Gene Expression Profiling of Philadelphia Chromosome(Ph) Negative CD34+ Hematopoietic Stem and Progenitor Cells of Patients with Ph Positive CML in Complete Molecular Remission during Therapy with Imatinib.

Treatment of chronic myelogenous leukemia (CML) with Imatinib has shown its efficacy and superiority to conventional therapies. Beside inhibition of BCR-ABL Imatinib also has inhibitory effects on other tyrosine kinases such as wild-type ABL, c-KIT and platelet-derived growth factor receptors (PDGF-R) alpha and beta which play a role in differentiation and proliferation of hematopoietic stem cells and are involved in DNA repair mechanisms. Inhibition of these genes might therefore result in functional disturbance of hematopoiesis or even in secondary karyotypic abnormalities. Therefore, we compared gene expression profiles of immunomagnetically isolated CD34+ bone marrow (BM) cells from six healthy volunteers with Ph negative CD34+ cells from BM of eight patients (6 males, 2 females; median age: 52 years, range: 25–68 years) with Ph positive CML in chronic phase who reached major molecular remission during imatinib therapy (400 mg/day; 28 months (range: 11–39 months)). Cytogenetics, FISH analysis and quantitative real-time RT-PCR for BCR-ABL transcripts showed that all patients were in complete cytogenetic and in major molecular remission ( ≥ 3 log reduction of the BCR-ABL/G6PDH ratio in comparison to pretreatment value). CD34+ cells (median: 5 x 10⁵; range 2 x 10⁵–1.8 x 10⁶) were isolated immunomagnetically from bone marrow with a purity >98%. Total RNA (median: 550 ng; range: 130 – 990 ng) from isolated CD34+ cells was used to generate biotin-labelled cRNA (median: 7.5 μg; range: 2.6–12.3 μg). Labeled cRNA was hybridized to Affymetrix HG-Focus GeneChips covering 8793 genes representing a broad spectrum of different functional groups. For normalization and data analysis we used the statistical scripting language “R”. Raw data were normalized using a method of variance stabilizing transformations (VSN). To compare the normalized data from CD34+ cells of imatinib-treated patients and healthy volunteers we used the Significance Analysis of Microarrays (SAM) algorithm v1.21. We found that CD34+ cells of patients after reaching major molecular remission during first-line treatment with 400 mg imatinib per day showed no significantly differentially expressed genes in comparison to CD34+ cells of healthy volunteers in vivo. There was no evidence for transcriptional changes of genes involved in DNA repair or oncogenesis supporting the view that secondary chromosomal aberrations in Ph negative hematopoiesis observed during imatinib therapy might not be induced by the tyrosine kinase inhibitor itself. Our data suggest that imatinib as first-line therapy might not result in a major functional disturbance of Ph negative hematopoiesis.