Introduction

Breast cancer is the most frequently diagnosed malignancy in women in many populations [1], and the incidence of breast carcinoma has increased by 2.5% over the last 50 years [2]. Many risk factors have been associated with the pathogenesis of this disease; including family history, hormones, cigarette smoking and alcohol consumption [3], but the molecular mechanisms related to the breast carcinogenesis remain poorly understood. In Argentina, deaths caused by breast carcinoma raise up to 21.8 per every 100,000 women, the second place in Latin America, and represent 5,400 deaths per year [4].

Currently a large number of infectious agents have been described to either cause or contribute to specific human cancers [5]. Indeed, 10–15% of the cancers worldwide can be linked to viral infections [6]. Many studies have recently suggested that certain viruses might be involved in the pathogenesis of breast cancer, such as specific types of Human papillomavirus (HPV), Epstein Barr virus (EBV) and Mouse mammary tumor virus (MMTV), but viral involvement in breast carcinogenesis still remains controversial. While some groups have suggested that high risk HPV could be implicated in breast tumor development [3], [7]–[22], other groups detracted this proposal [23]–[26]. However, HPV immortalization of normal breast epithelium has been demonstrated in vitro [27], raising the possibility that HPV might be etiologically related to a proportion of breast cancers.

Human papillomaviruses (HPVs) species, family Papillomaviridae, genus Alphapapillomavirus, are small DNA viruses that infect epithelial cells of the skin and mucosa. HPV is the causal agent of cervix-uterine cancer and anogenital malignancies. However, HPV has been detected in extragenital tumors such as oral, esophageal, tonsillar, laryngea and lungs [1]. The HPV types that infect the genital mucosa can be divided into two groups. The high-risk types, including HPV16, 18, 31 and others, are frequently found in cervical cancers. By contrast, the low-risk types, including HPV6 and 11, also infect the genital epithelia, but are rarely detected in malignancies [28]

Considering the controversial reports on the association of HPV in breast carcinomas, the aim of this study was to explore HPV presence in this neoplasia among a female Argentinean population. Also to examine HPV genotype, particularly those of high risk and correlate their presence with clinical characteristics. This study demonstrated for the first time in Argentina the presence of HPV in a proportion of the malignant breast tissues, with a prevalence of low risk HPV11, followed by high risk HPV16.

Materials and Methods

Results and Discussion

Patients’ clinical characteristics and HPV association are shown in Table 1. Sixteen out of 61 patients (26%) were HPV positive for MY09/MY11 and GP5+/GP6+ amplification. Our results confirm and broaden earlier reports that also described a similar proportion of positive cases, such as the 23% HPV association with invasive ductal carcinoma, reported by Heng B et al in Australia [9], the 21% described in Japan by Khan et al [7] or the 25.9% HPV positive cases in Iran [16]. In line with these, Li et al conducted a meta-analysis and revealed that 24.5% of the breast carcinoma cases were associated with HPV, of which 32.4% occurred in Asia and 12.9% in Europe [12]. Increased HPV incidence in breast cancer was observed in Australia, were both Glenn et al [17] and Antonsson et al [22] described HPV DNA prevalence in the breast cancer samples of 50%. Conversely, only a 6.5% HPV association was described in China [21].

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Table 1. HPV status according to clinical parameters in breast carcinoma cases.

https://doi.org/10.1371/journal.pone.0061613.t001

In Latin American population, Cantu de León et al described a 29.4% of HPV association with Mexican breast carcinoma [11] and Aguayo et al, an 8.7% of HPV16 presence in Chile [14]. In contrast, others studies performed in our region, in Brazil [25] and Mexico [26], described no evidence of HPV association with breast carcinoma. The findings reported by our study also support the notion for a viral association with breast carcinoma. In fact, in Argentina our group has previously also described EBV presence in 31% breast carcinoma patients, with a particular latency profile, not already observed in EBV-associated tumors [30]. Interestingly, 1/61 patients of this analyzed series harboured both HPV and EBV, pointing out that both viruses might contribute to the neoplasic process.

In this report, clinical parameters such as tumor histology, axillary lymph node status or estrogen and progesterone receptors were not statistically associated with HPV presence (p>0.05 χ² test), in accordance with previous reports [7], [11], [14], [15]. However, Kroupis et al described the only breast cancer series harbouring high-risk HPV DNA sequences related to clinical parameters; in which those HPV+ cases displayed less estrogen-receptor and were proliferative [31]. Even though in the present study HPV presence was not statistically significant among histological tumor types, it is noteworthy that both papillary breast carcinoma cases, a rare histologic type, were HPV positive.

At present, about 130 HPV types were identified by their sequence of the gene encoding the major capsid protein L1 isolated from HPV associated diseases. Moreover, they can be also classified into high- and low-risk types depending upon their oncogenic potential. This is shown most clearly in the genital tract, in which there is regular or sporadic infection with about 30–40 types. These can be divided into those predominately associated with benign anogenital warts or condylomata, low-risk HPV types 6, 11 and their relatives, and those associated with anogenital cancers and the precursor lesions (intraepithelial neoplasia), particularly of the cervix, HPV 16, 18, 31, 33, 35, 45, and minor types. The most important players of cervical cancers are HPV 16, found in 50–70% of cases, and HPV 18, found in 7–20% of cases [32]. When HPV typing was performed by sequence analysis in a subgroup of 9 cases of our series, 4 cases were characterized as HPV 11, 3 as HPV 16 and 1 as HPV13. Notably, 1 case showed a co-infection with HPV 11 together with HPV 87. The presence of HPV11, a low risk type, was very remarkable since mostly high risk HPV 16 and 18 types were reported in breast carcinoma [17], [21], [22]. In Iran, high-risk HPV genotypes in breast cancer patients were the predominant types, but other genotypes (HPV-6, HPV-11, HPV-15, HPV-23, and HPV-124) were also detected [16]. Only de Villiers et al, in a German series, demonstrated that the most prevalent type in both breast carcinomas and nipples from the same patient was HPV 11, followed by HPV 6 [3]. In Argentina, the prevalence of HPV16 in high grade intraepithelial cervical lesions samples is 48.5% and 16.9% for HPV18; instead, invasive cervical cancer shows a prevalence of 59.5% for HPV16 and 17.6% for HPV18 [33].

HPV types in Latin American breast carcinoma patients, displayed prevalence for high risk HPV. In fact, in Chile only the HPV-16 genotype was present in positive breast carcinomas without co-infections with other HPV genotypes [14], while in Mexico, 66.6% were positive for HPV 16, 20% for HPV 18, and 13.4% were positive for both. High risk HPV types also prevail in breast carcinoma from other population. Damin et al [15] reported 56% HPV 16 and 40% HPV 18. In line with this, Li et al [12], in a meta-analysis, found that the four most commonly identified HPV types, in order of decreased prevalence, were HPV33, 18, 16, and 35. In Japan, the most frequently detected HPV genotype was HPV 16 (92%), followed by HPV 6 (46%), HPV 18 (12%), and HPV 33 (4%) [7]. Kroupis et al [31] identified a total of 21 high-risk viruses: 67% HPV 16, 14% HPV 59, 10% HPV 58, 5% HPV 73 and 5% HPV 82.

HPV analysis performed so far in this report enables exclusively evaluation of the presence of viral DNA, but do not inform on the possible viral activity or infection productivity. Therefore, evaluation of E6 and E7 mRNA in high risk genotypes may represent a good marker for HPV involvement in malignant transformation. In fact, a strong association between high risk mRNA-HPV presence and risk of neoplastic progression in cervical lesions has been described [34], [35]. In consequence, E6 and E7 mRNA-HPV presence in high risk phenotypes was suggested as a tool for early identification of cervical lesions that would evolve toward neoplastic transformation. Furthermore, HPV transcriptional activity was also found in most oropharyngeal squamous cell carcinoma [36]. E6 and E7 mRNA from high risk HPV16 and HPV18 evaluated in 9 cases with quality RNA from our series by RT-PCR were negative, demonstrating no transcriptional activity of high risk HPV types, as previously observed in Italy [37].

Although the route of transmission for the virus has not been determined, women positive for both breast and cervical cancers were found to be infected with the same HPV type in both tumors [37]. Furthermore, women with squamous precancer and women with glandular precancer in the cervix had a significantly higher risk of malignant breast tumors than the general female population from Norway [18]. Unfortunately, in our series HPV data in cervix tissue were not available to give rise to this assumption.

Acknowledgments

P Ch. and M.V.P. are members of the National Research Council (CONICET), Research Career Programme and M.L. is a National Research Council (CONICET) doctoral fellow.