Molecular monitoring by CDKN2A/p16INK4A and RB1 gene methylation in breast cancer

Sep 21, 2026

Prospective study evaluates methylation dynamics of CDKN2A/p16INK4A and RB1 genes as potential biomarkers for breast cancer monitoring. DNA methylation profiles were compared using cell-free DNA from blood collected on the day of total mastectomy and one year after surgery, alongside tumor and non-tumor tissue samples. Sodium bisulfite conversion and polymerase chain reaction with electrophoresis quantified methylation changes. Results show gene methylation in tumor and non-tumor tissues and cfDNA shifts across time points. Findings suggest cfDNA methylation detection may precede visible disease and support recurrence prediction.

LanguageEnglish

CategoryResearch & Report

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Pages5

Reading Time13 min

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Summary

Objective

Methods

Results

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Introduction

Methods

Study design

Data collection and ethical aspects

Results

Discussion

ORIGINAL ARTICLE [https://doi.org/10.1590/1806-9282.20231358](https://doi.org/10.1590/1806-9282.20231358) Molecular monitoring by CDKN2A/p16INK4A and RB1 gene methylation in breast cancer Luiz Fernando de Queiroz1 , Marcelo Soares da Mota e Silva2* , Fernando Colonna Rosman2 , Siane Lopes Bittencourt Rosas3 , Heitor Siffert Pereira de Souza3 , Maria da Glória da Costa Carvalho2 SUMMARY OBJECTIVE: This prospective study aimed to provide a comprehensive analysis of the methylation status of two pivotal genes, CDKN2A/p16INK4A (cyclin-dependent kinase inhibitor 2A) and RB1 (retinoblastoma transcriptional corepressor 1), in breast cancer patients. METHODS: Samples were obtained from 15 women diagnosed with breast cancer and who underwent a total mastectomy. DNA was extracted from the tumor, non-tumor tissue, and peripheral blood (circulating cell-free DNA) . The methylation pattern of cell-free DNA extracted from blood collected on the day of mastectomy was compared with the methylation pattern of cell-free DNA from blood collected 1 year post-surgery. The methylation analysis was carried out by sodium bisulfite conversion and polymerase chain reaction, followed by electrophoresis. RESULTS: Methylation of CDKN2A/p16INK4A was identified in 13 tumor samples and 12 non-tumor tissue samples. Two patients exhibited CDKN2A/ p16INK4A methylation in the cell-free DNA of the first blood collection, while another showed methylation only in the cell-free DNA of the subsequent blood collection. Regarding RB1, 11 tumors and 8 non-tumor tissue samples presented methylation of the gene. CONCLUSION: This study presents a novel approach for monitoring breast cancer patients through the analysis of cell-free DNA methylation. This analysis can detect changes in methylation patterns before any visible sign of cancer appears in breast tissue and could help predict the recurrence of malignant breast tumors. KEYWORDS: Retinoblastoma. Breast neoplasms. DNA methylation. INTRODUCTION Activation of oncogenes and inactivation of tumor suppressor genes are genetic oscillations related to cancer development 1,2 . DNA methylation, which is an epigenetic mechanism, can influence the expression of tumor suppressors and genes related to uncontrolled cell proliferation. The mechanism can inactivate gene expression and consequently prevent protein synthesis. The CDKN2A/p16INK4A (cyclin-dependent kinase inhibitor 2A), located at chromosome 9 (9p21.3), is a tumor suppressor gene that encodesp16INK4A, a protein involved in cell cycle regulation1,3,4. Thep16INK4A participates in the G1-to-S-phase checkpoint and may interrupt the cell cycle in response to various stressors, leading to the inhibition of cell proliferation1,5 . Additionally, p16INK4A promotes apoptosis of tumor cells and can increase sensitivity to chemotherapy in breast cancer3. An investigation found a positive correlation between hypermethylation of CDKN2A/p16INK4A and breast cancer progression and also verified that CDKN2A/p16INK4A hypermethylation impacts the tumor’s grade and stage3. The RB1 (retinoblastoma transcriptional corepressor 1) is also a tumor suppressor gene and is located on chromosome 13 (13q14.2) . RB1 encodes the retinoblastoma protein (Rb), and alterations in its tumor suppressor pathway can be considered a potential risk for the development of breast cancer6-8. RB1 can be inactivated by several mechanisms, including alterations in phosphorylation, viral oncoproteins, and promoter hypermethylation7. The analysis of methylation may be performed in circulating cell-free DNA (cfDNA) using liquid biopsy, a promising method for early detection and monitoring of breast cancer9. Liquid biopsy is non-invasive and easy to perform, and analysis 1 Universidade Federal do Rio de Janeiro, Faculty of Medicine, Postgraduate Program in Pathological Anatomy, Department of Pathology – Rio de Janeiro (RJ), Brazil. 2 Universidade Federal do Rio de Janeiro, Faculty of M

FAQs

What was the objective of this prospective study?

To comprehensively analyze the methylation status of CDKN2A/p16INK4A and RB1 in breast cancer patients using tissue and circulating cell-free DNA.

How were samples collected and methylation measured?

Cell-free DNA was taken from blood collected on the day of mastectomy and again one year post-surgery; DNA methylation was assessed using sodium bisulfite conversion, PCR, and electrophoresis. Tumor and non-tumor tissue were also analyzed.

What did the study find regarding cfDNA methylation over time?

CDKN2A/p16INK4A methylation appeared in most tumor and non-tumor tissue samples, and it was detected in cfDNA at different time points among patients. RB1 methylation was also observed in tumor and non-tumor tissues.

Sep 21, 2026

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