RUNX3 promoter hypermethylation is frequent in leukaemia cell lines and associated with acute myeloid leukaemia inv(16) subtype.

Correlative and functional studies support the involvement of the RUNX gene family in haematological malignancies. To elucidate the role of epigenetics in RUNX inactivation, we evaluated promoter DNA methylation of RUNX1, 2, and 3 in 23 leukaemia cell lines and samples from acute myeloid leukaemia (AML), acute lymphocytic leukaemia (ALL) and myelodysplatic syndromes (MDS) patients. RUNX1 and RUNX2 gene promoters were mostly unmethylated in cell lines and clinical samples. Hypermethylation of RUNX3 was frequent among cell lines (74%) and highly variable among patient samples, with clear association to cytogenetic status. High frequency of RUNX3 hypermethylation (85% of the 20 studied cases) was found in AML patients with inv(16)(p13.1q22) compared to other AML subtypes (31% of the other 49 cases). RUNX3 hypermethylation was also frequent in ALL (100% of the six cases) but low in MDS (21%). In support of a functional role, hypermethylation of RUNX3 was correlated with low levels of protein, and treatment of cell lines with the DNA demethylating agent, decitabine, resulted in mRNA re-expression. Furthermore, relapse-free survival of non-inv(16)(p13.1q22) AML patients without RUNX3 methylation was significantly better (P = 0·016) than that of methylated cases. These results suggest that RUNX3 silencing is an important event in inv(16)(p13.1q22) leukaemias.

Analysis of class I and II histone deacetylase gene expression in human leukemia.

Histone deacetylase (HDAC) inhibitors are well-characterized anti-leukemia agents and HDAC gene expression deregulation has been reported in various types of cancers. This study sought to characterize HDAC gene expression patterns in several types of leukemia. To do so, a systematic study was performed of the mRNA expression of all drug-targetable HDACs for which reagents were available. This was done by real-time PCR in 24 leukemia cell lines and 39 leukemia patients, which included AML, MDS and CLL patients, some of whom received HDAC inhibitor treatment. Among the samples analyzed, there was no discernible pattern in HDAC expression. HDAC expression was generally increased in CLL patients, except for HDAC2 and HDAC4. HDAC expression was also generally increased in VPA-treated MOLT4 cells. However, this increased expression was not seen in AML patients treated with vorinostat. In summary, increased HDAC expression was noted in CLL patients in general, but the HDAC expression patterns in myeloid malignancies appear to be heterogeneous, which implies that the role of HDACs in leukemia may be related to global expression or protein function rather than specific expression patterns.

Randomized open-label phase II study of decitabine in patients with low- or intermediate-risk myelodysplastic syndromes.

PURPOSE: This open-label, randomized phase II trial assessed efficacy and tolerability of two low-dose regimens of subcutaneous (SC) decitabine in patients with low- or intermediate-1-risk myelodysplastic syndrome (MDS). PATIENTS AND METHODS: Patients received decitabine 20 mg/m(2) SC per day for 3 consecutive days on days 1, 2, and 3 every 28 days (schedule A) or 20 mg/m(2) SC per day once every 7 days on days 1, 8, and 15 every 28 days (schedule B) for up to 1 year. Primary efficacy end point was overall improvement rate (OIR: complete remission [CR], partial remission [PR], marrow CR [mCR], or hematologic improvement [HI]). Secondary end points were HI, transfusion independence, cytogenetic response, overall survival (OS), and time to acute myeloid leukemia or death. RESULTS: Efficacy and safety populations were identical: schedule A, n = 43; schedule B, n = 22. Median time from MDS diagnosis to treatment was 3.6 months; 89% had de novo MDS. The trial was terminated early on achievement of protocol-defined OIR superiority of schedule A over schedule B; OIR was 23% for schedule A (seven CRs, three HIs) and 23% for schedule B (one mCR, one PR, three HIs). No differences were observed in secondary end points. Median OS was not reached; approximately 70% of patients were alive at 500 days. Patients in schedule A (67%) and schedule B (59%) were RBC/platelet independent on study. The most frequent drug-related adverse events overall were neutropenia (28% v 36%), anemia (23% v 18%), and thrombocytopenia (16% v 32%). CONCLUSION: In this phase II study, low-dose decitabine showed promising results in patients with low- or intermediate-1-risk MDS.

Gene Expression and Serum Cytokine Profiling of Low Stage CLL Identify WNT/PCP, Flt-3L/Flt-3 and CXCL9/CXCR3 as Regulators of Cell Proliferation, Survival and Migration.

Gene expression profiling (GEP) of 8 stage 0/I untreated Chronic Lymphocytic Leukemia (CLL) patients showed over-expression of Frizzled 3 (FZD3)/ROR-1 receptor tyrosine kinase (RTK), FLT-3 RTK and CXCR3 G-protein coupled receptor (GPCR). RT-PCR of 24 genes in 21 patients of the WNT pathway corroborated the GEP. Transforming growth factorß, fibromodulin, TGFßRIII and SMAD2 are also over-expressed by GEP. Serum cytokine profiling of 26 low stage patients showed elevation of IFNγ, CSF3, Flt-3L and insulin-like growth factor binding protein 4. In order to ascertain why CLL cells grow poorly in culture, a GEP of 4 CLL patients cells at 0 hr and 24 hr in culture demonstrated over expression of CXCL5, CCL2 and CXCL3, that may recruit immune cells for survival. Treatment with thalidomide, an immunomodulatory agent, showed elevation of CCL5 by GEP but was not cytotoxic to CLL cells. Our data suggest an interplay of several oncogenic pathways, cytokines and immune cells that promote a survival program in CLL.