Original Article
A pilot study on DNA hypermethylation status in promoter region of P16 gene in patients with sporadic breast cancer

https://doi.org/10.1016/j.mjafi.2021.07.011Get rights and content

Abstract

Background

Epigenetic modification of cancer-related genes plays a role over and above their genetic alterations and contributes to the tumor initiation and progression of breast cancer. Promoter methylation of tumor suppressor genes is one such epigenetic modification, which can be potential biomarker. In this study, promoter methylation status of p16 gene was studied in blood samples of patients with breast carcinoma.

Methods

Seventy-five patients, freshly diagnosed with carcinoma of breast and 20 age and sex matched healthy control subjects were recruited for the study. DNA extracted from EDTA blood sample was bisulfite converted and subjected to methylation-specific PCR to amplify the p16 promoter region.

Results

Out of 75 patients, 25 (33%) patients showed hypermethylation in promoter region of p16 gene, which was statistically significant in comparison with the control group (p < 0.05). In subgroup analysis, lymph node involvement, cancer grade, and histopathological finding did not show any difference with methylation status of p16 promoter.

Conclusion

Significant hypermethylation of p16 promoter region in the blood of histopathologically proven cases of breast cancer was observed suggesting promoter hypermethylation of p16 may be a possible mechanism accounting for sporadic carcinoma of breast.

Introduction

Carcinoma breast is one of the common cancers seen across the world and is the fifth leading cause of cancer deaths worldwide.1,2 Women from both developed and developing countries have the risk of developing breast cancer.2 Over the last decade, the incidence of breast cancer is the leading cause of cancer among the Indian population.3 Breast cancer is a multistep process resulting from the accumulation of genetic mutations leading to dysfunction of critical genes involved in biological process. Besides genetic alterations, it has become apparent over the years that epigenetic changes have significant effects on tumor initiation and progression. The aberrant methylation of the promoter region of a gene can lead to its silencing and contribute to the development of the tumor. Promoter methylation of various genes was explored previously as a biomarker of the disease.4 Hypermethylation of DNA occurs when a methyl group from S-adenosylmethionine is transferred to 5 methyl-Cytosine (5mC) as a new base on DNA. Promoter region CpG islands are usually differentially methylated in all normal tissues. The silencing of oncosuppressor genes may lead to the inactivation of the apoptotic pathway in different stages of cell cycles.5

The p16 gene encodes a cyclin-dependent kinase inhibitor, p16 INK4A, which regulates cell cycle at transition from G-1 to S-phase. It is located on chromosome 9p21.5 The transcripts of p16 INK4A gene translates into two functional proteins, p16 INK4A and p19 ARF. The promoter methylation of CpG island in p16 gene is associated with many tumors and has been seen in both human breast cancer cell lines and 20–30% of primary breast cancers. Thus, p16 silencing due to methylation is considered a possible contributor to breast tumor genesis. The studies have shown that use of DNA methylation inhibitors like 5-aza-2′-deoxycytidine can reactivate the repressed p16 gene, thus restoring the normal cell growth control.6,7

Most of the previous studies on promoter methylation of p16 gene were done using histopathological tissue. Evidence for an association between blood-based DNA methylation and breast cancer risk is inconsistent.8 Blood DNA methylation levels could be a replacement for breast tissue methylation;9, 10, 11, 12 moreover, there is the paucity on the availability on the Indian data. Hence, this study was undertaken to explore p16 gene hypermethylation on peripheral blood sample on 75 histo pathologically proven carcinoma of breast cases.

Section snippets

Materials and method

The study was conducted in the department of biochemistry of a tertiary care teaching hospital between October 2016 and October 2019. Sample size was calculated with 95% confidence interval estimation, 25.4% anticipated prevalence of methylation in histopathologically proven carcinoma of breast and 10% absolute error of margin by using formula: N = Z2 p (1-p)/d2. Thus, minimum sample size was calculated to be 72.03. But keeping in mind loss/attrition, data were collected for 75 patients. A

Patient characteristics

The age of patients included in the present study ranged between 26 and 81 years with the mean age of patient as 54.03 ± 11.20 years. Lymph node involvement was seen in 33 (44%) patients, and the remaining 42 (56%) patients had no lymph node involvement. Out of 75 patients, Grade I cancer was seen in 8 (10%) patients, Grade II cancer was seen in 47 (63%) patients, and Grade III cancer was seen in 20 (27%) patients. Sixty-four (85%) patients had ductal carcinoma of breast, and eleven (15%)

Discussion

Breast cancer is the most frequent cancer among women and is also the most important cause of cancer-related deaths among women.13,14 The CpG islands are located in the promoter region of most genes and are normally unmethylated. However, in cancer cells, the aberrant hypermethylation of the promoter regions is associated with the decrease in expression of these genes. The promoter hypermethylation of tumor suppressor gene like p16 may lead to inactivation of tumor suppressor function in

Conclusion

Statistically significant methylation in blood of histopathologically proven sporadic cases of breast cancer was observed in 33% patients in promoter region of p16 gene suggesting that epigenetic modification may be a possible underlying mechanism accounting for sporadic carcinoma of breast. Ease of collection of blood sample in comparison with tissue may make it a non-invasive marker for screening of breast cancer. Loss of p16 gene function due to its promoter methylation in sporadic breast

Acknowledgment

This article is based on Armed Forces Medical Research Committee Project No 4818/2016 granted by the office of the Director General Armed Forces Medical Services and Defence Research Development Organization, Government of India.

Disclosure of competing interest

The authors have none to declare.

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