Human papillomavirus detected in female breast carcinomas in Japan

Abstract To investigate the aetiological role of human papillomavirus (HPV) in breast cancer, we examined the presence, genotype, viral load, and physical status of HPV in 124 Japanese female patients with breast carcinoma. Human papillomavirus presence was examined by PCR using SPF10 primers, and primer sets targeting the E6 region of HPV-16, -18, and -33. The INNO-LiPA HPV genotyping kit was used to determine genotype. Human papillomavirus DNA was detected in 26 (21%) breast carcinomas. The most frequently detected HPV genotype was HPV-16 (92%), followed by HPV-6 (46%), HPV-18 (12%), and HPV-33 (4%). In 11 normal epithelium specimens adjacent to 11 HPV-16-positive carcinomas, 7 were HPV-16-positive. However, none of the normal breast tissue specimens adjacent to HPV-negative breast carcinomas were HPV-positive. The real-time PCR analysis suggested the presence of integrated form of viral DNA in all HPV-16-positive samples, and estimated viral load was low with a geometric mean of 5.4 copies per 104 cells. In conclusion, although HPV DNA was detected in 26 (21%) breast carcinomas and, in all HPV-16-positive cases, the HPV genome was considered integrated into the host genome, their low viral loads suggest it is unlikely that integrated HPV is aetiologically involved in the development of Japanese breast carcinomas that we examined. Main Breast cancer is one of the most prevalent malignancies throughout the world. It is the leading female cancer worldwide and comes second after stomach cancer in Japan (Parkin et al, 1999). Recent studies have suggested that some types of viruses, especially human papillomavirus (HPV), might be involved in the pathogenesis of breast cancer (Liu et al, 2001; Tsai et al, 2007). Molecular and epidemiological studies have shown that a persistent infection with high-risk HPV is the most important risk factor for both cervical cancer and its precursors (Cuschieri et al, 2005; Steenbergen et al, 2005). A number of studies have reported HPV DNA detection in extragenital cancers, although the aetiological involvement of HPV in those malignancies is still controversial (Gillison and Shah, 2003). Recently, it has been shown that HPV types 16 and 18 can immortalize normal breast epithelium (Band et al, 1990; Wazer et al, 1995). This raised the possibility that HPV may be aetiologically related to some cases of breast cancer. However, unlike cervical carcinoma, which is almost always associated with HPV, the causal role of HPV infection in the development of breast carcinoma remains controversial. Di Lonardo et al (1992) were the first to report the relationship between HPV and breast cancer, demonstrating HPV-16 DNA in 29.4% of breast carcinomas, using PCR with primer sets specific for HPV-11, -16, and -18 genotypes. A study in Norway, which used primers specific for HPV-11, -16, -18, and -33, in addition to GP5+/6+ primers, detected HPV in 46% of breast carcinomas. In this previous study, only HPV-16 was detected (Hennig et al, 1999). Another European study showed the presence of high-risk HPVs in 15% of invasive breast carcinomas in Greece (Kroupis et al, 2006). A Chinese study also found that 35% of breast carcinomas examined were HPV-16-positive, using type-specific primers for HPV-16 and consensus primers for the HPV L1 gene (Liu et al, 2001). In addition, a study in Korea reported HPV DNA presence in breast carcinomas (6.5%) but not in intraductal papilloma (Choi et al, 2007). Furthermore, a study in Brazil demonstrated HPV-16 and HPV-18 in 14 and 10% of breast carcinomas, respectively, using genotype-specific primers (Damin et al, 2004). In addition, an Australian study reported by Kan et al (2005) used primers specific for HPV-16, -18, and -33, and demonstrated the presence of HPV-18 in 48% of breast cancer specimens. However, neither HPV-16 nor HPV-33 was detected in their study. Another study conducted in China and Japan, which used primers for HPV-16, -18, and -33, detected HPV-33 in 41.7 and 11.1% of breast carcinomas in two countries, respectively (Yu et al, 1999). By examining nipple and breast cancer specimens, de Villiers et al (2005) detected high-risk and low-risk HPVs in 86% of them. They used GP5+/6+, FAP primers for cutaneous-type HPVs, and CP primers. In contrast, several other studies failed to demonstrate HPV DNA in tumors of the breast (Wrede et al, 1992; Lindel et al, 2007). In malignant transformation of the uterine cervical epithelia, the integration of high-risk HPV DNA into the host genome is considered an important step (zur Hausen, 1991). Viral DNA integration leads to frequent disruption of the HPV-16 E2 gene (Kalantari et al, 1998), a negative regulator of the E6/E7 promoter, resulting in the upregulation of the transcription of the E6 and E7 oncogenes. Human papillomavirus-16 E6 and E7 oncogenes interfere with the normal cell cycle by targeting the p53 and pRb tumor suppressor proteins, respectively (Vousden, 1993). Human papillomavirus-16 DNA integration into the host genome possibly provides a selective growth advantage to the host cell (Jeon et al, 1995; Jeon and Lambert, 1995). When considering the aetiological role of HPV in non-genital cancers, an important question that should be addressed is the integration of HPV into the host genome. Venuti et al (2000) found that 43% of HPV-16-positive laryngeal carcinomas had HPV-16 DNA integrated into the host genome. Koskinen et al (2003) also reported that 65% of HPV-16-positive head and neck carcinomas had integrated HPV-16 DNA. Recent studies conducted by our laboratory also showed that most HPV-16-positive esophageal and lung carcinomas had integrated HPV-16 DNA (Aguayo et al, 2007; Shuyama et al, 2007). To our knowledge, however, none of the published studies have investigated the possibility of integration of HPV in breast cancer tissues. Considering the controversial reports on the aetiology of HPV in breast carcinomas, the aim of this study was to examine the presence, genotype, viral load, and physical status of HPV, particularly high-risk HPV, among breast carcinomas in Japan, where breast cancer risk is low when compared with Europe and North America (Parkin et al, 2002), and to investigate its aetiological role. Materials and methods Study subjects A total of 124 formalin-fixed and paraffin-embedded breast cancer specimens consisting of 42 cases from Sagara Hospital (Kagoshima, Japan), diagnosed during the period between 2000 and 2001, and 82 cases from Kagoshima University Hospital (Kagoshima, Japan), diagnosed during the period between 2000 and 2006, were examined in this study. In addition, as positive controls, we examined formalin-fixed and paraffin-embedded specimens from six cervical cancer cases diagnosed at Kagoshima University Hospital. Clinicopathological information, including the expression of oestrogen and progesterone receptors, reproductive histories, and family history of breast cancer, was obtained from medical and pathol (...truncated)