Journal of Medical Virology 83:2157–2163 (2011) High Prevalence of Human Papillomaviruses in Fresh Frozen Breast Cancer Samples Annika Antonsson, 1 Terrence P. Spurr, 1 Alice C. Chen, 1 Glenn D. Francis, 2 Nigel A.J. McMillan, 1 Nicholas A. Saunders, 1 Michael Law, 3 and Ian C. Bennett 3 * ,y 1 University of Queensland, Diamantina Institute for Cancer, Immunology and Metabolic Medicine, Princess Alexandra Hospital, Woolloongabba, Brisbane, Queensland, Australia 2 Pathology Queensland, Princess Alexandra Hospital, Woolloongabba, Brisbane, Queensland, Australia 3 Breast & Endocrine Unit, Department of Surgery, Princess Alexandra Hospital, Woolloongabba, Brisbane, Queensland, Australia While the etiology of breast cancer remains enigmatic, some recent reports have examined the role of human papillomavirus (HPV) in breast carcinogenesis. The purpose of this study was to determine the prevalence of HPV in breast cancer tissue using PCR analysis and sequencing. Fifty-four (54) fresh frozen breast cancers samples that were removed from a co- hort of breast cancer patients were analyzed. Samples were tested for HPV using compre- hensive PCR primers, and in situ hybridization was performed on paraffin embedded tissue sections. Findings were correlated with clinical and pathological characteristics. The HPV DNA prevalence in the breast cancer samples was 50% (27/54) with sequence analysis indicating all cases to be positive for HPV-18 type. While HPV patients were slightly younger, no correla- tion was noted for menopausal status or family history. HPV positive tumors were smaller with earlier T staging and demonstrated lesser nodal involvement compared to HPV negative can- cers. In situ hybridization analyses proved neg- ative. The high proportion of HPV positive breast cancers detected in this series using fresh frozen tissues cannot be dismissed, how- ever the role of HPV in breast carcinogenesis remains unclear and may ultimately be ascer- tained by monitoring future breast cancer inci- dence amongst women vaccinated against high risk HPV types. J. Med. Virol. 83:2157– 2163, 2011. ß 2011 Wiley Periodicals, Inc. KEY WORDS: breast cancer; human papillo- mavirus (HPV); polymerase chain reaction (PCR); in situ hybridization INTRODUCTION The papillomaviruses are small, double-stranded DNA viruses belonging to the Papillomaviridae- family. So far, more than 110 different HPV types have been fully characterized, and in addition, several papillomavirus types have been isolated from a number of vertebrate species [de Villiers et al., 2004]. Papillomaviruses can be grouped according to tissue tropism with HPV types found in mucosal lesion being referred to as mucosal or genital types, while HPV types found in skin are called skin or cutaneous types. Mucosal HPV types that are found preferentially in cervical and other anogenital cancers have been desig- nated high-risk types. These high-risk HPVs have been identified as the causative agent in 99.7% or more of cervical cancers [Walboomers et al., 1999] and have also been detected in more than 50% of other anogenital cancers [zur Hausen, 1996]. The most prevalent high-risk HPV types are HPV-16 and HPV- 18, which account for 70% of the cancer cases, with another 10 types making up the other 30%. These Grant sponsor: Centres for Health Research, Princess Alex- andra Hospital Cancer Collaborative Group; Grant sponsor: Mazda Foundation (to A.A.); Grant sponsor: Prostate Cancer Foundation of Australia (to A.A.). There are no conflicts of interest. Ethical approval was obtained through the Princess Alexan- dra Hospital Ethics Committee. y Associate Professor. *Correspondence to: Ian C. Bennett, University of Queensland Department of Surgery, Princess Alexandra Hospital, Wooloon- gabba, Brisbane, Australia. E-mail: iancben@bigpond.com Accepted 8 August 2011 DOI 10.1002/jmv.22223 Published online in Wiley Online Library (wileyonlinelibrary.com). ß 2011 WILEY PERIODICALS, INC.