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Purpose: To develop and apply real time PCR assays for the diagnosis and risk assessment of MDS. Background: MDS are a heterogenous group of clonal hematopoietic disorders characterized by defects in the maturation of progenitor cells that result in ineffective hematopoiesis and variable risk of progression to acute leukemia. The incidence of MDS is increasing in the US population. Thus, detection and quantitation of early diagnostic biologic markers to identify MDS, particularly those which are more likely to progress to acute leukemia are important. Aberrant methylation of 5’ gene promoter regions associated with gene silencing is an epigenetic phenomenon involving many genes and is observed in almost all cancer types. Studies from our group and others have shown multiple genes which regulate cytokine signaling are inactivated through hypermethylation of their promoters in various tumor types including lymphoma/ leukemias. Methodology: Based on our preliminary findings we selected four potentially useful genes, three in the JAK-STAT pathway (SHP1, SOCS1, SOCS3) and one regulating the cell cycle (p15) and developed semiquantitative real time PCR assays to determine levels of methylation of these genes, and calculated the quantitative ratios (QRs) of methylated gene/MYOD1. We obtained bone marrow (n=45) or peripheral blood (n=5) specimens from 50 MDS patients from the USA and Japan. and control marrow and blood specimens from healthy volunteers or hematologic patients without cancer (n=65). Results: Using the WHO Classification, the MDS patients (median age 68 years, range 34-96) were divided into low risk (n=18), intermediate risk (n=15), or high risk (n=17) categories. Control samples were either negative for methylation or had very low levels. In general there were increasing frequencies and levels of methylation for all four genes between low, intermediate and high risk categories. The QRs for all 4 genes were significantly greater in the high risk compared to the low risk group, and the ratios of p15 and SHP1 were significantly greater in the high risk group compared to the intermediate group. In addition, the mean number of genes methylated per sample were significantly different in the three groups. Conclusions: Our results indicate that application of real time assays for multiple methylated genes may aid the diagnosis, prognosis and clinical management of MDS.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]