The authors have declared that no competing interests exist.
Conceived and designed the experiments: ALPS MVP PC. Performed the experiments: MAL RGL MC. Analyzed the data: HG PMV PG. Contributed reagents/materials/analysis tools: HG. Wrote the paper: ALPS PC.
The etiology and the molecular mechanisms related to breast carcinogenesis remain poorly understood. Some recent reports have examined the role of
Sixty one fresh frozen breast cancers samples were analyzed. Samples were tested for HPV by PCR, and products were automatically sequenced. Findings were correlated with clinical and pathological characteristics.
The HPV DNA prevalence in the breast cancer samples was 26% (16/61). Clinical parameters were not statistically associated with HPV presence (p>0.05 χ2 test). Sequence analysis in a subgroup of cases indicates the prevalence of low risk HPV11, followed by high risk HPV16. We found no HPV transcriptional activity.
The present study demonstrated for the first time in Argentina the presence of HPV in a proportion of the malignant breast tissues. This finding suggests that HPV may have a biological significance in breast carcinogenesis.
Breast cancer is the most frequently diagnosed malignancy in women in many populations
Currently a large number of infectious agents have been described to either cause or contribute to specific human 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
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.
The study was conducted on 61 fresh tissue biopsies of breast carcinoma, collected without any preselection criteria from the pathological archives of the Pathology Service of M. Villegas de Martinez Hospital. Tumors, which were typed according to the American Joint Committee on Cancer, included: 43 invasive ductal carcinomas, 10 invasive lobular, 2 papillary, 2 tubular, 1 adenocystic, 1 mucinous, 1 aprocrine and 1 comedocarcinoma. Patients’ age ranged from 35 to 92 years (median age 57 years).
This study has the approval of the Institutional Review Board and the Ethics Board of both M. Villegas de Martinez Hospital and Ricardo Gutierrez Children Hospital and is also in accordance with the Helsinki Declaration of 1975. A written informed consent was obtained from every patient.
DNA was extracted from tumor fresh biopsies using QIAamp DNA Mini Kit (QIAGEN GmbH, Hilden, Germany) following manufacturer’s instructions.
The presence of HPV DNA sequences was verified by amplification with two sets of primers. A first round with degenerated primers MY09, 5′-CGTCCMARRGGAWACTGATC-3′ and MY11, 5′-GCMCAGGGWCATAAYAATGG -3′, amplifying 450 bp long fragment in highly conserved region in L1 gene; and a second round with consensus primers GP5+, 5′-TTTGTTACTGTGGTAGATACTAC- 3′ and GP6+,
In a separate reaction tube, a set of primers for the beta-globin gene,
HPV PCR products were separated by electrophoresis in 2% agarose gel stained with ethidium bromide and purified with QIAEXII gel extraction kit (Qiagen GmbH) according to manufacturer’s instructions. These purified PCR products were directly sequenced using Big Dye Terminator v3.1 kit (Applied Biosystems, Foster City, CA) in an automated Genetic Analyzer 3130×l (Applied Biosystems). HPV sequences were compared with published ones of known HPV types. At least two independent sequencing reactions were performed with the inner primers to confirm each sequence.
Total RNA in fresh biopsies was extracted using Trizol according to manufacturer instructions. In order to confirm efficient extraction, the quality of RNA from each sample was assessed by RT-PCR amplification of the ubiquitously expressed phosphoglycerate kinase gene (PGK), which acted as a control to ensure that only RNA was amplified (247 pb fragment), along with the absence of contaminating DNA (additional 600 bp fragment). Good quality RNA samples were chosen and 2 ug were used for cDNA synthesis using Superscript II RT kit (Invitrogen Inc., California, USA) according to the manufacturer’s instructions. Amplification was performed with specific primers (kindly given by Dr Luis Jave Suárez) for E6 HPV16 fwd 5′
Statistical analysis was performed using GraphPad Prism 4 software (GraphPad Software, Inc., San Diego California USA). Fisher’s exact test or Chi square tests (χ2) were used for statistical analysis when appropriate.
Patients’ clinical characteristics and HPV association are shown in
Breast carcinoma characteristics | N | HPV status | ||
Invasive ductal | 43 | 12 | 28 | 0.2487 |
Invasive lobular | 10 | 1 | 11 | |
Papillary | 2 | 2 | 100 | |
Tubular | 2 | 1 | 50 | |
Adenocystic | 1 | 0 | 0 | |
Mucinous | 1 | 0 | 0 | |
Apocrine | 1 | 0 | 0 | |
Comedocarcinoma | 1 | 0 | 0 | |
Positive | 32 | 10 | 31 | 0.6956 |
Negative | 10 | 2 | 20 | |
Positive | 14 | 4 | 29 | 1.0000 |
Negative | 22 | 6 | 27 |
In Latin American population, Cantu de León et al described a 29.4% of HPV association with Mexican breast carcinoma
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
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
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
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
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
The present study demonstrated for the first time in Argentina the presence of HPV in a significant proportion of the malignant breast tissues analyzed. Presence of low risk types, together with the absence of transcriptional activity for high risk types suggest that HPV presence may have a biological significance in breast carcinogenesis as a contributing factor to tumor development, but not as the tumor driven force. It should be pointed out that these observations need additional studies to be substantiated, especially by monitoring future breast cancer incidence amongst women vaccinated against high risk HPV type. Meanwhile, the present results may represent an important issue in virus-associated cancer, which represent potential tools for the development of specific therapies leading for patients with breast cancer.
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.