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Identification of genes that are differentially expressed in leukemias through combination of cDNA subtraction and microarray analysis

The goal of these studies is to identify genes that are preferentially expressed in leukemias by using a combination of cDNA library subtraction and microarray analysis. A human leukemia cDNA library was constructed by subtracting a normal bone marrow library from a library derived from a pool of the most common AML FAB subtypes (M1, M2, M4, M5). Prescreening of the library showed enrichment of leukemia associated genes and a high complexity, as evidenced by the low frequency of repeated isolation of the same clones. For microarray analysis of the subtracted cDNA library, 2500 clones from the subtracted library were randomly picked, PCR amplified and arrayed onto glass slides as multiple replicates. To determine tissue distribution of the putative leukemia associated genes, the arrayed cDNA clones were used as targets to be hybridized with first strand cDNA probes that included 40 leukemia probes and 40 probes from different normal tissues. Leukemia probes were generated from cryopreserved samples obtained at the time of diagnosis from AML patients with poor outcome (patients who failed to achieve complete remission following conventional chemotherapy or relapsed within 6 month after remission) or good outcome (patients who achieved long term remission). To compare the gene expression profile between the diagnosis and relapse, probes were generated from AML samples obtained at the time of relapse. In addition, probes were derived from patients with CML in CP or BC, ALL at time of diagnosis, CLL at diagnosis or with refractory disease. To analyze gene expression during hematopoietic differentiation probes were generated from >95% pure fractions of CD34+, CD2+, CD14+, CD15+ and CD19+ cells derived from healthy individuals. Following microarray analysis, genes of potential interest were sequenced. Results identified 27 genes differentially overexpressed in leukemias including 22 known genes and 5 novel cDNA sequences. 24 genes were differentially underexpressed in leukemias, including 20 known genes and 4 novel cDNA sequences. Studies are in progress to correlate expression of these genes with prognostic factors and response to therapy (DFS and OS) and to confirm expression profile and prognostic relevance of these genes using Real-time PCR with a large panel of leukemia samples (n=120).

leukemia; gene expression; cDNA; microarrays

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