The etiological role of HPV or EBV in some extragenital tumors is under intense debate. In this report, the presence of HPV and EBV was evaluated in BCs from Chile, a Latin American country from the Pacific Ocean area. Our findings showed that HPV and EBV prevalence in BCs was relatively low since these viruses were present in 8.7% and 6.5% of breast tumors, respectively. The high-risk HPV-16, the genotype most frequently detected in cervix uterine cancer and extragenital malignancies in Chile and in the world [9, 10] was the only genotype identified in HPV positive BCs. However, the presence of HPV genomes is not a sufficient condition to establish a causal relationship. For this reason, we investigated the physical status of HPV and the presence of E6/E7 transcripts into the BC tumors. Thus, we found that HPV-16 was integrated into the host genome in all of the cases.
Other authors have analyzed HPV in BC specimens, suggesting a causal association. Hennig et al. proposed that HPV can be transported by circulation (bloodstream/lymphatic system) from cervix to the breast . In addition, it has been reported the presence of HPV-6/11 and other papillomaviruses in nipple and tumoral tissue from patients with BC, even though high-risk HPV was present in a low proportion of cases . In Asia, it has been reported a 20% of high-risk HPV in breast tumors from Japanese women and HPV-16 was the most frequent HPV genotype that was integrated into the host genome with a low viral load . In Latin America, only three studies have been reported: in Brazil it was reported HPV-16/18 presence in 24.8% of BCs  and recently it was published that 79 analyzed BCs were negative for HPV-6, 11, 16 and 18 . In Mexico, HPV was found in 29.4% of BC, being HPV-16 the most prevalent genotype . However, in the same country, HPV was detected in 10% of 60 BCs . In this last study, HPV was found integrated with a low viral load. In Oceania (Australia), it has been reported a study using In situ PCR for the successful detection of HPV in BCs. High-risk HPV was detected in the nucleus of 11/43 (25.6%) BCs . On the other hand, HPV-18 genotype was frequently found in Australian BCs .
The loss of E2 gene function by HPV integration is known to be one of the major genetic events facilitating transformation and transition to malignancy . The E2 loss allows abnormal expression of E6 and E7 oncoproteins . The mechanism of high-risk HPV-mediated oncogenesis involves the interaction of E6 oncoprotein with the p53 tumor suppressor protein and the E6-associated protein for induction of proteasomal-dependant p53 degradation . In addition, the high-risk HPV E7 binding to pRb protein induces E2F releasing and overexpression of p16INK4a by a negative feedback mechanism . High-risk HPVs are frequently integrated in high-grade cervical intraepithelial neoplasia lesions and low-risk HPVs are frequently present in an episomal form in low-grade or benign lesions . The analytical approach used in this report, considers HPV-16 integrated when E2 gene is not amplified using quantitative real-time PCR. This is an indirect method, based in the frequent E2 disruption when HPV is integrated into the host genome. However, HPV is able to integrate disrupting other regions as E1 . So, when E2 is successfully amplified is not possible discard HPV-16 integration. However, we were unable to amplify a fragment of E2 gene in four cases that previously were positive for HPV-16 presence. Thus, E2 loss by integration is the biological possibility that explains these results.
Even though, HPV-16 was present and integrated in BCs, we were unable to detect E6 and E7 transcript expressions in the specimens. The successful amplification of a fragment of β2-microglobulin was used as control of cDNA preparation and amplification from paraffin-embedded tissues. It has been reported that in general HPV load in extragenital tumors is heterogeneous and lower that in cervical cancer [13, 24, 25] so it was expected that E6/E7 transcript levels were very low too, in concordance with the viral load. However, the absence of detectable levels of E6/E7 transcripts is a hallmark of absence of functional activity in HPV-16 positive specimens. The RNA obtained from paraffin-embedded cervical carcinomas was used as control, and we were able to detect E6/E7 transcripts in all of them (data not shown). Curiously, this is the first study that addresses the expression of E6 and E7 transcripts in breast carcinomas, so we cannot compare our results with any previous published report. On the other hand, the viral load in three specimens (higher than unity) is compatible with an eventual direct carcinogenic role of HPV, although the absence of E6/E7 expression casts doubts about this notion. To this respect, it is possible to speculate the possibility of a "hit and run" mechanism of HPV action, however if HPV is related to the initiation, promotion or progression of breast carcinogenesis is unknown. Because PCR method is unable to determine if HPV localizes specifically in the tumoral tissue, the detected viral load in 3/4 cases allow us to speculate that at least tumoral cells are positive for HPV-16. Anyway, we cannot deny the possibility that HPV infection occurred after clonal expansion and cancer development, involving both tumoral and non-tumoral tissue. In addition, it is known that one HPV genome per cell is enough for neoplastic transformation as occur in SiHa cells harboring 1-2 HPV-16 copies/cell. Around the world, the HPV prevalence in BCs has been shown to be highly variable, ranging from 0 to 86% . This difference may be explained by variations in the methodological approaches used to detect HPV. Moreover, PCR protocols showing a diverse sensitivity and specificity are currently used to determine HPV presence in tumoral tissue and a "gold standard" protocol has not been defined. However, our analytical approach has been widely used by us and was previously reported by others .
On the other hand, there is a relative consensus that for detection of EBV latent infection, the "gold standard" method is ISH for EBER-1 . Interestingly, we detected three positive specimens using qRT-PCR for EBNA-1 and using ISH for EBER-1, the cases were negative. This discrepancy might be caused by differences in sensitivity of these methods or very low levels of EBER-1 expression in EBV infected cells. EBERs are necessary for the maintenance of malignant phenotypes of B lymphocyte cells but is unknown if are ever expressed in BC cells . Recently, it has been reported a 55% of EBV presence in BCs from India . Interestingly, the authors evaluated EBNA-1 expression using immunohistochemistry (IHC) and they compared with serology in women with cancer and controls. Unfortunately, the authors did not compare their results with EBER-1 detection in the same specimens. In BC, since the first study in 1993 reporting no detection of EBV in BCs , EBV presence has been consistently reported in a plethora of other studies [29, 31–36]. However, EBV has not been detected in other reports around the world [37–40]. In this study, using a Cox-regression model we found a statistically significant association between EBV EBNA-1 positivity and poor survival. Even though the sample size was low, this data is in agreement with a previous report where a relationship between poor survival and EBV presence was found . The molecular mechanism involved in poor survival in EBV- associated BC remains to be investigated. It is necessary to appoint that is not strictly necessary that a virus be directly involved as etiological agent to produce molecular alterations in the behavior of some tumor or progression of the disease. In fact, HCMV, another persistent virus, has been detected in some tumors where the virus is able to alter the host response, specifically through immune response modulation . However, how HPV or EBV may potentially be involved in tumor modulation and consequently are associated with outcome or prognostic warrants more investigation.
In conclusion, we reported here that HPV and EBV prevalence in breast cancer from Chile is relatively low, thus a possible direct etiological role of these virus is unlikely. However, additional studies are warranted to elucidate the function of HPV/EBV in a subset of breast carcinomas from Chile.