METHYLATION STATUS OF BCL6 DISTINGUISHES DNA SAMPLES FROM BENIGN AND MALIGNANT LYMPHOPROLIFERATIVE DISORDERS

Abstract

INTRODUCTION: Deregulation of DNA methylation occurs early in neoplastic transformation. Increased cytosine methylation leads to chromatin compaction with gene inactivation, and hypermethylated DNA in condensed chromatin may be less susceptible to mutation and translocation. Cytosine methylation sites in non‐malignant cells are highly regulated, in contrast to the regional hyper‐ and global hypomethylation frequently found in lymphoid and other neoplasms. This preliminary study assessed the methylation status of BCL6 upstream from exon 1. BCL6 encodes a transcription factor essential for normal germinal centre formation and for B cell development after antigen stimulation. It is a repressor of gene transcription but its target genes are ill‐defined. In some categories of B cell non‐Hodgkin lymphoma (B‐NHL), somatic mutation and translocation of BCL6 are frequent.METHODS: DNA was extracted from 60 lymphoproliferative disorders (LPD) comprising 24 benign LPD, 30 B‐NHL (low and high grade) and 6 T cell neoplasms. The level of BCL6 methylation was assessed by Southern hybridization: DNA was digested with HpaII and MspI (methylation sensitive and insensitive respectively), and restricted BCL6 fragments were detected with a 32P‐dCTP radiolabelled 4 kb SacI BCL6 probe that hybridized to exon 1 and 5′ flanking sequences. Following autoradiography, the size and relative density of hybridized fragments was used to assign methylation status.RESULTS: HpaII digested DNA from benign LPDs was characterised by fragments of approximately 0.9 kb, 3.6 kb, 3.9 kb, 4.6 kb and 4.9 kb, and using GenBank sequence NT5962, 3 methylated MspI restriction sites were predicted. Of 22/24 (92%) HpaII digested DNA samples from benign LPDs, the 3.6 kb, 3.9 kb, 4.6 kb and 4.9 kb fragments all had either similar density, or the latter two fragment sizes had relatively greater density. 28/30 (93%) HpaII digested DNA samples from B‐NHL had a decrease in relative density of the 4.6 kb and 4.9 kb fragments compared to the 3.6 kb and 3.9 kb fragments, and one sample had a 3.3 kb fragment. Of the 6 HpaII digested DNA samples from T cell neoplasms, 3 samples had fragments larger than 4.9 kb, 1 sample had 4.6 kb and 4.9 kb fragments having less density than the 3.6 kb and 3.9 kb fragments, and 2 samples had findings indistinguishable from benign LPD samples.CONCLUSION: This study of the methylation status of the upstream flanking region of BCL6 indicates there may be alternative deregulatory mechanisms leading to hypomethylation in the majority of B‐NHL and hypermethylation in some T cell neoplasms. Whether hypomethylation of BCL6 in B‐NHL predisposes to somatic mutation and translocation of this gene remains to be investigated.