profile. Many risk factors have been associated with the pathogenesis of this disease, including family history, hormone levels, cigarette smoking and alcohol consumption. During the past few decades, in addition to known factors, the factor of virus infections has been raised.
Human papillomavirus (HPV) is a small, circular, double-stranded DNA virus that is believed to be an important factor in the pathogenesis of infecting and transforming epithelium to particular benign and malignant lesions in humans. Approximately 120 HPV subtypes have been isolated from humans . High-risk HPV (HR-HPV) encodes a series of proteins, E6 and E7 oncoproteins, that have been associated with cell transformations, which lead to genomic instability that can result in malignancy [3–6]. HR-HPV infection has been thought to be the main cause of human cervical cancers , a substantial proportion of other anogenital cancers [8,9], and oropharyngeal cancers [10–12]. Recently, HPV infection is reported to be associated with lung cancers . The first evidence that revealed that HPV might be involved in BC was provided by Di Lonardo et al. in 1992 who demonstrated HPV 16 DNA in 29.4% of paraffin-embedded tissues (PET) of BC by polymerase chain reaction (PCR) using HPV 11, 16 and 18 primers . Many studies have reported HR-HPV infections in BC specimens from diverse populations across the world [15–17]. Koilocyte-like cells were not only observed in HPV positive breast cancer specimens but also observed in some HPV positive normal breast tissue specimens . However, several studies failed to detect HPV in BC tissues [19–27]. Because study designs, involved populations, and HPV detection methods were heterogeneous, the role of HPV in BC remains controversial.
To clarify the relationship between HPV and BC, we performed a case-control study to investigate the presence of HPV in BC tissue, normal specimens adjacent to carcinomas and breast tissue of breast benign disease (BBD) using PCR and analyzed the association between HPV infection and the risk of BC progression in Chinese women.
Materials and Methods
187 sets of BC PET including carcinoma and normal breast tissue adjacent to tumors and 92 BBD were used in this study. Patients were pathologically confirmed without restriction in regard to age and histological type and were consecutively recruited from Beijing Chao-Yang Hospital of Capital Medical University between June 2009 and July 2014. Those who had a history of cancer, metastasized cancer from other organs or neo-adjuvant treatments were excluded. All subjects were genetically unrelated ethnic Han Chinese women. At recruitment, personal data from each participant about demographic information and clinicopathological characteristics were collected. Informed consent was obtained from all participants. The oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2) statuses of BC patients were also abstracted from the medical records. Written consent had been obtained from every participant. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital, Capital Medical University.
DNA Extraction and Quality Control
Three cuts of 4-μm-thick sections of PET were cut and put into 1.5 ml microtubes. Positive controls (HPV 16 infected SiHa cells and HPV 18 infected HeLa cells embedded in paraffin) and negative controls (blank paraffin block) were also used for quality control. Total DNA from PET was extracted from each subject using the TaKaRa DEXPAT kit (Code No: 9091, TaKaRa, Dalian, China) or Universal Genomic DNA Extraction Kit Ver.3.0 (Code No: 9765, TaKaRa, Dalian, China) according to the instructions of the manufacturer. Amplification of a 268 bp fragment of the β-globin gene was used to assess the quality of DNA in PETs. The primers were GH20 (5′-GAA GAG CCA AGG ACA GGT AC-3′) and PC04 (5′-CAA CTT CAT CCA CGT TCA CC-3′). All samples were positive for β-globin gene, indicating that it was available for the following analysis.
Two independent PCRs were applied to detect HPV in tissue. Nested PCR was conducted to amplify fragments in highly conserved regions in the L1 gene. MY09/11 primers were used to amplify 450 bp fragment of L1 region and followed by the secondary PCR using the GP5+/6+ primers to amplify 140 bp inner product [28,29]. Primer sequences were MY09: 5′-CGT CCM ARR GGA WAC TGA TC-3′, MY11: 5′-GCM CAG GGW CAT AAY AAT GG-3′, GP5+: 5′-TTT GTT ACT GTG GTA GAT ACT AC-3′, GP6+: 5′-GAA AAA TAA ACT GTA AAT CAT ATT C-3′. Successfully amplified HPV PCR products were purified and sequenced by an automated sequencer ABI 3730xl (Applied Biosystems, Foster city, CA). The results were be evaluated by NCBI BLAST program. To verify the results independently, we conducted another PCR to amplify the HPV E6/E7 gene of HPV 16 and 18 using type-specific primers (type-specific PCR, TS PCR). The following were primer sequences : HPV16 E6: forward 5'-CTG CAA GCA ACA GTT ACT GCG ACG-3', reverse 5'-CAT ACA TCG ACC GGT CCA CC-3', product of 315 bp; HPV 18 E7: forward 5'-GAG CCG AAC CAC AAC GTC AC-3', reverse 5'-GGA TGC ACA CCA CGG ACA CA-3', product of 152 bp.
We examined 279 female breast tumor cases including BC and BBD, aged from 18 to 83 years. Mean age of BC and BBD groups were 57.1 and 36.5 (P < 0.001), respectively. The BMI of the BC group (BMI = 24.1) was higher than that of BBD (BMI = 22.3) (P < 0.001). The characteristics of the included subjects are summarized in Table 1. Of the total of 279 participants, 148 had invasive ductal carcinoma (IDC), three had ductal carcinoma in situ, five had invasive lobular carcinoma, two had mucinous carcinoma, 29 had unspecified invasive carcinoma, 53 had fibroadenoma, 24 had adenosis, ten had intraductal papilloma, and five had cystic mastopathy. In the BC group, the positive rate of lymph node metastasis, ER, PR and Her2 expression were 38.0%, 72.2%, 69.5% and 86.6%, respectively.
Results of HPV-DNA infected status in all specimen were confirmed by PCR using nested PCR and TS PCR, respectively. All samples were positive for β-globin, indicating adequate quality of DNA. The results of HPV positive controls and negative controls revealed that the PCR system worked well, and there was no evidence of contamination. Three out of 187 BC specimen were positive for HPV-DNA. Among them, two IDC were infected with HPV6 and HPV18, respectively; while one unspecified invasive carcinoma was infected with HPV16. However, HPV-DNA was not detected in all normal breast tissue adjacent to tumors and BBD specimen. PCR products of representative samples are shown in Fig 1. The samples were tested twice by different people, and the results were concordant.
M: Marker (TaKaRa DL2000); Lane1-5: nested PCR products; Lane 6–11: type-specific PCR products; Lane 1, 6 and 9 are positive controls; Lane 5, 8 and 11 show negative controls. Lanes 2–4 show positive bands of 140 bp, Lane 7 and 10 show positive bands of 315 bp and 152 bp, respectively.
Because Band  reported that products of HPV 16 and 18 could induce immortalization in human breast epithelial cells and Di Lonardo  demonstrated that 29.4% of BC patients had HPV infection, several studies have explored the association of HPV infection and BC risk. If HPV is infected in BC, there should be some differences in HPV infection status in normal tissues adjacent to and in BC in the same patient. The prevalence of HPV existing in BC varies greatly worldwide. Two published meta-analyses show that HPV infection is associated with BC risk [15,16]. Recently, several studies indicated that HPV infection is not associated with BC risk. In this study, to avoid random error of sampling, we detected HPV prevalence in breast tissue from BC and paired normal breast tissue adjacent to tumors, and BBD to investigate the role of HPV infection in breast cancer progression. During HPV infection and integration into the host genome, the L1 gene is frequently lost . Therefore, not only nested PCR of L1 gene but also E6/E7 region TS PCR were performed to detect HPV in the present study. With strict quality control, three HPV-DNA infection was found in 279 participants’ tissues including 187 sets of BC tissue and normal breast tissue adjacent to tumors, and 92 BBD tissues.
Characteristics of the published studies of HPV and BC risk with case-control setting are shown in Table 2 [18,27,33–51]. A literature review of eligible previous studies indicated a high heterogeneity of HPV prevalence in BCs and BBDs across divergent areas that vary from 2.0% to 60.0% and 0.0% to 19.0%, respectively. Another study reported HPV prevalence was highest especially for studies published between 2000 and 2005 (37.28%, 95% CI = 31.67–43.16%) . Most of published studies used PCR based method to detect HPV status in BC tissues, which have a higher rate of false positives. Different positive detection rates and amplification performances were reported in comparing various primer sets . HPV viral load may be very low in breast tissue compared with cervical cancer tissue , and sample preparation, DNA isolation and PCR amplification could introduce contamination. All of these factors have been indicated that tissue block section and biopsy preparation may affect HPV detection rates. Because nested PCR is a contamination-prone method, we think that rare HPV infection in our present study is reliable. Three studies that also reported negative HPV infection were conducted in USA, north-west China and Spain. Two of them used TS PCR which is able to test HPV 6, 11, 16 and 18 to detect HPV in PET samples in BC and BBD [33,41], the other used LiPA HPV Genotyping kit . In one study, HPV was detected using ISH in three (10%) BBD samples, but all of the positive specimens became negative after being frozen for 3 months -70°C . In summary, a lack of HPV in both BC and BBD has been reported by three studies using a total of 152 BC and 47 BBD.
In total, only 7 studies have published HPV status in BC and non-cancer controls with case-control design in female Chinese [34,38,40,41,45,49,50]. In 1992, Yu et al reported 15 of 34 BC infected with HPV 33 and both 2 benign and atypical ductal hyperplasia showed negative HPV infection using TS PCR and dot blot hybridization which could detect HPV 16, 18 and 33 . He et al in 2009 using HPV 16 E6 region TS PCR and Mou et al in 2011 using MY09/MY11 and GP5+/GP6+ primers PCR found that 24 of 50 BC, 1 of 20 normal breast tissue samples and 4 of 62 BC, none of 46 normal breast tissues infected with HPV, respectively [38,40]. Liang et al in 2013 found 48 of 224 and 6 of 37 breast fibroadenomas were HPV infection with hybrid capture 2 assay and no high-risk HPVs prevalence difference were detected between malignant tissues and controls (P = 0.468) . Using Sequenom MassARRay platform, Peng et al in 2014 reported 2 of 100 BC and 0 of 50 BBD infected with HPV . Fu et al also compared whether HPV58 present in BC and BBD . However, Chang et al in 2012 reported none of 48 BC and 30 BBD infected with HPV using E6/E7 region TS PCR and ISH . However, only two of these 7 studies using another method to perform the experiment again to verify the results [41,50].
Although HPVs infecting epithelial mucosa or cutaneous surfaces, the possible mechanisms by which HPVs are transmitted to the breast are not clear. HPV virions are known to be released when the envelope of cells desquamate. Therefore, it is assumed that cell surface to surface contact, mainly during sexual activities, play a very important role in HPV transmission , as a transmission route of HPV infection in oral carcinogenesis by oral sexual behaviour . Some authors suggested that a haematogenic and/or lymphatic transfer of viruses from one organ to another . Several studies also indicated HPV exist in nipple tissues and breast milk, suggesting that HPV may transfer from nipple to lactiferous ducts . Cells with HPV infection are eliminated by the immune system, and only long-term infections would cause pathogenesis. If HPV plays a role in BC carcinogenesis, HPV would be found in some precancerous lesions or normal tissue. Therefore, it is very important to test HPV status in BC with different stages of progression. Unfortunately, none of the published studies has strictly design to observe HPV infection in BC progression, such as using BC, BC adjacent normal tissue in the same patient and BBD. Although several studies reported higher HPV prevalence in BC compared with BBD, it is too early to conclude that HPV has a causal role in BC.
In summary, the present study using BC, BC adjacent normal tissue in the same patient and BBD examined HPV status in various stage of BC progression, with strictly quality control, we conclude that rare breast tumor infected with HPV and suppose that HPV infection is not associated with BC in Chinese women.
Conceived and designed the experiments: KZ. Performed the experiments: JL. Analyzed the data: JD. Contributed reagents/materials/analysis tools: KZ. Wrote the paper: JL JD KZ.
- 1. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA: a cancer journal for clinicians. 2014;64(1):9–29. pmid:24399786.
- 2. Bernard HU, Burk RD, Chen Z, van Doorslaer K, zur Hausen H, de Villiers EM. Classification of papillomaviruses (PVs) based on 189 PV types and proposal of taxonomic amendments. Virology. 2010;401(1):70–9. pmid:20206957; PubMed Central PMCID: PMC3400342.
- 3. Doorbar J. Papillomavirus life cycle organization and biomarker selection. Disease markers. 2007;23(4):297–313. pmid:17627064; PubMed Central PMCID: PMC3851388.
- 4. Munger K, Werness BA, Dyson N, Phelps WC, Harlow E, Howley PM. Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product. The EMBO journal. 1989;8(13):4099–105. pmid:2556261; PubMed Central PMCID: PMC401588.
- 5. Werness BA, Levine AJ, Howley PM. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science. 1990;248(4951):76–9. pmid:2157286.
- 6. Stanley MA, Pett MR, Coleman N. HPV: from infection to cancer. Biochemical Society transactions. 2007;35(Pt 6):1456–60. pmid:18031245.
- 7. Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. The Journal of pathology. 1999;189(1):12–9. pmid:10451482.
- 8. Steenbergen RD, de Wilde J, Wilting SM, Brink AA, Snijders PJ, Meijer CJ. HPV-mediated transformation of the anogenital tract. Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology. 2005;32 Suppl 1:S25–33. pmid:15753009.
- 9. Heideman DA, Waterboer T, Pawlita M, Delis-van Diemen P, Nindl I, Leijte JA, et al. Human papillomavirus-16 is the predominant type etiologically involved in penile squamous cell carcinoma. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2007;25(29):4550–6. pmid:17925550.
- 10. Kreimer AR, Clifford GM, Boyle P, Franceschi S. Human papillomavirus types in head and neck squamous cell carcinomas worldwide: a systematic review. Cancer epidemiology, biomarkers & prevention: a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology. 2005;14(2):467–75. pmid:15734974.
- 11. Hobbs CG, Sterne JA, Bailey M, Heyderman RS, Birchall MA, Thomas SJ. Human papillomavirus and head and neck cancer: a systematic review and meta-analysis. Clinical otolaryngology: official journal of ENT-UK; official journal of Netherlands Society for Oto-Rhino-Laryngology & Cervico-Facial Surgery. 2006;31(4):259–66. pmid:16911640.
- 12. Syrjanen S, Lodi G, von Bultzingslowen I, Aliko A, Arduino P, Campisi G, et al. Human papillomaviruses in oral carcinoma and oral potentially malignant disorders: a systematic review. Oral diseases. 2011;17 Suppl 1:58–72. pmid:21382139.
- 13. Zhai K, Ding J, Shi HZ. HPV and lung cancer risk: A meta-analysis. Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology. 2015;63C:84–90. pmid:25315992.
- 14. Di Lonardo A, Venuti A, Marcante ML. Human papillomavirus in breast cancer. Breast cancer research and treatment. 1992;21(2):95–100. pmid:1320958.
- 15. Li N, Bi X, Zhang Y, Zhao P, Zheng T, Dai M. Human papillomavirus infection and sporadic breast carcinoma risk: a meta-analysis. Breast cancer research and treatment. 2011;126(2):515–20. Epub 2010/08/27. pmid:20740311; PubMed Central PMCID: PMCPmc3164261.
- 16. Simoes PW, Medeiros LR, Simoes Pires PD, Edelweiss MI, Rosa DD, Silva FR, et al. Prevalence of human papillomavirus in breast cancer: a systematic review. International journal of gynecological cancer: official journal of the International Gynecological Cancer Society. 2012;22(3):343–7. Epub 2012/01/05. pmid:22214962.
- 17. Piana AF, Sotgiu G, Muroni MR, Cossu-Rocca P, Castiglia P, De Miglio MR. HPV infection and triple-negative breast cancers: an Italian case-control study. Virology journal. 2014;11(1):190. pmid:25413873; PubMed Central PMCID: PMC4243283.
- 18. Heng B, Glenn WK, Ye Y, Tran B, Delprado W, Lutze-Mann L, et al. Human papilloma virus is associated with breast cancer. British journal of cancer. 2009;101(8):1345–50. Epub 2009/09/03. pmid:19724278; PubMed Central PMCID: PMCPmc2737128.
- 19. Wrede D, Luqmani YA, Coombes RC, Vousden KH. Absence of HPV 16 and 18 DNA in breast cancer. British journal of cancer. 1992;65(6):891–4. pmid:1319728; PubMed Central PMCID: PMC1977759.
- 20. Lindel K, Forster A, Altermatt HJ, Greiner R, Gruber G. Breast cancer and human papillomavirus (HPV) infection: no evidence of a viral etiology in a group of Swiss women. Breast (Edinburgh, Scotland). 2007;16(2):172–7. pmid:17088061.
- 21. de Cremoux P, Thioux M, Lebigot I, Sigal-Zafrani B, Salmon R, Sastre-Garau X, et al. No evidence of human papillomavirus DNA sequences in invasive breast carcinoma. Breast cancer research and treatment. 2008;109(1):55–8. pmid:17624590.
- 22. Gopalkrishna V, Singh UR, Sodhani P, Sharma JK, Hedau ST, Mandal AK, et al. Absence of human papillomavirus DNA in breast cancer as revealed by polymerase chain reaction. Breast cancer research and treatment. 1996;39(2):197–202. pmid:8872328.
- 23. Czerwenka K, Heuss F, Hosmann JW, Manavi M, Lu Y, Jelincic D, et al. Human papilloma virus DNA: a factor in the pathogenesis of mammary Paget's disease? Breast cancer research and treatment. 1996;41(1):51–7. pmid:8932876.
- 24. Hachana M, Ziadi S, Amara K, Toumi I, Korbi S, Trimeche M. No evidence of human papillomavirus DNA in breast carcinoma in Tunisian patients. Breast (Edinburgh, Scotland). 2010;19(6):541–4. pmid:20547456.
- 25. Hedau S, Kumar U, Hussain S, Shukla S, Pande S, Jain N, et al. Breast cancer and human papillomavirus infection: no evidence of HPV etiology of breast cancer in Indian women. BMC cancer. 2011;11:27. pmid:21247504; PubMed Central PMCID: PMC3036645.
- 26. Silva RG Jr., da Silva BB. No evidence for an association of human papillomavirus and breast carcinoma. Breast cancer research and treatment. 2011;125(1):261–4. pmid:20734131.
- 27. Vernet-Tomas M, Mena M, Alemany L, Bravo I, De Sanjose S, Nicolau P, et al. Human papillomavirus and breast cancer: no evidence of association in a Spanish set of cases. Anticancer research. 2015;35(2):851–6. pmid:25667466.
- 28. de Roda Husman AM, Walboomers JM, van den Brule AJ, Meijer CJ, Snijders PJ. The use of general primers GP5 and GP6 elongated at their 3' ends with adjacent highly conserved sequences improves human papillomavirus detection by PCR. The Journal of general virology. 1995;76 (Pt 4):1057–62. pmid:9049358.
- 29. Gravitt PE, Peyton CL, Alessi TQ, Wheeler CM, Coutlee F, Hildesheim A, et al. Improved amplification of genital human papillomaviruses. Journal of clinical microbiology. 2000;38(1):357–61. pmid:10618116; PubMed Central PMCID: PMC88724.
- 30. Wang Y, Wang A, Jiang R, Pan H, Huang B, Lu Y, et al. Human papillomavirus type 16 and 18 infection is associated with lung cancer patients from the central part of China. Oncology reports. 2008;20(2):333–9. pmid:18636194.
- 31. Band V, Zajchowski D, Kulesa V, Sager R. Human papilloma virus DNAs immortalize normal human mammary epithelial cells and reduce their growth factor requirements. Proceedings of the National Academy of Sciences of the United States of America. 1990;87(1):463–7. pmid:2153303; PubMed Central PMCID: PMC53284.
- 32. Tjalma WA, Depuydt CE. Cervical cancer screening: which HPV test should be used—L1 or E6/E7? European journal of obstetrics, gynecology, and reproductive biology. 2013;170(1):45–6. pmid:23932300.
- 33. Bratthauer GL, Tavassoli FA, O'Leary TJ. Etiology of breast carcinoma: no apparent role for papillomavirus types 6/11/16/18. Pathology, research and practice. 1992;188(3):384–6. pmid:1320761.
- 34. Yu Y, Morimoto T, Sasa M, Okazaki K, Harada Y, Fujiwara T, et al. HPV33 DNA in premalignant and malignant breast lesions in Chinese and Japanese populations. Anticancer research. 1999;19(6b):5057–61. Epub 2000/03/04. pmid:10697510.
- 35. Damin AP, Karam R, Zettler CG, Caleffi M, Alexandre CO. Evidence for an association of human papillomavirus and breast carcinomas. Breast cancer research and treatment. 2004;84(2):131–7. Epub 2004/03/05. pmid:14999143.
- 36. Tsai JH, Tsai CH, Cheng MH, Lin SJ, Xu FL, Yang CC. Association of viral factors with non-familial breast cancer in Taiwan by comparison with non-cancerous, fibroadenoma, and thyroid tumor tissues. Journal of medical virology. 2005;75(2):276–81. Epub 2004/12/17. pmid:15602723.
- 37. de Leon DC, Montiel DP, Nemcova J, Mykyskova I, Turcios E, Villavicencio V, et al. Human papillomavirus (HPV) in breast tumors: prevalence in a group of Mexican patients. BMC cancer. 2009;9:26. Epub 2009/01/24. pmid:19161629; PubMed Central PMCID: PMCPmc2636825.
- 38. He Q, Zhang SQ, Chu YL, Jia XL, Wang XL. The correlations between HPV16 infection and expressions of c-erbB-2 and bcl-2 in breast carcinoma. Molecular biology reports. 2009;36(4):807–12. pmid:18427947.
- 39. Mendizabal-Ruiz AP, Morales JA, Ramirez-Jirano LJ, Padilla-Rosas M, Moran-Moguel MC, Montoya-Fuentes H. Low frequency of human papillomavirus DNA in breast cancer tissue. Breast cancer research and treatment. 2009;114(1):189–94. Epub 2008/04/01. pmid:18373273.
- 40. Mou X, Chen L, Liu F, Shen Y, Wang H, Li Y, et al. Low prevalence of human papillomavirus (HPV) in Chinese patients with breast cancer. The Journal of international medical research. 2011;39(5):1636–44. Epub 2011/11/29. pmid:22117964.
- 41. Chang P, Wang T, Yao Q, Lv Y, Zhang J, Guo W, et al. Absence of human papillomavirus in patients with breast cancer in north-west China. Medical oncology (Northwood, London, England). 2012;29(2):521–5. Epub 2011/04/19. pmid:21499926.
- 42. Frega A, Lorenzon L, Bononi M, De Cesare A, Ciardi A, Lombardi D, et al. Evaluation of E6 and E7 mRNA expression in HPV DNA positive breast cancer. European journal of gynaecological oncology. 2012;33(2):164–7. Epub 2012/05/23. pmid:22611956.
- 43. Glenn WK, Heng B, Delprado W, Iacopetta B, Whitaker NJ, Lawson JS. Epstein-Barr virus, human papillomavirus and mouse mammary tumour virus as multiple viruses in breast cancer. PloS one. 2012;7(11):e48788. Epub 2012/11/28. pmid:23183846; PubMed Central PMCID: PMCPmc3501510.
- 44. Sigaroodi A, Nadji SA, Naghshvar F, Nategh R, Emami H, Velayati AA. Human papillomavirus is associated with breast cancer in the north part of Iran. TheScientificWorldJournal. 2012;2012:837191. Epub 2012/05/09. pmid:22566779; PubMed Central PMCID: PMCPmc3329875.
- 45. Liang W, Wang J, Wang C, Lv Y, Gao H, Zhang K, et al. Detection of high-risk human papillomaviruses in fresh breast cancer samples using the hybrid capture 2 assay. Journal of medical virology. 2013;85(12):2087–92. Epub 2013/08/21. pmid:23959946.
- 46. Ali SH, Al-Alwan NA, Al-Alwany SH. Detection and genotyping of human papillomavirus in breast cancer tissues from Iraqi patients. Eastern Mediterranean health journal = La revue de sante de la Mediterranee orientale = al-Majallah al-sihhiyah li-sharq al-mutawassit. 2014;20(6):372–7. Epub 2014/06/25. pmid:24960513.
- 47. Ahangar-Oskouee M, Shahmahmoodi S, Jalilvand S, Mahmoodi M, Ziaee AA, Esmaeili HA, et al. No Detection of 'High-risk' Human Papillomaviruses in a Group of Iranian Women with Breast Cancer. Asian Pacific journal of cancer prevention: APJCP. 2014;15(9):4061–5. Epub 2014/06/18. pmid:24935597.
- 48. Manzouri L, Salehi R, Shariatpanahi S, Rezaie P. Prevalence of human papilloma virus among women with breast cancer since 2005–2009 in Isfahan. Advanced biomedical research. 2014;3:75. Epub 2014/03/15. pmid:24627883; PubMed Central PMCID: PMCPmc3950802.
- 49. Peng J, Wang T, Zhu H, Guo J, Li K, Yao Q, et al. Multiplex PCR/mass spectrometry screening of biological carcinogenic agents in human mammary tumors. Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology. 2014;61(2):255–9. pmid:25088618.
- 50. Fu L, Wang D, Shah W, Wang Y, Zhang G, He J. Association of human papillomavirus type 58 with breast cancer in Shaanxi province of China. Journal of medical virology. 2015;87(6):1034–40. pmid:25676062.
- 51. Choi YL, Cho EY, Kim JH, Nam SJ, Oh YL, Song SY, et al. Detection of human papillomavirus DNA by DNA chip in breast carcinomas of Korean women. Tumour biology: the journal of the International Society for Oncodevelopmental Biology and Medicine. 2007;28(6):327–32. Epub 2008/04/09. pmid:18391549.
- 52. Chaiwongkot A, Pientong C, Ekalaksananan T, Kongyingyoes B, Thinkhamrop J, Yuenyao P, et al. Evaluation of primers and PCR performance on HPV DNA screening in normal and low grade abnormal cervical cells. Asian Pacific journal of cancer prevention: APJCP. 2007;8(2):279–82. pmid:17696746.
- 53. Khan NA, Castillo A, Koriyama C, Kijima Y, Umekita Y, Ohi Y, et al. Human papillomavirus detected in female breast carcinomas in Japan. British journal of cancer. 2008;99(3):408–14. Epub 2008/07/24. pmid:18648364; PubMed Central PMCID: PMCPmc2527789.
- 54. Lawson JS, Glenn WK, Heng B, Ye Y, Tran B, Lutze-Mann L, et al. Koilocytes indicate a role for human papilloma virus in breast cancer. British journal of cancer. 2009;101(8):1351–6. Epub 2009/09/24. pmid:19773762; PubMed Central PMCID: PMCPmc2768439.
- 55. D'Souza G, Agrawal Y, Halpern J, Bodison S, Gillison ML. Oral sexual behaviors associated with prevalent oral human papillomavirus infection. The Journal of infectious diseases. 2009;199(9):1263–9. pmid:19320589.
- 56. Widschwendter A, Brunhuber T, Wiedemair A, Mueller-Holzner E, Marth C. Detection of human papillomavirus DNA in breast cancer of patients with cervical cancer history. Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology. 2004;31(4):292–7. pmid:15494272.
- 57. de Villiers EM, Sandstrom RE, zur Hausen H, Buck CE. Presence of papillomavirus sequences in condylomatous lesions of the mamillae and in invasive carcinoma of the breast. Breast cancer research: BCR. 2005;7(1):R1–11. pmid:15642157; PubMed Central PMCID: PMC1064094.