Multiplex PCR/mass spectrometry screening of biological carcinogenic agents in human mammary tumors

doi:10.1016/j.jcv.2014.07.010

Journal of Clinical Virology

Volume 61, Issue 2, October 2014, Pages 255-259

https://doi.org/10.1016/j.jcv.2014.07.010 Get rights and content

Highlights

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We analyzed the prevalence of 30 oncogenic HPVs, EBV, KSHV and polyomaviruses.
•
Our results support an etiologic role for EBV in breast cancer pathogenesis.
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We developed a 10-plex analysis of six polyomaviruses, EBV and KSHV.

Abstract

Background

While many studies have suggested a possible link between breast cancer pathogenesis and infection by viruses, the role of viruses in breast carcinogenesis remains controversial.

Objectives

We analyzed the prevalence of 30 oncogenic human papillomaviruses (HPVs), Epstein–Barr virus (EBV), Kaposi's sarcoma herpes virus (KSHV) and six polyomaviruses in breast tumor specimens.

Study design

We analyzed breast specimens from 100 breast cancer patients (group 1) and 50 benign breast disease patients (group 2) from Shaanxi Province in China. We also screened for the viruses in blood samples from the patients and 96 female blood donor volunteers (group 3).

Results

EBV, Merkel cell polyomavirus (MCPyV) and HPV-18 were detected in 60, 14 and 2 breast cancer patients, respectively, and EBV and MCPyV were detected in 16 and 1 benign breast disease patients, respectively. EBV and MCPyV were more prevalent in group 1 than in group 2 (EBV: 60.0% vs. 32.0%, p = 0.0012; MCPyV: 14.0% vs. 2.0%; p = 0.02). In contrast, there was no difference in the prevalence of EBV and MCPyV in blood samples between group 1 and group 2, group 1 and group 3. EBV was detected in malignant breast tissue and its presence was confined to the malignant cells using in situ hybridization.

Conclusions

We found that EBV and MCPyV were more prevalent in the tumors of women with breast cancer than in samples from women with benign breast disease.

Our results support an etiologic role for EBV in breast cancer pathogenesis in Chinese patients.

Section snippets

Background

Breast cancer is the most common type of cancer in women worldwide, with an estimated 1.38 million new cases and over 450,000 deaths annually worldwide [1]. While 5–10% of breast cancer cases are caused by hereditary mutations [2], [3], the etiology of the majority of sporadic breast cancers remains enigmatic. Many studies have suggested a possible link between breast cancer pathogenesis and viral infection, particularly human papillomavirus (HPV), Epstein–Barr virus (EBV), simian virus 40

Objective

The objective of the present study was to explore the prevalence of 30 oncogenic HPVs, SV40, BKPyV, JCPyV, WUPyV, KIPyV, MCPyV, EBV and KSHV in tumor specimens from 100 breast cancer patients and 50 benign breast disease patients from Shaanxi Province of China using MS-HPV and MS-PEK.

Sample preparation

Sample sets were collected from 100 female breast cancer patients and 50 female benign breast disease patients who received surgery at the Department of Vascular and Endocrine Surgery, Xijing hospital, the Fourth Military Medical University, China, between October 2010 and October 2011. Exclusion criteria were systemic therapy prior to surgery, bilateral breast cancer, metastatic or recurrent disease, and cancer of other origin. Adjuvant therapy was based on current National Comprehensive

Sensitivity and specificity of the MS-PEK assay

The specificity of the MS-PEK method was analyzed using 1000 plasmids/reaction with specific inserts of each target virus. The results showed that this method accurately identified SV40, BKPyV, JCPyV, WUPyV, KIPyV, MCPyV, EBV and KSHV (data not shown). Using a standard 10-fold serial dilution of 1–10,000 plasmids/reaction, we determined that the detection limit per PCR reaction was approximately 10 copies (Fig. S1). Throughout the PCR-MS testing, there were no results from the negative

Discussion

In this study, we found that EBV was more prevalent among women with breast cancer than women with benign breast disease. Labrecque et al. detected EBV in 21% (19/91) samples from breast cancer patients using PCR targeting a region in the EBV BamHIW major repeat sequence [7]. In another study, Bonnet et al. detected EBV by PCR in 51% of the breast cancer specimens, while the virus was not detected in healthy tissue adjacent to the tumor. The presence of the EBV in breast cancers was confirmed

Funding

This work was supported by the Ministry of Science and Technology of the People's Republic of China (2011ZX10004-001), the National Natural Science Fund (81272899), Institute of Pathogen Biology, CAMS&PUMC (2013IPB102), the Shaanxi Fund (2013K12-03-03) and Xi’an Found (SF1323(3), the Discipline booster plan of Xi Jing Hospital (XJZT12Z07).

Competing interests

None declared.

Ethical approval

The study has been approved by the Xijing Hospital Ethics Committee. Written informed consent was obtained from each patient, the parents of the minors/children participants or blood donor volunteer.

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The mean age (±SD) was 49 ± 16 in papilloma and 52 ± 14 in BC patients, respectively. We found high prevalence of HPV in papilloma and carcinoma: 29.6% (n = 8) and 44.4% (n = 8), respectively. The most common type identified in breast lesions was HPV-11, and the others were HPV- 6, -11, -39, and -82. Cancer-involved samples were more contaminated by HPV in comparison to normal-adjacent tissues (p = 0.016). In HPV positive cancer-involved cases, hormone receptors were found to be more positive than HER2-Neu (p = 0.035).
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348 of the 508 (68.5%) specimens from the hospitalized children with diarrhea contained at least 1 common enteric virus. STLPyV was identified in 11 specimens in the case group (2.2%), among which 4 specimens were negative for those common enteric viruses. STLPyV was not more prevalent among the case group than the control group (2.2% versus 3.0%; p = 0.50, χ² test). In case group, when common enteric viruses’ positive and negative groups were compared, the difference in detection rate of STLPyV was not statistically significant (2.5% versus 2.0%; p = 0.98, χ² test). Two whole genome sequences of STLPyV were obtained.
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¹: ¹These authors contributed equally.

View full text

Multiplex PCR/mass spectrometry screening of biological carcinogenic agents in human mammary tumors

Highlights

Abstract

Background

Objectives

Study design

Results

Conclusions

Section snippets

Background

Objective

Sample preparation

Sensitivity and specificity of the MS-PEK assay

Discussion

Funding

Competing interests

Ethical approval

Breast

Cancer Lett

Curr Opin Virol

J Clin Virol

Virol J

Cancer Lett

Lab Invest

Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008

Int J Cancer

Hereditary breast cancer: new genetic developments, new therapeutic avenues

Hum Genet

Breast and ovarian cancer risks due to inherited mutations in BRCA1 and BRCA2

Science

Evidence for a role of the Simian Virus 40 in human breast carcinomas

Breast Cancer Res Treat

The sero-epidemiology of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) in adults with cancer in Uganda

Int J Cancer

Human papilloma virus is associated with breast cancer

Br J Cancer

Epstein–Barr virus in epithelial cell tumors: a breast cancer study

Cancer Res

Viruses and human breast cancer

Future Microbiol

Role of viruses in the development of breast cancer

Infect Agent Cancer

Are viruses associated with human breast cancer? Scrutinizing the molecular evidence

Breast Cancer Res Treat

The possible involvement of virus in breast cancer

J Cancer Res Clin Oncol