Breast Cancer Res Treat (2012) 133:969977 DOI 10.

1007/s10549-011-1876-5

PRECLINICAL STUDY

Investigation of human JC and BK polyomaviruses in breast carcinomas

Mohamed Hachana Khaled Amara Sonia Ziadi Riadh Ben Gacem Sadok Korbi Mounir Trimeche

Received: 16 February 2011 / Accepted: 4 November 2011 / Published online: 23 November 2011 Springer Science+Business Media, LLC. 2011

Abstract We have previously showed the presence of the simian virus 40 (SV40) and the mouse mammary tumor virus (MMTV)-like in a signicant proportions of Tunisian breast carcinomas. However, to date there are no published studies concerning evaluation of the possible implication of the human polyomaviruses JC (JCV) and BK (BKV) in breast carcinomas. The presence of JCV and BKV DNA was investigated by PCR in a 123 primary breast carcinomas and matched adjacent non-tumor breast tissues. The results were correlated to clinicopathological and virological parameters. JCV T-antigen DNA was detected in 23% of breast carcinoma cases; however, all cases were negative for BKV. JCV T antigen PCR products were further conrmed as authentic JCV genome by direct sequencing. JCV was found in invasive ductal carcinomas (28/112 cases) but not in invasive lobular carcinomas (0/5) or medullary carcinomas (0/6). JCV DNA presence correlates inversely with the expression of estrogen (P = 0.022) and progesterone (P = 0.008) receptors. JCV DNA presence correlates also with triple negative phenotype (P = 0.021). With regard to virological data, a trend toward an inverse correlation was noted between the presence of JCV and SV40 (P = 0.06). Moreover, signicant correlation was found between multiple viral infection (JCV, and/or SV40, and/or MMTV-like in the same tumor) and triple negative phenotype (P = 0.001) and also with p53 accumulation (P = 0.028). To the best of our knowledge, this is the rst study demonstrating the presence of JCV in a subset of breast carcinomas. Also our results suggest that
M. Hachana (&) K. Amara S. Ziadi R. B. Gacem S. Korbi M. Trimeche Department of Pathology, Farhat Hached Hospital, 4000 Sousse, Tunisia e-mail: m_trimech@yahoo.fr

triple negative breast carcinomas are viral-related tumors. Keywords Breast cancer Polyomaviruses BK virus JC virus Tunisia

Introduction Breast carcinoma is a very common malignancy in women and it is well recognized as a heterogeneous condition and a major cause of death among women [32, 41]. There is a general agreement that inherited and genetic factors inuence breast cancer development, however, the potential role of other environmental contributors remains unclear. Although there are recognized factors that increase the risk of breast cancer, the etiology of this cancer remains largely unknown. The human polyomaviruses JC (JCV) and BK (BKV) are oncogenic in animal models and readily transform animal and human cells in vitro [39]. A major mechanism by which this is achieved is through the action of the transforming proteins of the early region. In particular, the large T-antigen act as a transcription factor and interacts with several cellular proteins, including the 2 tumor suppressor proteins, pRb and p53, key players in cell cycle progression [40]. Infection with JCV and BKV is prevalent in the human population, although its mode of transmission is not clear [2, 3, 19]. BKV and JCV establish subclinical infections in immunocompetent hosts, but can produce pathologic effects in immunocompromised individuals by destroying infected cells [22]. Genomic sequences of these two viruses have been reported in different human tumor types. Indeed, several

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studies have demonstrated that JCV was found in a large percentage of brain tumors, such as astrocytomas, oligoastrocytomas, glioblastomas, and ependymomas [5, 11, 24, 27, 30], in colorectal cancers [5, 11] and more recently in gastric cancers [28, 35]. Similarly, BKV has been detected in different human tumors including Kaposis sarcoma, brain tumors, and urinary tract tumors [8, 9, 16]. The aim of our present study was to evaluate the possible implication of JCV and BKV in breast carcinogenesis. For this issue, we examined the presence of these viruses in a series of 123 cases of human breast carcinomas using polymerase chain reaction (PCR). We also investigated the relationship between these viruses and several clinicopathological data, immunohistochemical expression status of progesterone and estrogen receptors, HER2, and p53.

Materials and methods Patients and samples For this study, a total of 123 Tunisian patients with sporadic breast carcinomas were randomly selected from the tumors bank retained by the Department of pathology at the Farhat-Hached Hospital in Sousse, Tunisia. The cases were diagnosed between 1995 and 2006. All cases were selected on the basis of availability of paired frozen samples of breast cancer tissue and matched adjacent normal breast tissue obtained at the time of primary surgery before any treatment. The clinicopathological characteristics and immunohistochemical status of estrogen and progesterone receptors, HER2, and p53 expression of these cases was reported in our previous report [20]. According to the WHO Classication [37], the histopathological type was ductal in 112 cases, lobular in 5 cases, and medullary in 6 cases. The clinical staging was done by the TNM staging system [33] and the malignancy stage of the inltrating carcinomas was scored according to the modied ScarffBloomRichardson (SBR) classication [13]. Survival data were available for 84 patients. PCR DNA was extracted from frozen tissues according to protocol described in our previous study [21]. Extracted DNAs were assessed for their suitability for PCR analysis by a control reaction designed to amplify a fragment of 268 bp of the b-globin gene as described previously [34]. The presence of JCV and BKV was analyzed by PCR using the primers set PEP1 (50 -AGTCTTTAGGGTCTTC TACC-30 ) and PEP2 (50 -GGTGCCAACCTATGGAA CAG-30 ), which amplify a 173 bp sequence in the NH2-terminal region of the JCV T antigen, and the primers

set BES-3 (50 -AATATTATGCCCAGCACACATG-30 ) and BES-6 (50 -CTTTCCCTCTGATCTACACCAG-30 ), which amplify a 151 bp sequence in the DNA binding region of the BKV T antigen [4, 36]. The PCR reactions were performed using 200 ng of DNA template in a total volume of 25 ll, containing 0.2 lM of each sense and antisense primers, 10 mM TrisHCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 mM of each dNTP, and 1 U of Taq DNA TM polymerase (Promega, Madison, USA) in a PTC 200 DNA engine thermal cycler (MJ Research, Watertown, USA). Cycling conditions were as follows: denaturation at 95C for 5 min, followed by 35 cycles of 1 min at 95C, for 1 min at the specic annealing temperature (56 and 57C, respectively for JCV and BKV), and for 1 min at 72C. The reaction was nished with a 5 min extension at 72C. All steps of the PCR procedure were conducted in parallel with positive and negative controls. Negative controls were genomic DNA extracted from normal breast tissues. Positive controls for PCR reactions were plasmids containing cloned JCV (pBRJC-MAD-1) and BKV (pBRBKVDunlop) genomes (kindly provided by Dr. Regis A. Vilchez, Baylor College of Medicine, Houston, Texas, USA). PCR products were analyzed by electrophoresis on 2% agarose gels containing ethidium bromide. The electrophoretic pattern was photographed under ultraviolet light with the Gel Doc 2000 System (Bio-Rad, Marnes-laCoquette, France). PCR experiments for each case were repeated 4 times. Samples were considered to contain viral DNA if they exhibited detectable signals at least three times under ethidium bromide staining conditions. Standard precautions were taken to guard against PCR contamination. In addition, DNA extraction, PCR, and gel electrophoresis were performed in a separate room in the laboratories. Furthermore, to rule out a possible contamination of our samples by plasmids containing JCV and/or BKV sequences, a primer pair (50 -GCTCACGCTGTAGGTATCTC-30 and 50 -TCTAGTGTAGCCGTAGTTAG-30 ) previously described by Carbone et al. [7] was used. This primer pair amplies a 241 bp portion of the pUC origin of replication present in pBRJC-MAD-1, pBRBKV-Dunlop and in virtually all plasmids that are propagated in Escherichia coli. DNA direct sequencing for PCR products PCR products were also sequenced to conrm their identity; in fact there is some sequence similarity between SV40, BK virus, and JCV polyomaviruses [42]. All positive PCR products were puried from agarose gels using Wizard SV Gel and PCR Clean-Up Purication kit (Promega) and sequenced using an ABI PRISM BigDye Terminator v. 1.1 cycle sequencing kit on an ABI PRISM 3100 Avant Genetic Analyzer (Applied Biosystems).

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All the sequencing data obtained were analyzed for homology using the nucleotidenucleotide BLAST search feature located on the National Center for Biotechnology Information Web site. Furthermore, these data were aligned with JCV (GenBank Accession Nos. NC_001699), BKV (GenBank Accession Nos. NC_001538), and SV40 sequences (GenBank Accession Nos. AY538779) using BLAST and ClustalW (version 1.83) programs. Statistical analysis Relationships between the presence of these viruses and clinicopathological parameters, immunohistochemical ndings were evaluated by the v2 test or Fishers exact test as appropriate. Overall survival was measured from the day of randomization until death due to any cause. Surviving patients were censored at the day of the last contact. Disease- free survival was measured from randomization until local recurrence, distant relapse, occurrence of contralateral breast cancer or second primary tumor or death without relapse, whichever occurred rst. Times to event distributions were estimated using KaplanMeier curves. All analyses were carried out using the SPSS software package (Chicago, IL, USA). For all of the tests, probability values of P \ 0.05 were regarded as statistically signicant.

Fig. 1 Representative ethidium bromide-stained agarose gel electrophoresis after specic PCR of a b-globin gene, b human polyomavirus JC (JCV) and c human polyomavirus BK (BKV). Cases 97, 12, and 161 show positivity in tumor (T) but not in normal (N) breast tissues. Cases 3 and 39 lack bands in both tumor and normal breast tissues, indicating the absence of JCV DNA sequence in these cases. Lanes M show 50 bp ladder (Promega); lanes P represent positive controls; lanes B represent negative control

Direct DNA sequencing for PCR products Results Viral detection The quality of extracted DNA was assessed by performing b-globin DNA PCR. All samples showed adequate DNA quality for further DNA amplication procedures (Fig. 1a). Each case was analyzed for the presence of JCV and BKV. Appropriate primers were used to amplify a band that had an appropriate size compared to the positive controls; however, none of the breast carcinoma tissue samples examined yielded a PCR product which indicated the presence of BKV DNA (Fig. 1c). In contrast, JCV DNA was found in 23% (28/123) of these cases (Fig. 1b). Whereas, none of the adjacent non-tumor breast tissue samples was positive for these viruses (Fig. 1b). To check for plasmid contaminations and eliminate the possibility of false-positive, all JCV-positive breast samples were tested by PCR using an additional primer set that amplies a 241 bp sequence of the pUC origin of replication present in pBRJC-MAD-1. This analysis showed that all breast DNA samples were negative and only the positive control (plasmid pBRJC-MAD-1) showed positive signal (Fig. 2). PCR products were identied as authentic amplicons of the JCV (and not the BKV or SV40 species of polyomavirus) genome by DNA sequencing. Although there were several point mismatches no signicant differences in the DNA sequences were observed between examined breast carcinoma cases (Fig. 3). Clinicopathological correlations The comparison of the clinicopathological and immunohistochemical data between the JCV-positive and -negative breast cancer cases is summarized in Table 1. Overall, no correlation was found between the detection of JCV and patients age, histological type, histological grade, lymph node status, or tumor size. Immunohistochemical overexpression of HER2 and accumulation of p53 did not vary signicantly between JCV-positive and -negative cases. With regard to hormonal receptors, JCV presence correlates inversely with the expression of estrogen (P = 0.022) and progesterone (P = 0.008) receptors. We also noted a signicant correlation between JCV presence and triple negative (ER-, PR-, HER2-) breast carcinomas (P = 0.021).

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Fig. 2 Ethidium bromide-stained agarose gel showing representative examples of PCR experiment using a set of primers amplifying a 241 bp fragment of the pUC origin of replication present in pBRJCMAD-1 and in virtually all plasmids that are propagated in E. coli. This experiment was used to investigate a possible contamination of

our DNA breast carcinomas samples by JCV genome present in the pBRJC-MAD-1 plasmid. Lanes MW shows 50 bp DNA ladder; lane B represents negative control. All JCV-positive (lanes 36, 69, 97, 12, 163, and 161) cases showed negative results, and only the positive control (plasmid pBRJC-MAD-1) showed positive signal (lane T)

Fig. 3 Multiple nucleotide alignment of the JCV T antigen sequences from ten Tunisian human breast carcinomas (indicated by their respective Nos.) with JCV sequences retrieved from GenBank database (Accession Nos. NC_001699). Sequences were aligned and then manually adjusted using CLUSTAL W, version 1.83

Survival data were available for 83 patients with a median follow-up period of 59 months, which ranged from 4 to 143 months. Overall and disease-free survival were compared between JCV PCR-positive and

-negative cases (Fig. 4). No signicant differences in overall or disease-free survival rates were seen between patients according to the JCV positivity and negativity.

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Breast Cancer Res Treat (2012) 133:969977 Table 1 Correlation between the presence of JCV DNA and clinicopathological characteristics of breast carcinomas
Number of JCV-positive cases Age (years) \35 3550 [50 Histological type Invasive ductal carcinomas Invasive lobular carcinomas Medullary carcinomas Histological gradeb Grade I Grade II Grade III Tumor size (mm) B20 [20 Lymph node metastasesc Negative Positive p53d Negative Positive Estrogen receptord Negative Positive Progesterone receptord Negative Positive HER2d Negative Positive Triple negativee No Yes Simian virus 40 (SV40) Negative Positive MMTV-like Negative Positive
a

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Number of JCV-negative cases

P valuea

0.579 4 15 9 28 0 0 5 10 13 4 26 20 6 14 14 21 7 21 7 20 8 16 12 26 2 24 4 11 43 41 0.169 84 5 6 0.235 19 33 32 0.312 22 73 0.805 61 16 0.522 54 41 0.022 48 47 0.008 44 51 0.726 71 24 75 20 73 22 82 13 1 0.06 0.021

simian virus 40 (SV40) and the mouse mammary tumor virus (MMTV)-like [20]. We found a trend toward an inverse correlation between the presence of JCV and SV40 in breast carcinomas (P = 0.06; Table 1). Moreover, simultaneous multiple viral infections, dened by the presence of more than one viral agent in the same tumor, were noted in 10 breast carcinoma cases. Indeed, simultaneous JCV and SV40 infection was noted in two cases, JCV and MMTV-like infection in three cases, SV40 and MMTV-like infection in ve cases, and only one patient was infected with the three viruses simultaneously (Fig. 5). The comparison of the clinicopathological characteristics between the two groups of breast carcinomas, with and without viral infection is summarized in Table 2. This analysis revealed a signicant correlation between viral infection dened by the presence of one or more viruses (JCV, and/or SV40, and/or MMTV-like) in the same tumor, and triple negative phenotype (ER-, PR-, HER2-) (P = 0.001), and also with p53 accumulation (P = 0.028).

Discussion JCV and BKV are two human polyomaviruses widespread among general population, with a large portion of people (6080%) exhibiting specic antibodies. These viruses remain in a latent stage throughout life and can reactivate under immunosuppressive conditions inducing multifocal leukoencephalopathy for JCV and nephropathy, respectively, for BKV [2, 3, 19]. Recently, links have been suggested between JCV and various types of human cancers, such as brain tumors [40], colon cancers [14, 25, 38], esophageal cancers [12], gastric cancers [28, 35], and lung cancers [1, 28]. Likewise, BKV sequences have been reported to be present in different human tumors including Kaposis sarcoma, brain tumors, and tumors of the urinary tract [8, 9, 16]. However, to our knowledge, to date no study has been conducted to investigate the presence of JCV and BKV in breast carcinomas. For this issue we evaluated the prevalence of JCV and BKV in Tunisian patients with breast carcinomas and we analyzed their relations to clinicopathological features. In our study, BKV was not detected in any breast cancer cases; this nding indicates that BKV is unlikely to play a role in the breast carcinogenesis. In contrast, JCV T antigen DNA sequences were detected by PCR in 23% of cases, exclusively in the tumor tissues. In our experiments, PCR contamination is improbable since DNA extraction, amplication and analyse of PCR products by electrophoresis were performed in different areas. In addition, negative control was included in every reaction and no amplication was shown. Moreover, to rule out a possible contamination of our breast samples by

P-values were calculated by v2 or Fishers exact tests (two-sided) and were considered to be statistically signicant for P \ 0.05; bold numbers indicate signicant correlations

b Scarff-Bloom and Richardson classication was performed only in invasive ductal carcinoma cases c d e

Twenty cases did not benet from the lymph nodes resection Immunohistochemical data were reported previously [20] Simultaneous negativity for estrogen and progesterone receptors, and HER2

We also assessed the relation between JCV and two others viruses also found positives previously in some breast carcinoma cases in the same Tunisian series, the

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974 Fig. 4 Kaplan-Meier estimates of overall survival (a) and disease-free survival (b) of breast carcinomas patients classied according to the status of JCV

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Fig. 5 A diagram summarizing the distribution of the number of positive cases for the JCV, the SV40 and the MMTV-like in a series of 123 breast carcinoma cases from Tunisia

plasmids containing JCV sequences, all our breast samples were tested by PCR assay amplifying DNA sequence present in common laboratory plasmids. This analysis showed that all breast samples were negative. This experiment rules out the possibility that our JCV-positive breast samples may be caused by plasmid contamination. Furthermore, direct sequencing of the PCR products reveled that the amplied DNA sequences found in the breast specimens were authentically of JCV and not of related viruses genome. To our knowledge, this is the rst demonstration of the presence of JCV DNA in breast cancer s. The presence of the JCV DNA in breast tumors but not in the adjacent normal breast tissues suggests a role of this virus in breast carcinogenesis. These observations should stimulate further investigations on the association of this oncogenic virus in other human populations and the investigation of pathways that may be deregulated by JCV in the tumor cells. Previous studies have suggested the link between JCV and various types of human cancers [10, 14, 15, 17, 18, 23, 26, 29, 31, 35], and has been reported that JCV T antigen bind and inactivate wild type of p53 [26, 29] and cause chromosomal instability [18, 29, 31], as well as the

stabilization of b-catenin [14, 17]. In contrast, in our study we do not nd a signicant correlation between the presence of JCV and aberrant accumulation of p53. This negative result could be due to the number of investigated cases or it could indicate that in the breast, the JCV interacts preferentially with other pathways than p53. Interestingly, we found a correlation between the presence of JCV and the negativity of expression of estrogen (P = 0.022) and progesterone (P = 0.008) receptors. Similar correlation was also reported for other viruses that have been related to breast cancer, such as Epstein-Barr virus (EBV) [6] and the MMTV like [20]. Additional studies looking at JCV are needed to clarify the genetic or the epigenetic mechanisms by which JCV interferes with the hormonal receptors expression in breast carcinomas. In the current study, no signicant correlation was found between JCV and the other clinicopathological parameters examined, including patients age, histological type and grade, lymph node metastasis, tumor size, HER2 overexpression, and survival rates. These ndings suggest the absence of a specic prole for JCV-related breast carcinomas. However, it is interesting to note that all the 28 JCV-positive breast carcinoma cases belong to the invasive breast ductal carcinoma type and none of the six medullary carcinomas or the ve invasive lobular carcinomas was positive (see Table 1). Additional attention should be devoted to the breast carcinoma histological type in future studies exploring more important number of cases and histological types to determine whether JCV is specically associated with the invasive breast ductal carcinoma type. In the current study, we also assessed the relation between JCV and two others viruses (SV40 and MMTVlike) also found positive previously in the same series of Tunisian breast carcinomas studies, [20]. We found a trend toward an inverse correlation between the presence of JCV and SV40 in breast carcinomas (see Table 1). This result indicates that the two polyomavirus JCV and SV40 are independent factors in breast carcinogenesis. Nevertheless, in our breast carcinoma series, we detected a simultaneous

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Breast Cancer Res Treat (2012) 133:969977 Table 2 Comparison of the clinicopathological characteristics between breast carcinomas with and without multiple viral infection Cases with viral infection Age (years) \35 3550 [50 Histological type Invasive ductal carcinoma Other types Histological gradeb Grade I Grade II Grade III Tumor size (mm) B 20 [ 20 Lymph node metastasesc Negative Positive p53d Negative Positive Estrogen receptord Negative Positive Progesterone receptord Negative Positive HER2d Negative Positive Triple negativee No Yes 48 10 35 23 43 22 56 9 0.001 0.036 39 19 26 39 0.003 35 23 34 31 0.370 26 32 42 23 0.028 13 45 40 9 13 52 41 13 0.480 0.743 11 21 24 13 22 21 0.823 56 2 56 9 0.042 8 24 26 7 34 24 0.478 Cases without viral infection P valuea

975

comparison of the clinicopathological characteristics between the two groups of breast carcinomas, with and without viral infection (one or more viruses in the same tumor), revealed a signicant correlation between the viral infection and the triple negative phenotype of tumors (ER-, PR-, HER2-). This observation suggests that triple negative breast carcinomas are, at least in part, viralrelated cancers. This observation is also very interesting in the future for the therapeutic management of this group of triple negative breast carcinomas for which hormonal therapy and anti-HER2 immunotherapy are not possible, and for which not much therapeutic arms are currently available. Indeed, these infectious agents, or their antigens, could be an interesting targets for new therapies. In conclusion, our study demonstrates for the rst time the presence of JCV DNA in a subset of breast carcinomas (23% of cases). JCV DNA was detected in the breast tumor tissues but not in the matched non-tumor breast tissues. We also demonstrated a correlation between JCV presence and the absence of hormonal receptors expression. These ndings suggest a role of JCV in breast carcinogenesis. Further studies are needed to elucidate the role of JCV infection in the breast cancer development or progression.
` re de Acknowledgments This study was supported by the Ministe rieur, de la Recherche Scientique et TechnollEnseignement Supe ` re de la Sante Publique of Tunisia. ogie and the Ministe Conict of interest The authors have no conicts of interest in relation to this article.

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Dened by the presence of one or more viruses (JC virus, simian virus 40, MMTV-like) in the same tumor P-values were calculated by Fishers exact tests (two-sided) and were considered to be statistically signicant for P \ 0.05; bold numbers indicate signicant correlations
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Scarff-Bloom and Richardson classication was performed only in invasive ductal carcinoma cases Twenty cases did not benet from the lymph nodes resection Immunohistochemical data were reported previously [20]

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