IT behind a platform for Translational Cancer Research - concept and objectives.

The German Consortium for Translational Cancer Research (DKTK) and the Rhine-Main Translational Cancer Research Network (RM-TCRN) are designed to exploit large population cohorts of cancer patients for the purpose of bio-banking, clinical trials, and clinical cancer registration. Hence, the success of these platforms is heavily dependent on the close interlinking of clinical data from cancer patients, information from study registries, and data from bio-banking systems of different laboratories and scientific institutions. This article referring to the poster discusses the main challenges of the platforms from an information technology point of view, legal and data security issues, and outlines an integrative IT-concept concerning a decentralized, distributed search approach where data management and search is in compliance with existing legislative rules.

Epigenetic dysregulation of GATA1 is involved in myelodysplastic syndromes dyserythropoiesis.

Myelodysplastic syndromes (MDS) are characterized by dyserythropoiesis resulting in anemia. This pathological hallmark is incompletely understood. Notch signaling has been linked to impaired erythropoietic and megakaryopoietic development of CD34+ progenitor cells, but its role in MDS is unclear. We have analyzed the transcriptional activity of Notch pathway elements and its association with the key erythroid factor globin transcription factor 1 (GATA1) and the apoptosis regulatory gene B-cell lymphoma-xl (BCLxl) in MDS. The methylation of GATA1 erythroid promoter CpG dinucleotides flanking cis-regulatory elements, including an N-box suppressor binding site for HES1 and a GATA-box binding site, was examined in normal and MDS erythropoiesis. We have generated a kinetic in vitro model of MDS erythropoiesis using CD34+ bone marrow cells from healthy donors (n = 7) and patients with MDS (low risk: RA/n = 6, RARS/n = 3; high risk: RAEB/n = 4, RAEB-T/n = 2). RNA expression of GATA1, BCLxl, DLK1, Notch1, HES1, and HERP2 was measured by real-time RT-PCR (qPCR). DNA methylation at seven CpG dinucleotides of the GATA1 gene promoter was quantitatively analyzed by pyrosequencing of bisulfite-treated genomic DNA at any specific time point. For the Notch pathway elements, no conclusive expression differences were found between MDS and normal erythropoiesis. But we found steadily up-regulated RNA expression of GATA1 and of BCLxl during late normal erythropoietic differentiation. In contrast, during MDS, erythropoiesis a loss of typical up-regulation of GATA1 and BCLxl was observed. Hypermethylation of CpG dinucleotides flanking the repressor HES1 binding site within the 5' region of GATA1 was detected particularly during late MDS erythropoiesis. Interestingly, decremental GATA1 promotor methylation values were seen during normal erythropoiesis matching GATA1 RNA up-regulation. Our data show that the critical erythropoietic transcription factor GATA1 as well as the antiapoptotic molecule BCLxl fails to be normally up-regulated during MDS erythropoiesis. The higher residual 5'-GATA1 methylation values in MDS erythropoiesis but decremental loss thereof in normal erythropoiesis suggest a gene dose effect for GATA1 during erythropoiesis being finely tuned by CpG methylation. Its dysregulation may contribute to the ineffective erythropoiesis observed in MDS.

Identification of defects in the transcriptional program during lineage-specific in vitro differentiation of CD34(+) cells selected from patients with both low- and high-risk myelodysplastic syndrome.

OBJECTIVE: Development of myelodysplastic syndrome (MDS) is suggested to follow a multistep pathogenesis and is characterized by accumulation of molecular defects of the hematopoietic stem/progenitor cells, resulting in aberrant differentiation and proliferation. MATERIALS AND METHODS: To detect alterations within the transcriptional program in MDS-derived CD34(+) cells during lineage-specific differentiation, we performed serial gene expression analysis of in vitro differentiated erythro-, granulo-, and megakaryopoietic cells using oligonucleotide microarrays (HG-U133A, Affymetrix, Santa Clara, CA, USA). For selected genes, expression data were confirmed using real-time polymerase chain reaction. RESULTS: We identified genes with altered expression during lineage-specific differentiation in either low- or high-risk MDS cells compared to the expression patterns of continuously up- or downregulated genes from the normal transcriptional program of hematopoiesis. In cluster analyses, we could show that MDS samples have a distinct expression pattern of a set of selected genes compared to normal cells, which allows prediction of the affiliation of a sample to one group. Furthermore, this study gives an overview of genes that are differentially expressed in MDS cells compared to normal hematopoiesis. CONCLUSION: Our data provide the first comprehensive transcriptional analysis of differentiating human CD34(+) cells derived from MDS patients compared to normal individuals. It gives new insights into the alteration of differentiation and proliferation of MDS stem cells.

Detection and HER2 expression of circulating tumor cells: prospective monitoring in breast cancer patients treated in the neoadjuvant GeparQuattro trial.

PURPOSE: This study was aimed at detecting and characterizing circulating tumor cells (CTC) before and after neoadjuvant therapy (NT) in the peripheral blood of patients with breast cancer. EXPERIMENTAL DESIGN: The clinical trial GeparQuattro incorporated NT approaches (epirubicin/cyclophosphamide prior to randomization to docetaxel alone, docetaxel in combination with capecitabine, or docetaxel followed by capecitabine) and additional trastuzumab treatment for patients with HER2-positive tumors. We used the Food and Drug Administration-approved CellSearch system for CTC detection and evaluation of HER2 expression and developed HER2 immunoscoring for CTC. RESULTS: We detected > or =1 CTC/7.5 mL in 46 of 213 patients (21.6%) before NT and in 22 of 207 patients (10.6%) after NT (P = 0.002). Twenty (15.0%) initially CTC-positive cases were CTC-negative after NT, whereas 11 (8.3%) cases were CTC-positive after NT, although no CTC could be found before NT. CTC detection did not correlate with primary tumor characteristics. Furthermore, there was no association between tumor response to NT and CTC detection. HER2-overexpressing CTC were observed in 14 of 58 CTC-positive patients (24.1%), including 8 patients with HER2-negative primary tumors and 3 patients after trastuzumab treatment. CTC scored HER2-negative or weakly HER2-positive before or after NT were present in 11 of 21 patients with HER2-positive primary tumors. HER2 overexpression on CTC was restricted to ductal carcinomas and associated with high tumor stage (P = 0.002). CONCLUSION: CTC number was low in patients with primary breast cancer. The decrease in CTC incidence during treatment was not correlated with standard clinical characteristics and primary tumor response. Information on the HER2 status of CTC might be helpful for stratification and monitoring of HER2-directed therapies.

Tumor-associated lymphocytes as an independent predictor of response to neoadjuvant chemotherapy in breast cancer.

PURPOSE Preclinical data suggest a contribution of the immune system to chemotherapy response. In this study, we investigated the prespecified hypothesis that the presence of a lymphocytic infiltrate in cancer tissue predicts the response to neoadjuvant chemotherapy. METHODS We investigated intratumoral and stromal lymphocytes in a total of 1,058 pretherapeutic breast cancer core biopsies from two neoadjuvant anthracycline/taxane-based studies (GeparDuo, n = 218, training cohort; and GeparTrio, n = 840, validation cohort). Molecular parameters of lymphocyte recruitment and activation were evaluated by kinetic polymerase chain reaction in 134 formalin-fixed, paraffin-embedded tumor samples. Results In a multivariate regression analysis including all known predictive clinicopathologic factors, the percentage of intratumoral lymphocytes was a significant independent parameter for pathologic complete response (pCR) in both cohorts (training cohort: P = .012; validation cohort: P = .001). Lymphocyte-predominant breast cancer responded, with pCR rates of 42% (training cohort) and 40% (validation cohort). In contrast, those tumors without any infiltrating lymphocytes had pCR rates of 3% (training cohort) and 7% (validation cohort). The expression of inflammatory marker genes and proteins was linked to the histopathologic infiltrate, and logistic regression showed a significant association of the T-cell-related markers CD3D and CXCL9 with pCR. CONCLUSION The presence of tumor-associated lymphocytes in breast cancer is a new independent predictor of response to anthracycline/taxane neoadjuvant chemotherapy and provides useful information for oncologists to identify a subgroup of patients with a high benefit from this type of chemotherapy.

Identification of biology-based breast cancer types with distinct predictive and prognostic features: role of steroid hormone and HER2 receptor expression in patients treated with neoadjuvant anthracycline/taxane-based chemotherapy.

INTRODUCTION: Reliable predictive and prognostic markers for routine diagnostic purposes are needed for breast cancer patients treated with neoadjuvant chemotherapy. We evaluated protein biomarkers in a cohort of 116 participants of the GeparDuo study on anthracycline/taxane-based neoadjuvant chemotherapy for operable breast cancer to test for associations with pathological complete response (pCR) and disease-free survival (DFS). Particularly, we evaluated if interactions between hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) expression might lead to a different clinical behavior of HR+/HER2+ co-expressing and HR+/HER2- tumors and whether subgroups of triple negative tumors might be identified by the help of Ki67 labeling index, cytokeratin 5/6 (CK5/6), as well as cyclooxygenase-2 (COX-2), and Y-box binding protein 1 (YB-1) expression. METHODS: Expression analysis was performed using immunohistochemistry and silver-enhanced in situ hybridization on tissue microarrays (TMAs) of pretherapeutic core biopsies. RESULTS: pCR rates were significantly different between the biology-based tumor types (P = 0.044) with HR+/HER2+ and HR-/HER2- tumors having higher pCR rates than HR+/HER2- tumors. Ki67 labeling index, confirmed as significant predictor of pCR in the whole cohort (P = 0.001), identified HR-/HER- (triple negative) carcinomas with a higher chance for a pCR (P = 0.006). Biology-based tumor type (P = 0.046 for HR+/HER2+ vs. HR+/HER2-), Ki67 labeling index (P = 0.028), and treatment arm (P = 0.036) were independent predictors of pCR in a multivariate model. DFS was different in the biology-based tumor types (P < 0.0001) with HR+/HER2- and HR+/HER2+ tumors having the best prognosis and HR-/HER2+ tumors showing the worst outcome. Biology-based tumor type was an independent prognostic factor for DFS in multivariate analysis (P < 0.001). CONCLUSIONS: Our data demonstrate that a biology-based breast cancer classification using estrogen receptor (ER), progesterone receptor (PgR), and HER2 bears independent predictive and prognostic potential. The HR+/HER2+ co-expressing carcinomas emerged as a group of tumors with a good response rate to neoadjuvant chemotherapy and a favorable prognosis. HR+/HER2- tumors had a good prognosis irrespective of a pCR, whereas patients with HR-/HER- and HR-/HER+ tumors, especially if they had not achieved a pCR, had an unfavorable prognosis and are in need of additional treatment options.

Prediction of qualitative outcome of oligonucleotide microarray hybridization by measurement of RNA integrity using the 2100 Bioanalyzer capillary electrophoresis system.

RNA quality is critical to achieve valid results in microarray experiments and to save resources. The RNA integrity number (RIN) can be measured with minimal sample consumption by microfluidics-based capillary electrophoresis. To determine whether RIN can predict the qualitative outcome of microarray hybridization, we measured RIN in total RNA samples from 484 different experiments by the 2100 Bioanalyzer system and correlated with the percentage of present calls (%pc) of downstream oligonucleotide microarrays. The correlation coefficient for RNA and %pc in all 408 samples for which the bioanalyzer algorithm was able to produce an RIN was 0.475 (p < 0.05), ranging from 0.039 to 0.673 for different tissue- and assay-type subgroups. Multivariate analysis found RIN to be the best predictor of microarray quality as assessed by %pc, outperforming the 28S to 18S ratio. For a %pc threshold of 25% and 35%, we determined optimal cut points for RIN at 7.15 and 8.05, respectively. Using the suggested cut points, RIN can support the final decision whether a certain RNA sample is appropriate for successful microarray hybridization.

Aberrant promotor methylation in MDS hematopoietic cells during in vitro lineage specific differentiation is differently associated with DNMT isoforms.

Aberrant promoter methylation may contribute to the hematopoietic disturbances in myelodysplastic syndromes (MDS). To explore a possible mechanism, we therefore analyzed expression of DNA methyltransferase (DNMT) subtypes kinetics and aberrant promoter methylation of key regulatory genes during MDS hematopoiesis. An in vitro model of MDS lineage-specific hematopoiesis was generated by culturing CD34+ cells from healthy donors (n=7) and MDS patients (low-risk: RA/n=6, RARS/n=3; high-risk: RAEB/n=4, RAEB-T/n=2) with EPO, TPO and GCSF. Promoter methylation analysis of key genes involved in the control of apoptosis (p73, survivin, DAPK), DNA-repair (hMLH1), differentiation (RARb, WT1) and cell cycle control (p14, p15, p16, CHK2) was performed by methylation specific PCR of bisulfite-treated genomic DNA. Expression of DNMT1, DNMT3a and DNMT3b was analyzed and correlated with gene promoter methylation for each lineage at different time points. DNMT expression (all isoforms) was increased during thrombopoiesis whereas elevated DNMT1 level were seen during erythropoiesis. Associations between aberrant promoter methylation and DNMT expression were found in high-risk MDS for all lineages and during erythropoiesis. Hypermethylation of p15, p16, p73, survivin, CHK2, RARb and DAPK were associated with elevated DNMT isoform expression. No general overexpression of DNMT subtype was detected during MDS hematopoiesis. However a negative association of DNMT3a and 3b expression with MDS disease risk (IPSS) could be observed. Our data indicate that all mammalian DNMT isoforms may be involved in the aberrantly methylated phenotype in MDS but seem also to be essential for the differentiation of normal hematopoietic stem cells. In particular elevated DNMT1 expression may in particular contribute to ineffective erythropoiesis in MDS.

DNA methylation profiling of myelodysplastic syndrome hematopoietic progenitor cells during in vitro lineage-specific differentiation.

Deregulated epigenetic mechanisms are likely involved in the pathogenesis of myelodysplastic syndromes (MDSs). Which genes are silenced by aberrant promotor methylation during MDS hematopoiesis has not been equivalently investigated. Using an in vitro differentiation model of human hematopoiesis, we generated defined differentiation stages (day 0, day 4, day 7, day 11) of erythro-, thrombo- and granulopoiesis from 13 MDS patients and seven healthy donors. Promotor methylation analysis of key regulatory genes involved in cell cycle control (p14, p15, p16, CHK2), DNA repair (hMLH1), apoptosis (p73, survivin, DAPK), and differentiation (RARb, WT1) was performed by methylation-specific polymerase chain reaction. Corresponding gene expression was analyzed by microarray (Affymetrix, HG-U133A). We provide evidence that p16, survivin, CHK2, and WT1 are affected by promotor hypermethylation in MDSs displaying a selective International Prognostic Scoring System risk association. A methylation-associated mRNA downregulation for specific hematopoietic lineages and differentiation stages is demonstrated for survivin, CHK2, and WT1. We identified a suppressed survivin mRNA expression in methylated samples during erythropoiesis, whereas WT1 and CHK2 methylation-related reduction of mRNA expression was found during granulopoiesis in all MDS risk types. Our data suggest that lineage-specific methylation-associated gene silencing of survivin, CHK2, and WT1 in MDS hematopoietic precursor cells may contribute to the MDS-specific phenotype

In vivo drug-response in patients with leukemic non-Hodgkin's lymphomas is associated with in vitro chemosensitivity and gene expression profiling.

Only a few approaches are available to address the mechanisms of cell death in vivo which are induced by anticancer treatment in patients with malignancies. In this study in vitro chemosensitivity testing of primary peripheral blood leukemic cells of five patients suffering from different leukemic non-Hodgkin's lymphomas was combined with the analysis of the in vivo rate of apoptosis by flow-cytometry (Annexin V and depolarisation of mitochondrial membrane potential (MMP) by JC-1). Furthermore, changes in expression patterns of apoptosis related proteins during chemotherapeutic treatment were detected by Western Blot. Gene expression profiling (HG-U133A, Affymetrix, Santa Clara, CA) was employed to identify common marker genes of in vivo drug response. In vitro chemosensitivity was tested using the cytotoxic agents which the patients were scheduled to receive and was strongly correlated with effective reduction of leukemic lymphoma cells in patients resulting in complete remissions in all five cases. Due to the rapid clearance of apoptotic tumor cells in vivo neither the analysis of the in vivo rate of apoptosis and depolarisation of MMP nor the assessment of expression of regulators of apoptosis showed concordant results concerning the drug response. However, assessment of gene expression during therapy could identify a set of 30 genes to significantly discriminate between samples from patients before treatment compared to samples from the same patients after receiving cytotoxic therapy. Among these 30 genes we found a high proportion of genes associated with apoptotic cell death, cell proliferation and cell cycle signalling including complement lysis inhibitor (clusterin/CLU), beta-catenin interacting protein (ICAT), peroxisome proliferator activated receptor alpha (PPARalpha), TNF alpha converting enzyme (ADAM17/TACE), homeo box A3 (HOX1), inositol polyphosphatase 5-phosphatase type IV (PPI5PIV) and inhibitor of p53 induced apoptosis alpha (IPIA-Alpha/NM23-H6). These results indicate that in vitro chemosensitivity testing and gene expression profiling can successfully be utilised to analyse in vivo drug response in patients with leukemic NHL's and can be used to explore new pathway models of drug-induced cell death in vivo which are independent of different lymphoma subtypes and different treatment regimens.

Transcriptional profiling of human hematopoiesis during in vitro lineage-specific differentiation.

To better understand the transcriptional program that a ccompanies orderly lineage-specific hematopoietic differentiation, we performed serial oligonucleotide microarray analysis of human normal CD34+ bone marrow cells during lineage-specific differentiation. CD34+ bone marrow cells isolated from healthy individuals were selectively stimulated in vitro with the cytokines erythropoietin (EPO), thrombopoietin (TPO), granulocyte colony-stimulating factor (G-CSF), and granulocyte macrophage colony-stimulating factor (GM-CSF). Cells from each of the lineages were harvested after 4, 7, and 11 days of culture for expression profiling. Gene expression was analyzed by oligonucleotide microarrays (HG-U133A; Affymetrix, Santa Clara, CA). Experiments were done in triplicates. We identified 258 genes that are consistently upregulated or downregulated during the course of lineage-specific differentiation within each specific lineage (horizontal change). In addition, we identified 52 genes that contributed to a specific expression profile, yielding a genetic signature specific for successive stages of differentiation within each of the three lineages. Analysis of horizontal changes selected 21 continuously upregulated genes for EPO-induced differentiation (including GTPase activator proteins RAP1GA1 and ARHGAP8, which regulate small Rho GTPases), 21 for G-CSF-induced/GM-CSF-induced differentiation, and 91 for TPO-induced differentiation (including DLK1, of which the role in normal hematopoiesis is not defined). During the lineage-specific differentiation, 58 (erythropoiesis), 30 (granulopoiesis), and 37 (thrombopoiesis) genes were significantly downregulated, respectively. The expression of selected genes was confirmed by real-time polymerase chain reaction. Our data encompass the first extensive transcriptional profile of human hematopoiesis during in vitro lineage-specific differentiation.

Clonal heterogeneity in growth kinetics of CD34+CD38- human cord blood cells in vitro is correlated with gene expression pattern and telomere length.

Human hematopoietic stem cells (HSCs) are characterized by an extensive proliferative capacity that decreases from fetal liver to cord blood (CB) to adult bone marrow. In previous studies, it was demonstrated that the proliferative capacity of individual CD34+CD38- HSC clones is correlated with their growth kinetics in vitro and that HSC turnover in vivo can be estimated by telomere-length measurements. The present study was aimed at the characterization of the clonal composition of CD34+CD38- human umbilical CB cells in terms of growth kinetics, telomere length, and gene expression profile. For this purpose, individual CD34+CD38- CB cells were sorted into 96-well plates containing serum-free medium supplemented with six growth factors. During expansion, cell numbers in each individual well were scored in 3-day intervals. Once sufficient cell numbers were achieved, telomere length was measured by flow fluorescence in situ hybridization (flow FISH). In a second set of experiments, gene expression and colony-forming capacity were analyzed in slowly growing clones as compared with fast-growing clones, using linear amplification and oligonucleotide microarrays (HG-U133A; Affymetrix). Individual CD34+CD38- cells from CB displayed an extensive functional heterogeneity in growth kinetics. Among highly proliferative clones, the most slowly growing clones were characterized by the longest telomeres. Furthermore, significant differences in gene expression were detected between slow- and fast-growing clones, whereas no significant difference in colony-forming capacity was observed. These data provide further evidence for a functional hierarchy in the human HSC compartment and suggest a link between telomere length and proliferation capacity of individual HSC clones.

Impact of persistent cytomegalovirus infection on human neuroblastoma cell gene expression.

In a model of human neuroblastoma (NB) cell lines persistently infected with human cytomegalovirus (HCMV) we previously showed that persistent HCMV infection is associated with an increased malignant phenotype, enhanced drug resistance, and invasive properties. To gain insights into the mechanisms of increased malignancy we analyzed the global changes in cellular gene expression induced by persistent HCMV infection of human neuroblastoma cells by use of high-density oligonucleotide microarrays (HG-U133A, Affymetrix) and RT-PCR. Comparing the gene expression of different NB cell lines with persistently infected cell sub-lines revealed 11 host cell genes regulated in a similar manner throughout all infected samples. Nine of these 11 genes may contribute to the previously observed changes in malignant phenotype of persistently HCMV infected NB cells by influencing invasive growth, apoptosis, angiogenesis, and proliferation. Thus, this work provides the basis for further functional studies.

Disruption of LTBP-4 function reduces TGF-beta activation and enhances BMP-4 signaling in the lung.

Disruption of latent TGF-beta binding protein (LTBP)-4 expression in the mouse leads to abnormal lung development and colorectal cancer. Lung fibroblasts from these mice produced decreased amounts of active TGF-beta, whereas secretion of latent TGF-beta was significantly increased. Expression and secretion of TGF-beta2 and -beta3 increased considerably. These results suggested that TGF-beta activation but not secretion would be severely impaired in LTBP-4 -/- fibroblasts. Microarrays revealed increased expression of bone morphogenic protein (BMP)-4 and decreased expression of its inhibitor gremlin. This finding was accompanied by enhanced expression of BMP-4 target genes, inhibitors of differentiation 1 and 2, and increased deposition of fibronectin-rich extracellular matrix. Accordingly, increased expression of BMP-4 and decreased expression of gremlin were observed in mouse lung. Transfection of LTBP-4 rescued the -/- fibroblast phenotype, while LTBP-1 was inefficient. Treatment with active TGF-beta1 rescued BMP-4 and gremlin expression to wild-type levels. Our results indicate that the lack of LTBP-4-mediated targeting and activation of TGF-beta1 leads to enhanced BMP-4 signaling in mouse lung.

Mechanisms of resistance to STI571 (Imatinib) in Philadelphia-chromosome positive acute lymphoblastic leukemia.

Resistance against STI571 (Imatinib) appears to be multifactorial, but the most likely mechanisms can be broadly categorized as interference with the pharmacologic activity of STI571 or genetic changes which alter the biologic behaviour of the leukemic cells. In Ph + ALL, responses to STI571 are not sustained, and in the overwhelming majority of patients development of resistance is rapid. Clinically, two types of resistance to STI571 can be distinguished: 'primary resistance', corresponding to a failure to achieve fewer than 5% blasts in the bone marrow, and 'secondary resistance' in patients with STI571-induced complete remission who relapse despite continued STI571 treatment. Attempts to identify mechanisms by which Ph + ALL acquire resistance to STI571 have already been successful. Mutations in the ATP binding site of ABL are frequent events which counteract the antileukemic effect of STI571. Gene expression profiling has been shown to discriminate between resistant and sensitive leukemic cells. Application of this technique has also generated several hypotheses regarding the ability of leukemic cells to bypass the BCR-ABL signal transduction pathway. This may result in the proliferation of Ph + leukemic cells even in the presence of STI571.

Inhibitory effect of imatinib on normal progenitor cells in vitro.

Imatinib is a novel tyrosine kinase inhibitor used for the treatment of Philadelphia chromosome-positive leukemias and other malignancies. Side effects are mostly moderate; however, a dose-dependent hematologic toxicity affecting all hematopoietic lineages is observed clinically. The aim of this study was to investigate the effect of imatinib on normal hematopoietic stem and progenitor cells in vitro. A dose-dependent decrease in proliferation potential was found when CD34+ cells were expanded in serum-free medium supplemented with 6 growth factors and imatinib. Functionally, a decrease in colony-forming capacity was observed under increasing doses of imatinib. However, no such effect on more primitive cobblestone area-forming cells was detectable. Both withdrawal of stem cell factor from our expansion cultures or functional inhibition of c-kit led to a similar degree of inhibition of expansion, whereas the effect of imatinib was substantially greater at all dose levels tested. These data suggest a significant inhibitory effect of imatinib on normal CD34+ progenitor (but not stem) cells that is largely independent of c-kit signaling.

Risk and prognosis of central nervous system leukemia in patients with Philadelphia chromosome-positive acute leukemias treated with imatinib mesylate.

PURPOSE: In patients with acute leukemias, a lymphoid phenotype, the presence of a Philadelphia chromosome (Ph), and inadequate central nervous system (CNS)-directed prophylactic therapy are risk factors for CNS involvement. Imatinib mesylate has promising single-agent antileukemic activity in patients with advanced Ph(+) acute leukemias. It was the aim of this analysis to determine the incidence of, and risk factors associated with, meningeal leukemia during imatinib monotherapy. STUDY DESIGN: We analyzed 107 consecutive patients with relapsed or refractory Ph(+) acute lymphoid leukemia (ALL; n = 65) or chronic myeloid leukemia blast crisis (n = 42) who were enrolled in successive Phase II trials of single-agent imatinib and who did not receive routine prophylactic intrathecal chemotherapy. RESULTS: CNS leukemia developed in 13 of 107 patients (12%) and was associated primarily with a lymphoid or bilineage phenotype (12 of 78; 15%) and with imatinib refractory Ph(+) ALL (5 of 19; 26%). Meningeal leukemia did not occur among patients who received prior prophylactic cranial irradiation. The median survival with combined CNS and systemic disease was 108 days (range, 58-141), with no patient surviving long term. In contrast, two of three patients with exclusively meningeal leukemia achieved prolonged molecular remissions with intrathecal chemotherapy, cranial irradiation, and continued imatinib. CONCLUSIONS: Patients with Ph(+) ALL are at considerable risk of meningeal leukemia during imatinib monotherapy and should routinely receive CNS prophylaxis. Although the prognosis with combined meningeal and systemic relapse is dismal, patients with an isolated meningeal relapse may still achieve sustained remissions. The optimal type of CNS-directed treatment and the extent of protection afforded by prophylactic cranial irradiation remain to be defined.

Presence of the BCR-ABL mutation Glu255Lys prior to STI571 (imatinib) treatment in patients with Ph+ acute lymphoblastic leukemia.

The tyrosine kinase inhibitor STI571 (imatinib) binds competitively to the adenosine triphosphate (ATP) binding site of the ABL kinase, thereby inhibiting auto- and substrate phosphorylation of the oncogenic protein BCR-ABL and preventing the activation of downstream signaling pathways. Comparative studies on leukemic cell samples obtained from chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) patients before and after treatment with STI571 reported point mutations in resistant samples after a short time of therapy. The aim of this study was to determine whether patients with Ph+ ALL in whom resistance developed as a consequence of the Glu255Lys mutation already harbored this subclone prior to STI571 treatment. First, the migration pattern of cDNAs from 30 bone marrow samples from patients with Ph+ ALL was analyzed by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Thereafter, detailed mutational analysis using genomic DNA was performed on initial STI571-naive bone marrow samples of 4 individuals with Ph+ ALL, for whom the mutation Glu255Lys in association with STI571 treatment had been shown. A 166-bp PCR fragment spanning from nucleotide (nt) 862 to nt 1027 was cloned, and 108 clones per sample were analyzed by direct sequencing. This more sensitive technique revealed the presence of the Glu255Lys mutation in 2 initial samples, one clone each. We identified for the first time the mutation Glu255Lys in STI571-naive leukemic samples of Ph+ ALL patients. The findings suggest that the mutation exists in a very small subpopulation of leukemic cells at the beginning of STI571 therapy.

Characterization of gene expression of CD34+ cells from normal and myelodysplastic bone marrow.

Gene patterns of expression in purified CD34(+) bone marrow cells from 7 patients with low-risk myelodysplastic syndrome (MDS) and 4 patients with high-risk MDS were compared with expression data from CD34(+) bone marrow cells from 4 healthy control subjects. CD34(+) cells were isolated by magnetic cell separation, and high-density oligonucleotide microarray analysis was performed. For confirmation, the expression of selected genes was analyzed by real-time polymerase chain reaction. Class membership prediction analysis selected 11 genes. Using the expression profile of these genes, we were able to discriminate patients with low-risk from patients with high-risk MDS and both patient groups from the control group by hierarchical clustering (Spearman confidence). The power of these 11 genes was verified by applying the algorithm to an unknown test set containing expression data from 8 additional patients with MDS (3 at low risk, 5 at high risk). Patients at low risk could be distinguished from those at high risk by clustering analysis. In low-risk MDS, we found that the retinoic-acid-induced gene (RAI3), the radiation-inducible, immediate-early response gene (IEX1), and the stress-induced phosphoprotein 1 (STIP1) were down-regulated. These data suggest that CD34(+) cells from patients with low-risk MDS lack defensive proteins, resulting in their susceptibility to cell damage. In summary, we propose that gene expression profiling may have clinical relevance for risk evaluation in MDS at the time of initial diagnosis. Furthermore, this study provides evidence that in MDS, hematopoietic stem cells accumulate defects that prevent normal hematopoiesis.