p16 Tumor Suppressor Gene Methylation in Diffuse Large B Cell Lymphoma: A Study of 88 Cases at Two Hospitals in the East Coast of Malaysia

Abstract

<br /> <strong><span style="font-size: small;">Introduction: </span></strong><em><span style="font-family: Times New Roman,Times New Roman; font-size: small;"><span style="font-family: Times New Roman,Times New Roman; font-size: small;">p16 </span></span></em><span style="font-family: Times New Roman,Times New Roman; font-size: small;"><span style="font-family: Times New Roman,Times New Roman; font-size: small;">gene plays an important role in the normal cell cycle regulation. Methylation of </span></span><em><span style="font-family: Times New Roman,Times New Roman; font-size: small;"><span style="font-family: Times New Roman,Times New Roman; font-size: small;">p16 </span></span></em><span style="font-family: Times New Roman,Times New Roman; font-size: small;"><span style="font-family: Times New Roman,Times New Roman; font-size: small;">has been reported to be one of the epigenetic events contributing to the pathogenesis of diffuse large B-cell lymphoma (DLBCL) which occurring at varying frequency. DLBCL is an aggressive and high-grade malignancy which accounts for approximately 30% of all non-Hodgkin lymphoma cases. However, little is known regarding the epigenetic alterations of </span></span><em><span style="font-family: Times New Roman,Times New Roman; font-size: small;"><span style="font-family: Times New Roman,Times New Roman; font-size: small;">p16 </span></span></em><span style="font-family: Times New Roman,Times New Roman; font-size: small;"><span style="font-family: Times New Roman,Times New Roman; font-size: small;">gene in DLBCL cases in Malaysia. Therefore, the objective of this study was to examine the status of </span></span><em><span style="font-family: Times New Roman,Times New Roman; font-size: small;"><span style="font-family: Times New Roman,Times New Roman; font-size: small;">p16 </span></span></em><span style="font-family: Times New Roman,Times New Roman; font-size: small;"><span style="font-family: Times New Roman,Times New Roman; font-size: small;">methylation in DLBCL. </span></span><strong><span style="font-size: small;">Methods: </span></strong><span style="font-family: Times New Roman,Times New Roman; font-size: small;"><span style="font-family: Times New Roman,Times New Roman; font-size: small;">A total of 88 formalin-fixed paraffin-embedded DLBCL tissues retrieved </span></span><span style="font-family: Times New Roman,Times New Roman; font-size: small;"><span style="font-family: Times New Roman,Times New Roman; font-size: small;">from two hospitals located in the east coast of Malaysia, namely Hospital Tengku Ampuan Afzan (HTAA) Pahang and Hospital Universiti Sains Malaysia (HUSM) Kelantan, were chosen for this study. DNA specimens were isolated and </span></span><span style="font-family: Times New Roman,Times New Roman; font-size: small;"><span style="font-family: Times New Roman,Times New Roman; font-size: small;">subsequently subjected to bisulfite treatment prior to methylation specific-PCR. Two pairs of primers were used to </span></span><span style="font-family: Times New Roman,Times New Roman; font-size: small;"><span style="font-family: Times New Roman,Times New Roman; font-size: small;">amplify methylated and unmethylated regions of </span></span><em><span style="font-family: Times New Roman,Times New Roman; font-size: small;"><span style="font-family: Times New Roman,Times New Roman; font-size: small;">p16 </span></span></em><span style="font-family: Times New Roman,Times New Roman; font-size: small;"><span style="font-family: Times New Roman,Times New Roman; font-size: small;">gene. The PCR products were then separated using agarose gel electrophoresis and visualised under UV illumination. SPSS version 12.0 was utilised to perform all statistical analysis. </span></span><strong><span style="font-size: small;">Result: </span></strong><em><span style="font-family: Times New Roman,Times New Roman; font-size: small;"><span style="font-family: Times New Roman,Times New Roman; font-size: small;">p16 </span></span></em><span style="font-family: Times New Roman,Times New Roman; font-size: small;"><span style="font-family: Times New Roman,Times New Roman; font-size: small;">methylation was detected in 65 of 88 (74%) samples. There was a significant association between </span></span><em><span style="font-family: Times New Roman,Times New Roman; font-size: small;"><span style="font-family: Times New Roman,Times New Roman; font-size: small;">p16 </span></span></em><span style="font-family: Times New Roman,Times New Roman; font-size: small;"><span style="font-family: Times New Roman,Times New Roman; font-size: small;">methylation status and patients aged >50 years old (p=0.04). </span></span><strong><span style="font-size: small;">Conclusion: </span></strong><span style="font-family: Times New Roman,Times New Roman; font-size: small;"><span style="font-family: Times New Roman,Times New Roman; font-size: small;">Our study demonstrated that methylation of </span></span><em><span style="font-family: Times New Roman,Times New Roman; font-size: small;"><span style="font-family: Times New Roman,Times New Roman; font-size: small;">p16 </span></span></em><span style="font-family: Times New Roman,Times New Roman; font-size: small;"><span style="font-family: Times New Roman,Times New Roman; font-size: small;">tumor suppressor gene in our DLBCL cases is common and significantly increased among patients aged 50 years </span></span><span style="font-family: Times New Roman,Times New Roman; font-size: small;"><span style="font-family: Times New Roman,Times New Roman; font-size: small;">and above. Aging is known to be an important risk factor in the development of cancers and we speculate that this might be due to the increased transformation of malignant cells in aging cell population. However, this has yet to be </span></span><span style="font-family: Times New Roman,Times New Roman; font-size: small;"><span style="font-family: Times New Roman,Times New Roman; font-size: small;">confirmed with further research and correlate the findings with clinicopathological parameters. </span></span>