Molecular prevalence of human papillomavirus infection among Iranian women with breast cancer

Abstract

BACKGROUND:

The etiology and molecular mechanisms involved in the development of breast cancer still remain poorly understood. Some epidemiological studies have shown an association between human papillomavirus (HPV) and breast cancer. However, the findings are controversial.

OBJECTIVE:

Our study was aimed to investigate the presence of HPV DNA in breast carcinomas of Iranian women.

METHODS:

In total, 72 samples of formalin-fixed paraffin-embedded (FFPE) tissues of breast cancer collected between December 2014 and April 2016 were examined. HPV DNA detection was performed by nested-PCR assay. Next, positive samples were subjected to genotyping by the CLART HPV2 microarray system. All statistical analysis was carried out using SPSS v.18.0.

RESULTS:

HPV DNA was detected in 4/72 (5.55%) samples. Clinical factors were not statistically associated with HPV presence. However, CLART HPV2 microarray assay failed to determine the genotype of any positive samples.

CONCLUSION:

The low frequency of HPV detected in our study does not support an association between breast carcinoma and HPV infection. However, it is possible that HPV may be responsible for breast carcinogenesis only in small percentage of all breast cancer.

1. Introduction

Worldwide, breast cancer is the second most frequent malignancy and the most common cancer leading to death of females, constituting 25% (1.67 million) of the overall new cancer cases (corresponding to an incidence rate of 43 per 100,000) and 521,900 deaths in 2012 around the world [1]. The total burden of breast cancer among women was increasing during the past 30 years in both developing and developed countries, and unfortunately, it is estimated that the global incidence of breast carcinoma is projected to double by the year 2030, and occur more commonly in younger females [2].

Cancer is the third mortality cause after cardiovascular events and traffic accidents in Iran, and each year more than 30,000 Iranian people die from cancer. It is expected that the incidence rate of cancer increases to be doubled in future decades in Iran [3]. According to estimates from the WHO/ICO Information Centre on HPV and Cervical Cancer, the incidence of breast cancer in Iranian women was 2.5 per 100,000 in 2015 [4]. Nearly all breast cancers are carcinomas; type of malignant tumors emerging from epithelial cells, and in Iran, the infiltrative ductal carcinoma is the most common type (77% of breast carcinomas) [5].

In general, the etiologic cause of this common cancer pointed to genetic, reproductive, environmental, and hormonal factors in which estrogen-related elements are amongst the most important reasons. It has reported that infectious agents are responsible for about 20%–25% of all human cancers cases worldwide [6]. Most of these agents are oncogenic viruses such as HPV, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), mouse mammary tumor virus (MMTV), and Epstein-Barr virus (EBV) [7–9].

HPV belongs to the Papillomaviridae family, a group of small, nonenveloped, double-stranded DNA viruses with an 8 Kb genome. To date, more than 200 genotypes of HPV have been recognized which are categorized as high-risk and low-risk groups according to their oncogenic potential [10,11]. The high-risk genotypes of HPV are associated with nearly 600,000 cases of different cancers worldwide annually, including cancers of the cervix, anal, oropharyngeal, vulvovaginal, penile, and head and neck squamous cell [12–14].

The first investigation on the relationship between HPV infection and breast cancer was carried out in Italy in 1992 [15]. As a consequence, numerous studies were initiated to investigate the presence of HPV DNA in breast cancer tissues. However, available data are inconclusive and it is still not clear whether HPV play a role in the carcinogenesis of breast cancer. The present study was conducted to help clarify these controversies. We aimed to investigate the presence of HPV DNA in breast tissues in a group of Iranian women with breast cancer and evaluate the relationship between HPV status and various clinical characteristics.

2. Materials and methods

2.1. Study population

A total of 72 FFPE samples were collected from histologically confirmed cases of breast carcinomas diagnosed in the Department of Pathology of Khatam-Al-Anbia Hospital (Tehran, Iran) during the period between December 2014 and April 2016. A self-made questionnaire containing following information filled for each patient: age, marriage status, cancer grade/type/malignancy, the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2). This cross-sectional study was performed according to the Declaration of Helsinki and was approved by the local ethics committee of the Iran University of Medical Sciences, Tehran, Iran.

2.2. DNA extraction

10-μm sections of tissue samples were cut from each FFPE blocks with a standard microtome and transferred into sterile microtubes. To minimize cross-contamination of samples, the microtome blade was changed for each block. In the next step, sections were deparaffinized by xylene and digested using tissue lysis buffer containing proteinase K. Afterwards, DNA was extracted by the HiPurA^TM Multi-Sample DNA Purification Kit (HiMedia, India) according to the manufacturer’s protocol. Quantity of extracted DNA was assessed by NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc. Waltham, MA, USA). For the assessment of DNA quality and integrity, the extracted genomic DNA was subjected to PCR amplification with specific primers for the human 𝛽-globin gene.

2.3. HPV Nested PCR

HPV detection was performed with the standard nested PCR assay, using a MY09/MY11 primer set as outer primers in the first round of PCR, and a GP5+/GP6+ primer set as inner primers in the second round. Table 1 shows characteristics of primers used in this study. DNA extracted from CaSki cell line and ddH2O were used as positive and negative controls, respectively.

For the first round of PCR, the amplification mixture contained 1X PCR buffer, 1.5 mM MgCl₂, 200 μM dNTPs, 50 pmole of each primer (MY09/MY11), 1 unit of Taq DNA polymerase, and 500 ng of DNA sample in a final volume of 50 μL. Amplifications were performed in a Bio-Rad T100^TM Thermal cycler using the following conditions: an initial denaturation for 5 minutes at 95 °C and then, 40 cycles of 95 °C for 1 minute, 56 °C for 1 minute and 72 °C for 1 minute; final extension at 72 °C for 10 minutes.

For the second round of PCR, the amplification mixture consisted of 1X PCR buffer, 2 mM MgCl₂, 200 μM of each dNTP, 50 pmole of each primer (GP5+/GP56+), 1 unit of Taq DNA polymerase, and 500 ng of DNA product from the first round of PCR in a final volume of 50 μL. The PCR was performed with the following cycling parameters: initial denaturation for 5 min at 95 °C, followed by 40 cycles of 95 °C for 1 minute, 52 °C for 1 minute and 72 °C for 1 minute; final extension at 72 °C for 4 minutes. For each round, PCR products were subjected to gel electrophoresis by using a 1.5% agarose gel and subsequently visualized by UV light after ethidium bromide staining.

2.4. CLART HPV2 genotyping

All positive samples from nested-PCR were subjected to genotyping by the CLART HPV2 (Genomica, Madrid, Spain), a semi-automated microarray assay based on PCR amplification of genotype specific HPV L1 fragments from 35 high-risk and low-risk HPV genotypes. All procedures were performed according to manufacturer’s protocol.

2.5. Data analysis

All statistical analysis was performed using the SPSS version 18.0 (SPSS Inc, USA), and P values less than 0.05 were considered to be statistically significant. P values were determined by the Mann-Whitney test for non-normally distributed variables. To assess the significant correlation between our results, Pearson Chi-square and Fisher’s exact test were used.

3. Results

A total of 72 FFPE samples of breast carcinoma were examined for the presence of HPV in our investigation. This study group consisted of women aged from 35 to 81 years (mean age 52.15 ± 10.63 years). Of these patients, 56 had invasive ductal carcinomas (77.8%), 13 had invasive lobular carcinomas (18.1%), 1 had invasive metaplastic carcinoma (1.4%), and 1 had invasive medullary carcinoma (1.4%). The positive rate of ER, PR, and Her2 expression were 43.1%, 44.4%, and 18.1%, respectively (Table 2).

All samples were positive for 𝛽-globin, indicating a good quality of DNA. Detection of HPV DNA was performed by a nested PCR method using MY and GP primers for all extracted DNAs. Overall, HPV DNA was found among 5.55% of the specimens (4/72). Fig. 1 shows the relationship between the age, HPV status and cancer grade in our patients. Results of nested PCR are presented in Fig. 2. With respect to the histological status, all 4 positive samples had invasive ductal carcinomas. Unfortunately, CLART HPV2 microarray assay failed to determine the genotype of any positive samples.

4. Discussion

The role of infection in development of several cancers has become well established during the past years. Among various viral agents, HPV is linked with a variety of cancers in human such as cervical cancer, anal cancer and a series of penile and oral cancers $[$ 12 $]$ . Despite some reports of an association between HPV and breast cancer [16–18], the role of HPV infection in breast cancer carcinogenesis still remains debated. The results of present study have shown a low frequency of HPV DNA (5.55%) in breast cancer specimens. Our results are consistent with the findings of other studies from different geographical regions which reported 4.4% and 3.57% of breast cancer tissues samples were positive in Mexican and Thai women, respectively [19,20]. Similarly, Choi and colleagues have performed a study on Korean women with breast carcinoma and found a detection rate of 6.5% for HPV[21]. However, our estimated prevalence was lower than that reported in other regions of Iran (18.2–34.6%) [22–24], proposing that demographic characteristics and genetic backgrounds may contribute to geographic dissimilarities. There is no international standard assay for HPV genotyping which can lead to variation in test results between laboratories in different regions of the world [25].

While the most commonly reported HPV genotypes were 16, 18, 11, 35, 31 and 33 in the previous studies, we found neither of high/ low-risk genotypes in our subjects. It is notable that the primers used in the nested PCR in our study were designed for targeting a region within the L1 region of HPV. Similarly in the CLART HPV2 microarray system, detecting and genotyping of HPV is usually performed by PCR amplification of a fragment located in the L1 region of the virus [26]. It should be noted that persistent infection with high risk genotypes of HPV generally are associated with integration of the HPV genome into the host genome, which usually results in the loss of L1 gene during this process [27]. Therefore, one possible explanation of the low detected HPV in our findings can be attributed to this phenomenon.

At present there are a number of commercially available methods for detection of HPV genotypes such as Digene Hybrid Capture 2 (HC2) HPV DNA Test (Qiagen, Gaithersburg, MD), INNO-LiPA (Innogenetics NV, Gent, Belgium), and Linear Array (LA) HPV Genotyping Test (Roche Molecular Systems, Alameda, CA). For comparison, different genotyping methods for HPV including Anyplex II HPV28 (Seegene, Seoul, Korea), CLART HPV2, MGP5+/6+ primer system were performed on FFPE samples in patients with suspected HPV infection and compared to a real-time PCR reference protocol [28]. The findings indicated that high sensitivity for FFPE specimens was obtained by the application of CLART HPV2 and Anyplex II HPV28. The CLART system from Genomica is approved in the European Union as one of the widely used diagnostic tool for detection and genotyping of HPV which is able to use FFPE tissues as specimen.

Fresh tissue specimens from breast cancer are considered as gold standard for the detection of HPV[29]. In addition, FFPE blocks have also been frequently used, which in some cases are related to limitations leading to discordance results. During formalin fixation, denaturation of most molecules can occur and usually, the extracted DNA from FFPE tissues are fragmented and in low concentrations. As another weakness, detection of some HPV genotypes particularly 42, 16, 18, 39, 56, 58, 59 and 66 in FFPE samples is associated with reduced sensitivity [29]. Because malignant form of breast cancer usually has a low prevalence, access to fresh tissue was very time consuming and limited in our study. For this reason, we have performed our survey using archival FFPE specimens.

In our study, the average age of patients diagnosed with breast cancer was 52.15 ± 10.63 years. It is notable that in all studies performed on patients with breast cancer in Iran, the mean age was 48.59 years (CI 95% from 46.47 and 50.72), and we found a significant relationship between these two averages (CI 95%, from 49.65 to 54.65, p = 0.006). More precisely, in this study, HPV positive patients with breast cancer have 34.10 to 66.39 years of age (CI 95%), which is consistent with all other reviews so far (CI 95% odds ratio 0.377, 0.294 to 0.483, p <0.001).

Our findings have revealed that all HPV-positive cases were associated with invasive ductal carcinoma, which was in accordance with others reports. All previous studies performed on patients with HPV-positive breast cancer around the world demonstrated that invasive ductal cell carcinomas is the most common type of breast cancer. It was hypothesized that the exposure of the mammary ducts to the external environment through the nipple areola complex can become an entry point for HPV infection [16]. This can be considered as an explanation why the most breast cancers originate from the mammary duct epithelia.

Hong et al. indicated the role of HPV in p53 gene mutation and in the occurrence and progress of invasive ductal carcinoma [30]. These clarifications further highlight a role for HPV genotypes in the pathogenesis of human breast cancer and offer the prospect prevention of breast cancer using an HPV vaccine. Long-standing follow up of vaccinated women at recommended age could demonstrate the validity of this assumption. It has shown that infection with high-risk HPV genotypes in breast carcinoma significantly correlates with worst prognosis of breast cancer [31]. Further genetic and epigenetic landscape of HPV is recommended to find responsible HPV genotypes that deteriorate breast cancer.

In summary, the findings of our study showed a low frequency of HPV DNA in breast cancer tissues, suggesting that HPV infection is not an important risk factor involved in breast carcinogenesis. However, it is still a controversial issue and not entirely clear. So that according to some previous works performed in this field, it is possible that infection with some high risk HPV genotypes may be associated with development of breast cancer. Hence further studies with different design and various specimens’ types will illuminate the potential role of HPV in breast cancer development.