Detection of human papillomavirus DNA in breast cancer of patients with cervical cancer history

Journal of Clinical Virology 31 (2004) 292–297

Detection of human papillomavirus DNA in breast cancer of patients with cervical cancer history Andreas Widschwendtera,∗ , Thomas Brunhuberb , Annemarie Wiedemaira , Elisabeth Mueller-Holznera , Christian Martha a

Department of Obstetrics and Gynecology, Innsbruck University Hospital, Anichstrasse 35, A-6020 Innsbruck, Austria b Department of Pathology, University of Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria Accepted 20 June 2004

Abstract Background: Recent studies have revealed a possible role for the human papillomavirus (HPV) in the pathogenesis of breast cancer. In this study, patients having both a history of invasive cervical cancer and breast cancer as second primary cancer were selected for enrolment in a study of breast carcinomas for the presence of HPV. Methods: Paraffin-embedded tissue from cervical cancer, pelvic lymph nodes, breast cancer and axillary lymph nodes of eleven patients were examined for the presence of HPV DNA using a polymerase chain reaction – enzyme immuno assay. DNA extraction was performed with the “QIAamp Tissue Kit” according to the manufacturer’s instructions. Additionally, serum samples taken between diagnosis of cervical and breast cancer, were analyzed for the presence of HPV DNA to examine a possible haematogenous spread of oncogenic HPV DNA. Results: All cervical carcinomas were HPV-positive. HPV DNA was detected in seven out of eleven cases in breast cancer and/or axillary lymph node tissue. Six patients had the same HPV type (HPV-16) in cervical cancer and in the corresponding breast cancer/lymph node tissue. In one case, the same HPV DNA type (HPV 16) was detected in cervical cancer, breast cancer and serum sample. Conclusion: These results suggest that HPV DNA might be transported from the original site of infection to the breast tissue by the bloodstream, and that it is possibly involved in the carcinogenesis of breast neoplasia in some patients. © 2004 Elsevier B.V. All rights reserved. Keywords: Human papillomavirus; HPV 16; Breast cancer; Cervical cancer; PCR

1. Introduction The association between human papillomavirus (HPV) and anogenital tumors, especially cervical cancer, is well documented. The relationship between genital HPV types and breast cancer is unclear. 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 etiologically related to some cases of breast cancer. There is some evidence that breast carcino∗

Corresponding author. Tel.: +43 512 504 3051; fax: +43 512 504 3055. E-mail address: [email protected] (A. Widschwendter). 1386-6532/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2004.06.009

genesis may in some cases be initiated or promoted by a viral agent. The relationship between virus and mammary tumors is known from induction of mammary cancer in mice with the mouse mammary tumor virus (MMTV) (Dickson, 1990; Medina, 1976). The intermediate DNA of MMTV can integrate into the genomes and perturb the activity of a protooncogene, inducing accelerated cell growth (Dickson, 1990). Particles similar to this mouse virus were also observed in human breast cancer tissue, indicating a possible viral etiology in human breast cancer (Vokaer, 1975). In 1992, Di Lonardo et al. (1992) demonstrated the presence of HPV type 16 in 29.4% of breast tumors and metastatic lymph nodes using polymerase chain reaction techniques. Recently, HPV 16 was identified in 46% of breast carcinomas of women

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treated for high-grade cervical intraepithelial neoplasia (CIN III) (Hennig et al., 1999e). In addition, HPV 33 was identified in breast tumors of 42% of Chinese and 11% of Japanese women with breast cancer (Yu et al., 1999). It has been shown that HPV 16 antibody seroprevalence in breast cancer patients is 10% (Strickler et al., 1998). Accumulating lines of evidence have revealed that there is tumor DNA in patients’ circulation. Such DNA can be detected in plasma or serum via specific genetic and epigenetic alterations of the primary tumor. Though the mechanism of this phenomenon is not clear, the presence of tumor DNA in the blood may be of diagnostic and prognostic value. Interestingly, viral DNA has been documented to occur as tumor DNA in the circulation of patients with primary tumors caused by viral infection. For example, there is a high frequency of hepatitis viral genomes and EpsteinBarr viral (EBV) DNA in the circulation of patients with hepatoma and nasopharyngeal cancer (NPC), respectively (Mutirangura et al., 1998; Takahashi et al., 1998). HPV DNA has been detected in sera of patients with cervical cancer and HPV associated head and neck squamous cell carcinoma, which may support the hypothesis of a possible haematogenous transfer of HPV from one organ to another (Capone et al., 2000; Pornthanakasem et al., 2001). In view of this and the finding of genital HPVs in malignancies at distant sites, such as bronchopulmonary cancer, breast cancer and Hodgkin’s lymphoma (Cheng et al., 2001; Hennig et al., 1999c,d,e; Yu et al., 1999),we studied breast cancer patients with a history of cervical cancer. The aim of the study was to investigate a correlation between HPV-associated cervical cancer, and a possible presence of HPV DNA in breast cancer of the same patient. To explore the hypothesis of a haematogenous spread of oncogenic HPV DNA to the breast, we also investigated the patients’ sera for the presence of HPV DNA. We furthermore measured IgG to HPV virus-like particles (VLPs) in the sera of the patients to assess exposure to HPV in the past.

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2. Materials and methods 2.1. Study subjects Patient selection was performed with the help of The Cancer Register of Tyrol. Patients with histologically verified invasive cervical cancer and with breast cancer as second primary cancer were selected. A total of 13 patients, all treated at the Department of Obstetrics and Gynecology, Innsbruck University Hospital, between 1988 and 1996, were included in this study. In the end, tissue and serum samples from eleven patients were available. Patients’ clinical and histopathological characteristics are listed in Table 1. Eleven breast cancer patients without history of cervical cancer served as control subjects. These patients were matched for age, tumor size and lymph node status. 2.2. Tumor specimens and serum samples Paraffin blocks from breast cancer, cervical cancer and lymph nodes were obtained from the Department of Pathology, University of Innsbruck. Five-␮m-thick sections were cut and collected in sterile tubes. To avoid contamination between various specimens, special care was taken to change gloves before cutting each block. The knife was changed before cutting each new tissue sample. The specimen holder of the microtome was washed several times with absolute alcohol between cuttings. No paraffin block containing samples of cervical cancer was cut on the microtome during the process of breast tissue sectioning. Serum samples obtained between the diagnosis of cervical cancer and breast cancer were taken for analysis. 2.3. DNA extraction and HPV detection Formalin-fixed paraffin-embedded tissues were processed with the “QIAamp Tissue Kit” according to the manufacturer’s instructions (Qiagen, Hilden, Germany). “QIAamp”

Table 1 Clinical and histopathological characteristics Patients

Cervical cancer

Breast cancer

Age

Stage

Pelvic LN

Histology

Grading

Age

Stage

Axillary LN

Histology

Grading

1 2 3 4 5 6 7 8 9 10 11

28 71 78 73 65 54 47 37 40 69 57

1b 1b 1a1 3b 2 1b 3b 1b 1b 3b 1b

Negative nss nss nss nss Negative nss Negative Negative nss Negative

Squamous Squamous Squamous Squamous Squamous Squamous Squamous Squamous Squamous Squamous Squamous

3 2 nd 2 nd 3 2 2 2 nd 3

33 71 78 74 68 54 53 37 45 69 57

1b 2 2 1b 2 4b 1 2 1b 3 1b

Negative Positive Positive Negative Negative Positive Negative Negative Negative Negative Positive

Lobular Lobular Lobular Papillary/DCIS Ductal Ductal Ductal Medullary Ductal Ductal/DCIS Ductal

3 2 2 2 1 2 2 3 2 1 3

LN, lymph node; ductal, invasive ductal cancer; lobular, invasive lobular cancer; papillary, invasive papillary cancer; medullary, invasive medullary cancer; DCIS, ductal cancer in situ; nss, not surgical staged; nd, not done.

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total DNA purification comprises cellular lysis, proteinase K digestion, adsorption of the cell lysate to the QIAamp silica membrane, washing steps and elution of the membranebound DNA (QIAamp Tissue Kit Handbook, Qiagen, Hilden, 1996). Serum samples were treated overnight with SDS and proteinase K at 55 ◦ C, followed by phenol/chloroform extraction and ethanol precipitation of DNA. As negative control for DNA isolation, paraffin sections without tissue were processed with the “QIAamp Tissue Kit” as described above. Additionally, the DNA isolation procedure was also performed with aqua instead of a specimen probe. These negative controls were used for PCR and enzyme immunoassay (EIA). After each DNA isolation the centrifuge was cleaned with a decontamination reagent. DNA isolation and PCR were performed in two different rooms. Ten ␮l of purified total cellular DNA were employed for PCR using consensus primers GP5+/bioGP6+ as described by Jacobs et al. (1995). These primers amplify a region of about 140 bp within the L1 open-reading frame of more than 37 different HPV types (Jacobs et al., 1997, 2000). The reaction mixture contained 50 mM KCl, 10 mM TrisHCl (pH 8.3), 3.5 mM MgCl2 , 200 ␮M of each dNTP, 1 U of thermo-stable DNA polymerase (Taq DNA Polymerase, Roche, Vienna, Austria) and 50 pmol each of the GP5+ (5TTTGTTACTGTGGTAGATATACTAC-3) and GP6+bio (5GAAAAATAAACTGTAAATCATATT-3) primers (MWGBiotech, Ebersberg, Germany). A 4-min denaturation step at 94 ◦ C was followed by 40 cycles of amplification in a PCR cycler (Gene Amp PCR System 9600, Perkin-Elmer, Norwalk, USA). Each cycle included a denaturation step at 94 ◦ C for 1 min, a primer-annealing step at 48 ◦ C for 2 min and a chain-elongation step at 72 ◦ C for 1.5 min. The final elongation step was prolonged by 4 min to ensure complete extension of the amplified DNA. In addition to the negative controls described above, a PCR negative control (10 ␮l aqua instead of DNA) was used for each PCR. To determine the HPV type, the PCR product was subjected to an enzyme immunoassay as described by Jacobs et al. (1997). Briefly, a streptavidin-coated microtiter plate (Roche, Vienna, Austria) was loaded with 5 ␮l of the amplified reaction mixture in 50 ␮l freshly prepared 1× SSC/0.5% Tween-20 solution (Merck, Darmstadt, Germany) and incubated at 37 ◦ C for 60 min to bind the biotinylated components. After three washing steps to remove unbound biotinylated PCR products, the DNA was denatured into single strands. A cocktail of digoxigenin-labeled oligonucleotides specific for 14 high-risk HPV types was then added, followed by hybridization for 1 h at 37 ◦ C. Unbound oligonucleotides were removed by washing. Detection of specifically bound digoxigenin-labeled probe was achieved by immunochemistry, and the optical density at 405 nm was subsequently measured at various times (Jacobs et al., 1997). HPV DNA typing demanded that the EIA be performed with each digoxigeninlabeled HPV type-specific oligonucleotide. Five ␮l purified total cellular DNA was used for ␤globin PCR to assess the quality of the DNA. The

primers used were: 5-CAACTTCATCCACGTTCACC-3 and 5-GAAGAGCCAAGGACAGGTAC-3 spanning 268 bp. ␤globin PCR was performed as described for the GP5+/ bioGP6+ primers with slight modifications. Annealing temperature was 55 ◦ C, and 1.5 mM MgCl2 was used in the reaction mixture instead of 3.5 mM MgCl2 . Amplified DNA was analyzed by agarose gel electrophoresis. 2.4. HPV–ELISA ELISA was performed for the analysis of antibodies to intact HPV 16, 18 and 33 virus-like particles in the sera of cervical and breast cancer patients as previously described (Heim et al., 2002).

3. Results Tissue sections taken from paraffin-embedded tissue from eleven breast cancer patients with cervical cancer in their history were available for analysis by PCR EIA for the presence of HPV. Tissue sections included breast carcinoma, cervical carcinoma, and axillary and pelvic lymph nodes. Time between diagnosis of cervical cancer and breast cancer was 0–61 months (median 19 months). DNA extracted from tissue sections and serum was positive for ␤-globin gene in all cases, indicating that quality and quantity of DNA were satisfactory. All investigated cervical cancer tissues were HPVpositive (Fig. 1). HPV-16 was the predominant HPV type (eight out of eleven samples). In two cases (non-metastatic), pelvic lymph nodes – available from five patients – were also HPV-16-positive. HPV DNA was detected in breast cancer and/or axillary lymph node tissue in seven out of eleven (64%) cases (Fig. 1). Six patients had the same HPV type (HPV-16) in cervical cancer, and in the corresponding breast cancer and/or axillary lymph node tissue (Table 2). HPV-VLP antibodies were detected in five out of eight patients. All five patients were HPV-16 antibody positive, and three patients additionally had HPV-18 and -31 antibodies. All HPV-16seropositive patients had the same HPV DNA type in cervical cancer and/or pelvic lymph node tissue (Table 2). In five patients, HPV DNA was found in serum samples taken between the diagnosis of cervical and breast cancer. The same HPV DNA type (HPV 16) in cervical cancer, breast cancer and serum sample was detected in one case. Five patients experienced a recurrence of breast cancer (patients 1, 3, 6, 7 and 9), one patient of cervical cancer (patient 5) and one patient of both carcinomas (patient 10). Five of six patients with breast cancer recurrence were HPV-positive, either in breast cancer or in axillary lymph node tissue or serum. Follow-up sera were taken at 3–12-month intervals. In patient 9, serum became high-risk HPV-positive 2 months before diagnosis of invasive breast cancer and was negative after treatment. In patient 7, serum was HPV DNA-negative after treatment of cervical cancer and turned positive 12 months before diagnosis of invasive breast cancer. Patient 7, who

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Fig. 1. Detection of HPV by general-primer-mediated PCR (GP-PCR). GP-PCR products after electrophoresis on a 1.5% agarose gel and ethidium bromide staining. (A) Cervical cancer tissue, (B) breast cancer tissue, (C) axillary lymph node tissue, (D) serum. Numbers indicate patients’ numbers. From patient 7 and 9, only tissue from recurrent breast cancer was available. bp, base pairs; mwm, molecular weight marker (pUCBM21-DNA cut with HpaII and DraI plus HindIII); +, positive control; −, negative control. Table 2 HPV DNA types in cervical cancer, pelvic lymph nodes (LN), breast cancer, axillary lymph nodes (LN), and serum and HPV antibodies in serum Patient

1 2 3 4 5 6 7 8 9 10 11 a b

Seruma HPV antibodies

HPV DNA Cervical cancer

Pelvic LN

Breast cancer

Axillary LN

Seruma

18/45 16 33/58 16 16 33/58 16 16 16 16 16

16 nss nss nss nss Negative nss Negative Negative nss 16

Negative 16 Negative 16 Negative 16 16b na Negativeb 16 Negative

Negative Negative Negative 16 Negative Negative 16 16 na na 16

16 na Negative 18 16/18 na 16 na 18 Negative Negative

16 na Negative 16/31 16 na Negative na 16/31 16/18 Negative

Serum samples taken between diagnosis of cervical and breast cancer. Tissue from recurrent breast cancer. nss, not surgical staged; na, not available.

experienced a recurrence of breast cancer 3 years after primary diagnosis, was HPV-positive in axillary lymph node tissue and recurrent tumor. In patient 5, who experienced a recurrence of cervical cancer, serum HPV DNA was detected 20 months before clinical diagnosis of recurrence. Serum had been HPV DNA-negative after initial treatment of cervical cancer. None of the 11 breast cancer patients without history of cervical cancer was HPV-positive, except one who showed

an HPV-positive breast carcinoma. This result was confirmed when DNA extraction and PCR were repeated. Serum samples from control patients before diagnosis were not available.

4. Discussion The association between human papillomavirus infection and the development of genital tumors is widely accepted

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(Walboomers et al., 1999). However, little is known about the influence of these viruses on carcinomas of other sites. Previous studies have demonstrated a higher incidence of certain second primary cancers in women with previous CIN III (Bjorge et al., 1995; Levi et al., 1999). To elucidate the association between HPV infection and breast cancer development, PCR EIA was used to examine the existence of HPV in breast tissue and axillary lymph nodes of breast cancer patients with cervical cancer in their history. Additionally, serum samples from these patients were analyzed for the presence of HPV DNA to examine a possible dissemination route of the oncogenic HPV DNA. Previous reports of HPV in breast cancer from unselected patients are conflicting. While some studies report HPVpositive breast cancer tissue ranging from 11% to 41% (Di Lonardo et al., 1992; Liu et al., 2001; Yu et al., 1999, 2000), others detected no HPV in breast cancer (Bratthauer et al., 1992; Gopalkrishna et al., 1996; Wrede et al., 1992). In women with a history of CIN III, HPV has been detected in various second primary cancers, e.g. bronchopulmonary cancer and breast cancer (Hennig et al., 1999a,d,e). The same increased cancer risk is observed among women with cancer of the vulva/vagina (Sturgeon et al., 1996). This indicates a possible common risk factor of oncogenic HPV DNA in developing certain second primary cancers after HPV-related primary neoplasia. In our study, HPV DNA was detected in seven out of eleven breast and/or axillary lymph node tissues in women with a clinical history of cervical cancer. Serum samples taken between diagnosis of cervical cancer and breast cancer revealed HPV DNA in five cases. The same HPV type in cervical cancer, breast cancer and serum sample was found in one patient. The detection of HPV DNA in serum samples from patients with HPV-associated diseases has been described (Capone et al., 2000; Dong et al., 2002). The biological role of circulating oncogenic DNA is still unclear. The so-called “Hypothesis of Genometastasis” has been proposed, which suggests that malignant transformation might develop as a result of transfection of susceptible cells in distant target organs with dominant oncogenes that circulate in the plasma and are derived from the primary tumor (GarciaOlmo and Garcia-Olmo, 2001). The genometastasis theory is supported by various lines of evidence previously reported. In an in vivo study, healthy rats having received plasma from a tumor-bearing rat showed the presence of the tumor marker gene in their lung DNA (Garcia-Olmo et al., 1999). Plasma from cancer patients used as culture medium has the ability to transform cells. When such cells are subcutaneously injected into syngeneic rats, tumors develop (Garcia-Olmo and Garcia-Olmo, 2001). Experiments performed by Anker et al. (1994) have shown that the supernatant of colorectal cancer cell cultures can transfect fibroblast cultures. In the light of these studies and of our results, it can be supposed that circulating HPV DNA plays a possible role in malignant transformation. The exact time of haematogenous spread of HPV DNA can not be determined. As the most likely time of HPV DNA release from the primary tumor is before or

at the time of clinical diagnosis, serum samples taken before diagnosis would reveal a higher HPV-positive rate. In vitro studies have shown that HPV DNA can immortalize normal breast epithelium (Band et al., 1990; Wazer et al., 1995). Additionally, a significantly lower expression of p53 and p21 proteins in HPV 16-positive breast carcinomas has been observed than in those that are HPV-negative (Hennig et al., 1999b). This confirms a possible etiological role of HPV in some cases of breast cancer. In previous studies, HPV DNA was detected in breast cancer tissue of patients without a known history of HPVassociated diseases (Di Lonardo et al., 1992; Liu et al., 2001; Yu et al., 1999, 2000). A prior HPV infection might have been undetected. This could explain the HPV-positive breast carcinoma in one patient without a history of cervical cancer in our study. As no serum samples before diagnosis of breast cancer were available from this patient, a possible route of infection could not be examined. In some patients, cervical cancer tissue, breast cancer tissue and serum sample showed a different HPV type. This could be due to an undetected double infection or a low viral load of the primary tumor. The advantage of serologic assays in comparison to DNA-based detection systems is that serology can assess current exposure as well as past HPV infection (Wideroff et al., 1999). In our study, HPV double infection, as revealed by HPV antibody detection, was observed in three cases. Besides HPV 16, we also detected HPV 18 and HPV 31 antibodies. The primary cervical carcinoma of these three cases contained only HPV type 16. These results could also explain the detection of different HPV types in various organs of one patient. Our results suggest that HPV DNA might be transported from the original site of infection to breast tissue by the bloodstream, and may possibly be involved in the carcinogenesis of breast neoplasia in some patients.

Acknowledgements We thank Ms. A. Sapinsky for skillful technical assistance. This work was supported by the “Medizinischer Forschungs Fonds Tirol” and the “Hans und Blanca Moser Stiftung”.

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