Distinct gene expression pattern of malignant hematopoietic stem and progenitor cells in polycythemia vera.

Polycythemia vera (PV) is a chronic myeloproliferative disorder with an expansion of multipotent hematopoietic progenitor cells. Although it is known that hematopoietic progenitors in PV are erythropoietin independent and hypersensitive to several cytokines, the molecular oncogenic mechanisms in PV are largely unknown. In this study, we examined gene expression profiles of CD34(+) cells from bone marrow of patients with de novo PV and from healthy volunteers to identify molecular changes associated with the malignant growth of hematopoietic stem and progenitor cells in this myeloproliferative disorder. Using cDNA arrays, we found significant differences (P < .01) in the expression of 107 genes. Proapoptotic genes (CASP2, CASP3, DAPK1, ALG2) were expressed at lower levels in PV-CD34(+) cells, reflecting a lower apoptotic activity. Fibrosis-stimulating growth factors (transforming growth factor beta1, transforming growth factor beta2, bone morphogenetic protein 2, and endothelial growth factor) were expressed at significantly higher levels in PV-CD34(+) cells. Furthermore, PV-CD34(+) cells overexpressed several receptors, protein kinases, and proteasome subunits, which might be targets for directed therapeutic approaches. It is interesting that three retinoid receptors were overexpressed in PV-CD34(+) cells--retinoic acid receptor beta (RARbeta), retinoid X receptor beta (RXRbeta), and cellular retinoic acid binding protein 2 (CRABP2). Using methylcellulose colony-forming assays, we found that the formation of erythroid colonies derived from PV hematopoietic progenitors was inhibited by all-trans-retinoic acid (ATRA), a natural ligand of those receptors, in a dose-dependent manner, showing a maximum inhibition of 89% at 10 microM; the growth of myelomonocytic colonies was not significantly affected. These data suggest that the use of ATRA could be of therapeutic benefit for patients with PV.

Distinct molecular phenotype of malignant CD34(+) hematopoietic stem and progenitor cells in chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) is a malignant disorder of the hematopoietic stem cell characterized by the BCR-ABL oncogene. We examined gene expression profiles of highly enriched CD34(+) hematopoietic stem and progenitor cells from patients with CML in chronic phase using cDNA arrays covering 1.185 genes. Comparing CML CD34(+) cells with normal CD34(+) cells, we found 158 genes which were significantly differentially expressed. Gene expression patterns reflected BCR-ABL-induced functional alterations such as increased cell-cycle and proteasome activity. Detoxification enzymes and DNA repair proteins were downregulated in CML CD34(+) cells, which might contribute to genetic instability. Decreased expression of junction plakoglobulin and CXC chemokine receptor 4 (CXCR-4) might facilitate the release of immature precursors from bone marrow in CML. GATA-2 was upregulated in CML CD34(+) cells, suggesting an increased self-renewal in comparison with normal CD34(+) cells. Moreover, we found upregulation of the proto-oncogene SKI and of receptors for neuromediators such as opioid mu1 receptor, GABA B receptor, adenosine A1 receptor, orexin 1 and 2 receptors and corticotropine-releasing hormone receptor. Treatment of CML progenitor cells with the selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) resulted in a dose-dependent significant inhibition of clonogenic growth by 40% at a concentration of 10(-5) M, which could be reversed by the equimolar addition of the receptor agonist 2-chloro-N6-cyclopentyladenosine (P<0.05). The incubation of normal progenitor cells with DPCPX resulted in an inhibition of clonogenic growth to a significantly lesser extent in comparison with CML cells (P<0.05), suggesting that the adenosine A1 receptor is of functional relevance in CML hematopoietic progenitor cells.

LightCycler-based quantitative real-time PCR monitoring of patients with follicular lymphoma receiving rituximab in combination with conventional or high-dose cytotoxic chemotherapy.

OBJECTIVES: Quantitative real-time polymerase chain reaction (qPCR) is a suitable method to measure residual disease in hematological malignancies. Our objective was to assess a LightCycler-based qPCR for t(14;18)(q32;q21)(IgH/bcl-2)-positive cells quantification in the context of clinical and morphopathological characteristics of patients with follicular lymphoma treated with rituximab (R) in combination with conventional or high-dose chemotherapy. METHODS: A total of 270 bone marrow (BM) and peripheral blood (PB) samples collected from 52 patients with follicular lymphoma at diagnosis or at relapse before or sequentially during therapy were examined by qPCR and nested-PCR. RESULTS: A greater amount of t(14;18)-positive cells was observed in BM in comparison with PB in 76% of paired samples. The presence and number of t(14;18)-positive cells in BM and PB correlated with lymphoma activity. Significantly higher numbers of lymphoma cells were found in patients under non-remission compared with patients in clinical remission. During non-remission, 10-fold higher numbers were measured at relapse than at diagnosis. During remission, significantly higher levels were found in partial compared with complete remission. During first-line therapy, R/cyclophosphamide/adriamycin/vincristine/prednisone (CHOP) had higher in vivo purging ability than R/fludarabine/mitoxantrone (FM). After R/high-dose cytosine-arabinoside and mitoxantrone (HAM) or R/carmustine/etoposide/cytarabine/melphalan (BEAM), the level of t(14;18)-positive cells dropped below the detection limit in 80% of patients. CONCLUSIONS: LightCycler qPCR is a reliable method for quantitative molecular monitoring of t(14;18)-positive cells in BM and PB of patients with follicular lymphoma. It reflects the clinical characteristics of patients and allows assessment of response to different treatment regimens on a molecular level.

Levels of minimal residual disease detected by quantitative molecular monitoring herald relapse in patients with multiple myeloma.

BACKGROUND AND OBJECTIVES: Detection of minimal residual disease (MRD) has helped to improve the treatment of patients with leukemia. At present MRD testing in patients with multiple myeloma (MM) is not applied as a standard diagnostic or prognostic method. DESIGN AND METHODS: Immunoglobulin heavy chain (IgH) polymerase chain reaction (PCR) using patient-specific TaqMan probes together with LightCycler technology was performed to quantify minimal residual disease in MM. Relative levels of clonotypic cells were assessed as IgH/2beta-actin ratios with a sensitivity of 10(-4) to 10(-5). RESULTS: Following stem cell transplantation, a significant reduction of clonotypic cells was observed in bone marrow (BM) and peripheral blood (PB) samples of 11 patients, comparing pre-treatment values with those of best response (median: 13% to 0.09% and 0.03% to 0%, respectively). In 5 patients with ongoing clinical remission IgH/2beta-actin ratios remained stable at a low level, while in 6 patients an increase to 2% in BM and 0.4% in PB was associated with progression of the disease. In 4 of these 6 patients the increase of clonotypic cells in PB was detectable a median of 3 months (range: 0.5-6) before relapse. Furthermore, time-to-progression of patients with pre-transplantation IgH/2b-actin ratios > 0.03% in BM was significantly shorter than that of patients with lower MRD levels. INTERPRETATION AND CONCLUSIONS: MRD in patients with MM can be quantified reliably using TaqMan chemistry adapted to the LightCycler system. Residual tumor cell levels before transplantation as well as results of sequential molecular monitoring are predictive of relapse.

Primary human CD34+ hematopoietic stem and progenitor cells express functionally active receptors of neuromediators.

Recently, overlapping molecular phenotypes of hematopoietic and neuropoietic cells were described in mice. Here, we examined primary human CD34(+) hematopoietic stem and progenitor cells applying specialized cDNA arrays, real-time reverse-transcriptase-polymerase chain reaction (RT-PCR), and fluorescent-activated cell sorter (FACS) analysis focusing on genes involved in neurobiologic functions. We found expression of vesicle fusion and motility genes, ligand- and voltage-gated ion channels, receptor kinases and phosphatases, and, most interestingly, mRNA as well as protein expression of G protein-coupled receptors of neuromediators (corticotropin-releasing hormone 1 [CRH 1] and CRH 2 receptors, orexin/hypocretin 1 and 2 receptors, GABAB receptor, adenosine A(2)B receptor, opioid kappa 1 and mu 1 receptors, and 5-HT 1F receptor). As shown by 2-color immunofluorescence, the protein expression of these receptors was higher in the more immature CD38(dim) than in the CD38(bright) subset within the CD34(+) population, and completely absent in fully differentiated blood cells, suggesting that those receptors play a role in developmentally early CD34(+) stem and progenitor cells. The intracellular concentration of cyclic adenosine monophosphate (cAMP) in CD34(+) cells was diminished significantly upon stimulation of either CRH or orexin receptors, indicating that those are functionally active and coupled to inhibitory G proteins in human hematopoietic cells. In conclusion, these findings suggest a molecular interrelation of neuronal and hematopoietic signaling mechanisms in humans.

Gene expression profiling identifies significant differences between the molecular phenotypes of bone marrow-derived and circulating human CD34+ hematopoietic stem cells.

CD34+ hematopoietic stem cells are used clinically to support cytotoxic therapy, and recent studies raised hope that they could even serve as a cellular source for nonhematopoietic tissue engineering. Here, we examined in 18 volunteers the gene expressions of 1185 genes in highly enriched bone marrow CD34+ (BM-CD34+) or granulocyte-colony-stimulating factor-mobilized peripheral blood CD34+ (PB-CD34+) cells by means of cDNA array technology to identify molecular causes underlying the functional differences between circulating and sedentary hematopoietic stem and progenitor cells. In total, 65 genes were significantly differentially expressed. Greater cell cycle and DNA synthesis activity of BM-CD34+ than PB-CD34+ cells were reflected by the 2- to 5-fold higher expression of 9 genes involved in cell cycle progression, 11 genes regulating DNA synthesis, and cell cycle-initiating transcription factor E2F-1. Conversely, 9 other transcription factors, including the differentiation blocking GATA2 and N-myc, were expressed 2 to 3 times higher in PB-CD34+ cells than in BM-CD34+ cells. Expression of 5 apoptosis driving genes was also 2 to 3 times greater in PB-CD34+ cells, reflecting a higher apoptotic activity. In summary, our study provides a gene expression profile of primary human CD34+ hematopoietic cells of the blood and marrow. Our data molecularly confirm and explain the finding that CD34+ cells residing in the bone marrow cycle more rapidly, whereas circulating CD34+ cells consist of a higher number of quiescent stem and progenitor cells. Moreover, our data provide novel molecular insight into stem cell physiology.