Endometrial cancer (EC) is the seventh most common cancer in women worldwide with an age-standardized incidence rate (ASR) of 8.4 per 100,000 women. According to the 2018 Indian statistics, 13328 women were diagnosed with EC and an estimated 5010 women succumbed to the disease accounting for an ASR of 2.3 per 100,000 women [1-3]. Though most of the patients are diagnosed in the early stages when the disease is still confined to the primary site, about 30% of the patients are diagnosed in late stages where the disease would have disseminated from the uterus [4]. Various genetic and epigenetic alterations are known to be involved in the initiation and progression of EC. Aberrant promoter hypermethylation of Tumor suppressor genes (TSGs) is known to be an early event in many cancers including EC. Promoter hypermethylation is associated with transcriptional silencing of several tumour suppressor genes [5]. Retinoids are known to play a role in tumor suppression upon interaction with retinoic acid receptors. The retinoic receptors are nuclear receptors and include three retinoic acid receptors (RARs) - RAR-α, RAR-β, and RAR-γ [6,7]. Suppression of expression and function of RAR-β, have been reported in various cancers [8-10]. One of the mechanisms of suppressing the expression of RAR-β is by the epigenetic mechanism of promoter hypermethylation where a methyl group is added to deoxycytidine residues in the CpG islands of its promoter. This methylation of all or a few of the cytosine in the promoter region renders the closure of the chromatin structure eventually resulting in transcriptional silencing of the gene [11-13]. In the present study, we have attempted to analyze the methylation pattern of the RAR-β gene in 78 endometrial cancers of patients of the Indian cohort and associated the methylation patterns with various clinicopathological parameters.

Sample collection: 78 histologically confirmed endometrial cancer samples were collected post-surgery from patients undergoing surgery for endometrial cancer. All the samples were immediately stored at -80⁰C until further processing.

Inclusion and exclusion criteria: Primary chemo naïve endometrial carcinoma patient samples were included in the study.

(n=60), serous (n=06), mucinous (n=02), clear cell (n=02) and poorly differentiated carcinoma (n=08). Six normal endometrial samples were analyzed for RAR-β methylation as a control cohort. 40 of the 78 samples analyzed (51.28%) showed methylation of the RAR-β gene. Among the subtypes, 50 % of the Endometroid, mucinous, clear cell, and poorly differentiated samples showed methylation for the RAR-β gene and 66.6% of the samples of the serous subtype showed methylation for the gene. About 50% of the patient in Stage I and II and about 60% of the patients of stage III and IV showed methylation for the gene respectively. 20/44, 11/19 and 9/15 samples of grades 1,2,3 showed methylation respectively.

68 of the 78 patients (87.18%) included in the study posted menopausal women and 52 % of these postmenopausal patients showed methylation for the gene.

One of the six normal endometrial samples analyzed showed positive for both unmethylated and unmethylated alleles.

Figure 1: (A) Representative Agarose gel image of the RAR-β methylation pattern observed in the endometrial carcinoma samples and (B) in normal endometrial samples.

(S: Endometrial carcinoma sample; N: Normal; U: Unmethylated; M: Methylated allele)

Cancers are known to evolve in a stepwise manner accumulating several genetic and epigenetic alterations [14,15]. One of the most well studied epigenetic alterations in the promoter methylation which plays significant roles in both normal and cancer cells. In normal cells, it is involved in processes such as chromatin organization, X-chromosome inactivation and genetic imprinting. On the contrary, in cancer cells aberrant hypermethylation is observed in the CpG islands of gene promoters which are involved in the regulation of cell cycle and apoptosis. This type of aberrant promoter hypermethylation leads to transcriptional gene silencing of several important tumour suppressor genes. Reports have also shown that the methylation patterns are gene-specific and cancer-specific and therefore specific methylation patterns may provide insights in unravelling specific molecular signatures of cancer [14-17].

The RAR-β gene is known to exhibit its tumour suppressor function by having significant roles in several important cellular pathways such as the apoptotic pathway and is known to inhibit cellular proliferation [18]. Aberrant hypermethylation of RAR-β has been studied across various cancer types.

Analysis of promoter hypermethylation pattern of tumor suppressor gene panels in larger sample cohorts will provide a deeper understanding of the underlying epigenetic alteration that contributes to endometrial carcinogenesis. These hypermethylation patterns can aid in the development of methylation-based biomarker assays that could aid in better diagnosis and management of the disease.

Nagaratna Shivanandappa: designed the study, Experimentation and data collection, manuscript writing, statistical analysis and manuscript editing Shalini N Swamy: experimental design, manuscript writing and editing, statistical analysis, Sandeep Kumar S: manuscript writing and editing, statistical analysis, Suma Sheshadri: data collection and sample screening, Pallavi V R: data collection and sample selection, Ramesh Gawari: designed the study, manuscript writing, editing, statistical analysis.