Detection of methylated ABL1 promoter in Philadelphia-negative myeloproliferative disorders.

A Aviram PhD, B Witenberg PhD, M Shaklai MD, D Blickstein MD

Institute of Hematology, Rabin Medical Center, Beilinsin Campus and Felsenstein Medical Research Center, Petach-Tikva, 49100, Israel.

Many tumor-suppressor genes are silenced in tumor cells by de-novo methylation of their promoter regions. This aberrant methylation is suggested to have a causal role in the pre-neoplastic stage of cancer progression. Others have shown methylation of CpG-rich islands at several sites in the proximal ABL1 promoter (Pa) on the Philadelphia (Ph) chromosome in chronic myeloid leukemia (CML). Two possible models may explain the origin of methylation on Pa. First, the occurrence of translocation 9;22 (t9;22) is an initial event proceeded by methylation of Pa. Second, methylation could be a stochastic event in a progenitor cell which will later acquire other mutations, i.e., t9;22. In this study we posed two questions: 1. What is the methylation status of Pa in Ph-neg (negative) myeloproliferative disorders (MPD) patients? 2. Is it possible that a search of Ph-neg MPD will shed light on the mechanism of translocation? For probing CpG methylation we adopted two different methodologies, site-methylation-sensitive restriction enzymes assay and methylation-specific PCR analysis following modification of DNA by bisulfite. We observed that twenty-two of 97 Ph-neg MPD patients expressed BCR-ABL transcripts. In some of the patients, PCR for BCR-ABL detection became negative on sequential analysis. Seven of the 97 patients studied possessed methylated Pa, but of these 7 patients only 2 expressed BCR-ABL transcripts. We concluded that 1. Ph-neg/BCR-ABL-pos (positive) MPD may be a distinct entity from Ph-pos MPD. 2. Aberrant methylation patterns could be an initial event triggering the occurrence of the translocation with its clinical expression. These findings may shed light on the pathogenesis and progression of MPD.

KEY WORDS: CpG methylation, myeloproliferative disorders.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Gene Expression, Part 2.