Title: Promoter Methylation of Selected Genes and Response to Azacitidine (AZ) Therapy in Patients with Non-BCR-ABL Myeloproliferative Disorders (MPDs)

Abstract

Abstract 4625 Background:Gene silencing via methylation of CpG islands in the promoter regions of many genes but specifically of APAF1, p15INK4B, p16INK4A, RARB, and CDH1 appears to play a role in pathogenesis of myeloid malignancies. Azacitidine (AZ) causes demethylation by inhibiting DNA methyltransferase and has already been shown to be an effective therapy for myelodysplastic syndromes. The demethylation induced by AZ is detectable in about 48 hours and increases significantly after 5 days of therapy. After that, the effect tends to plateau. Methods:We initiated a Phase 2 study of patients with non-BCR-ABL MPDs to determine clinical response to AZ therapy and correlate it with promoter DNA methylation and gene re-expression. The protocol was approved by the institutional IRB. Patients received AZ 75mg/m2 s/c for days 1–7 and repeated every 28 days for a minimum of 4 cycles. Responders were allowed to continue treatment until disease progression. Pretreatment and D 7 peripheral blood samples were analyzed for promoter methylation status and expression of the 5 genes mentioned above. Bisulfite conversion of DNA was followed by quantitative PCR using primers specific for methylated or for unmethylated promoter regions. For gene re-expression analysis, quantitative RT-PCR was performed with RNA isolated from the same patient samples and the same time points as the DNA methylation analyses. Results:Seven patients were enrolled before the study closed due to lack of accrual. The diagnoses were: Myelofibrosis (MF) 4, essential thrombocythemia 1, unclassified MPD with dysplasia 2. One patient with MF and one with unclassified MPD responded, the latter with normalization of marrow karyotype. Both responses were accompanied by significant decrease in APAF1 promoter methylation and surprisingly, an increase in promoter methylation of RARB. In three of the non-responders, APAF1 methylation increased. In patients with decreased Apaf1 methylation, a statistically significant increase in mRNA expression was observed. Conclusions:Within its limitations, this small trial shows that the methylation status of selected genes, particularly of APAF1 and RARB (inversely) is associated with response to treatment with azacitidine in patients with MPDs. In non-responders, Apaf1 methylation appears to increase. A larger study will be necessary to confirm these preliminary observations. Disclosures:Smith:Seattle Genetics, Inc.: Research Funding; Cephalon: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Spectrum: Consultancy; GSK: Speakers Bureau. Nand:Celgene Corporation: Research Funding.