Search

Additional file 3. Linear Regression

Additional file 2. Model Selection

Background and objectives Patient blood (more accurately: haemoglobin, Hb) management (PBM) aims to optimize endogenous Hb production and to minimize iatrogenic Hb loss while maintaining patient safety and optimal effectiveness of medical interventions. PBM was adopted as policy for patients by the World Health Organization (WHO), and, all the more, should be applied to healthy donors. Materials and methods Observational data from 489 bone marrow (BM) donors were retrospectively analysed, and principles of patient blood management were applied to healthy volunteer BM donations. Results and conclusion We managed to render BM aspiration safe for donors, notably completely avoiding the collection of autologous blood units and blood transfusions through iron management, establishment and curation of high-yield aspiration technique, limitation of collection volume to 1·5% of donor body weight and development of volume prediction algorithms for the requested cell dose.

[This corrects the article DOI: 10.1371/journal.pone.0179986.].

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression and their deregulation is involved in tumor development. Epigenetic gene silencing in cancer by DNA methylation contributes to the silencing of tumor-suppressor genes, including miRNAs. We have recently shown that the promoter of miR-708 is aberrantly methylated in chronic lymphocytic leukemia (CLL). To characterize the molecular signaling networks that are influenced by miR-708, we performed a luciferase-based screen evaluating the effects of ectopic miR-708 expression on leukemia-relevant signaling pathways. We found that miR-708 strongly repressed NF-κB signaling, a pathway known to be deregulated in CLL. Among the predicted miR-708 targets was IKKβ (inhibitor of kappa light polypeptide gene enhancer in B cells, kinase-β/IKBKB), a key kinase facilitating NF-κB signaling. We validated the interaction of miR-708 with the 3'-untranslated region of IKKβ and found that miR-708 overexpression represses endogenous IKKβ. Phosphorylation of the IKKβ target IκBα and expression of known NF-κB target genes were impaired by miR-708. Furthermore, we identified an enhancer region downstream of the miR-708 promoter that displays a distinct DNA methylation status in CLL. High enhancer methylation is significantly correlated with lower miR-708 expression and is predominantly found in patients with poor prognosis and shorter time to treatment. These results demonstrate that miR-708 regulates the NF-κB pathway by targeting IKKβ, and that methylation of a key enhancer region contributes to its suppression in CLL.

Background Red blood cell (RBC) depletion is a standard graft manipulation technique for ABO-incompatible bone marrow (BM) transplants. The BM processing module for Spectra Optia, “BMC”, was previously introduced. We here report the largest series to date of routine quality data after performing 50 clinical-scale RBC-depletions. Methods Fifty successive RBC-depletions from autologous (n = 5) and allogeneic (n = 45) BM transplants were performed with the Spectra Optia BMC apheresis suite. Product quality was assessed before and after processing for volume, RBC and leukocyte content; RBC-depletion and stem cell (CD34+ cells) recovery was calculated there from. Clinical engraftment data were collected from 26/45 allogeneic recipients. Results Median RBC removal was 98.2% (range 90.8–99.1%), median CD34+ cell recovery was 93.6%, minimum recovery being 72%, total product volume was reduced to 7.5% (range 4.7–23.0%). Products engrafted with expected probability and kinetics. Performance indicators were stable over time. Discussion Spectra Optia BMC is a robust and efficient technology for RBC-depletion and volume reduction of BM, providing near-complete RBC removal and excellent CD34+ cell recovery.

For better lung cancer diagnosis and therapy, early detection markers of tumor dissemination are urgently needed, as most lung cancers do not show symptoms until extensive metastasis formation has already taken place. Our previous studies showed that in non-small cell lung cancer (NSCLC) early tumor dissemination is associated with a loss of chromosome 4q12-q32 and the presence of disseminated tumor cells (DTC) in the bone marrow. In order to identify the potential target gene in this region, a screen for methylation-dependent expression was performed. Lung cancer cell lines showing a loss of 4q as well as a normal bronchial epithelial cell line as control were treated with 5-aza-2'-deoxycytidine (5-aza-CdR) followed by expression profiling. Seven genes within the 4q target region, which have been associated with a positive DTC status before were found to be regulated by hypermethylation. QRT-PCR in an independent sample set identified HERC5 as a potential target gene. Quantitative methylation analysis of these lung tissue samples revealed that HERC5 promoter hypermethylation was significantly associated with positive DTC status (p = 0.020) and occurrence of brain metastases (p = 0.015). In addition, hypermethylation of the HERC5 promoter in NSCLC was identified as a predictor for poor survival for Stage I adenocarcinoma patients (p = 0.022) and also for poor overall survival in metastatic lung cancer patients (p = 0.028). In conclusion, HERC5 may function as a prognostic marker and is associated with tumor dissemination in lung cancer.

Mobilization of hematopoietic stem cells (HSCs) from the bone marrow to the peripheral blood is a complex mechanism that involves adhesive and chemotactic interactions of HSCs as well as their bone marrow microenvironment. In addition to a number of non-genetic factors, genetic susceptibilities also contribute to the mobilization outcome. Identification of genetic factors associated with HSC yield is important to better understand the mechanism behind HSC mobilization. In the present study, we enrolled 148 Korean participants (56 healthy donors and 92 patients) undergoing HSC mobilization for allogeneic or autologous HSC transplantation. Among a total of 53 polymorphisms in 33 candidate genes, one polymorphism (rs11264422) in relaxin/insulin-like family peptide receptor 4 (RXFP4) gene was significantly associated with a higher HSC yield after mobilization in Koreans. However, in a set of 101 Europeans, no association was found between circulating CD34+ cell counts and rs11264422 genotype. Therefore, we suggest that the ethnic differences in subjects’ genetic background may be related to HSC mobilization. In conclusion, the relaxin—relaxin receptor axis may play an important role in HSC mobilization. We believe that the results of the current study could provide new insights for therapies that use relaxin and HSC populations, as well as a better understanding of HSC regulation and mobilization at the molecular level.

Despite some considerable progress in the therapy for chronic lymphocytic leukaemia (CLL) owing to fludarabine-based regimens and rituximab, no curative treatment is available so far. We conducted an explorative phase II study in patients with CLL, prolymphocytic leukaemia (PLL) and leukaemic lymphoplasmacytic lymphoma (LL) with the combination of fludarabine, epirubicin and rituximab (FER) to improve the complete remission (CR) rate and progression-free survival (PFS). Fludarabine 25 mg/m(2) was administered i.v. on days 1-5 and epirubicin 25 mg/m(2) i.v. on days 4 and 5, and rituximab was added at a dose of 375 mg/m(2) i.v. day 1 in the first cycle and at a dose of 500 mg/m(2) in all consecutive cycles. Patients exhibiting responsive disease after FER were eligible to receive maintenance therapy of up to 12 cycles of rituximab 375 mg/m(2) bimonthly. Forty-four patients (38 CLL, 4 PLL and 2 LL) with a median age of 65 yrs (43-84 yrs) were evaluable. Seventeen patients with CLL had stage Binet C, 14 Binet B and seven symptomatic or rapid progressive stage Binet A. Cytogenetic features showed normal karyotype in nine cases, an isolated deletion (del) 13q in 12 patients, trisomy 12 in 7, del 11 in two and del 17p in 4. Half of the patients (48%) had mutated IgVH genes. Treatment with FER achieved an overall response rate of 95%, including 63% CRs and 32% PRs. Haematological toxicity was considerable. After a median follow-up period of 34 months (range: 8-84 months), median PFS was 61 months and overall survival was yet not reached. All patients with PLL and LL achieved CR. The data support the high efficacy of the combination of rituximab with chemotherapy (FE) and are suggestive of possible benefit with rituximab maintenance therapy for PFS and DFS.

The clinical course of peripheral T-cell lymphoma (PTCL) is usually aggressive and the prognosis unfavorable. Therefore, there is a need for improvement of treatment options. Patients with newly diagnosed (n = 27) or refractory/relapsed (n = 11) PTCL received a combination of alemtuzumab, fludarabine, cyclophosphamide, and doxorubicin. The overall response rate (ORR) was 61%, with a complete response rate of 39%. In newly diagnosed patients the ORR was 63%, the median overall survival 25.9 months, and progression-free survival 11.8 months. In relapsed/refractory patients the median OS was 6.1 months. The most frequent grade 3/4 toxicities were leukopenia (95% of patients) and thrombocytopenia (58%). Cytomegalovirus (CMV) reactivation occurred in 12 patients, but only two had CMV disease. Treatment-related deaths occurred in six newly diagnosed patients and one with relapsed/refractory disease. In conclusion, Campath-FCD is active in PTCL but is associated with significant toxicity and is, therefore, not recommended for use or further study. Further studies are warranted to investigate other approaches to combining alemtuzumab with chemotherapy for the treatment of PTCL.

Individual response of disseminated cancer to chemotherapy is unpredictable. In vitro chemotherapy-induced apoptosis can be measured and might be a method to evaluate in vivo activity of tested drugs. In this report, tumor cells of a patient with signet cell carcinoma of the stomach and diffuse bone marrow infiltration were cultured and tested for in vitro chemosensitivity. The drugs gemcitabine, oxaliplatin and zoledronic acid were found to induce in vitro tumor cell apoptosis synergistically, and subsequently were used as combination chemotherapy regimen. An initially existing disseminated intravascular coagulopathy quickly resolved and after 6 months of treatment on ongoing complete response was induced, thus confirming the results of in vitro chemosensitivity testing.

CD4+ CD56+ neoplasia is a rare malignancy of unclarified origin. So far only 57 cases have been reported. We characterized in detail a case of CD4+ CD56+ malignancy with special emphasis on apoptosis induced by cytotoxic drugs and expression of sialyl Lewis X (CD15s). The disease was diagnosed in a 73-year-old female presenting with skin involvement, generalized lymphadenopathy and bone marrow infiltration. Treatment with cladribine/mitoxantrone induced a short-lasting partial response and the patient died 6 months after diagnosis. The neoplastic cells expressed CD4, CD56, HLA-DR, and CD15s. PCR for the T-cell receptor gamma chain revealed a polyclonal amplification product. In situ hybridization for Epstein-Barr Virus (EBV) was negative. Cytotoxic granule-associated proteins were not detected, consistent with the observation that the cells did not mediate cytotoxic activity against several target cells. Apoptosis of the tumor cells was inducible by anthracyclines and cladribine but not with gemcitabine. Combinations of cladribine or gemcitabine with anthracyclines however, resulted in synergistic effects on apoptosis. Expression of CD15s on the CD56+ cells was three times higher than on CD56+ cells from healthy controls. The results demonstrate that the features of the present case is in accordance with the diagnosis of CD4+ CD56+ malignancy. This is the first report demonstrating increased CD15s expression on a CD4+ CD56+ neoplasia, possibly explaining the frequent occurrence of the disease in the skin.

For better lung cancer diagnosis and therapy, early detection markers of tumor dissemination are urgently needed, as most lung cancers do not show symptoms until extensive metastasis formation has already taken place. Our previous studies showed that in non-small cell lung cancer (NSCLC) early tumor dissemination is associated with a loss of chromosome 4q12-q32 and the presence of disseminated tumor cells (DTC) in the bone marrow. In order to identify the potential target gene in this region, a screen for methylation-dependent expression was performed. Lung cancer cell lines showing a loss of 4q as well as a normal bronchial epithelial cell line as control were treated with 5-aza-2'-deoxycytidine (5-aza-CdR) followed by expression profiling. Seven genes within the 4q target region, which have been associated with a positive DTC status before were found to be regulated by hypermethylation. QRT-PCR in an independent sample set identified HERC5 as a potential target gene. Quantitative methylation analysis of these lung tissue samples revealed that HERC5 promoter hypermethylation was significantly associated with positive DTC status (p = 0.020) and occurrence of brain metastases (p = 0.015). In addition, hypermethylation of the HERC5 promoter in NSCLC was identified as a predictor for poor survival for Stage I adenocarcinoma patients (p = 0.022) and also for poor overall survival in metastatic lung cancer patients (p = 0.028). In conclusion, HERC5 may function as a prognostic marker and is associated with tumor dissemination in lung cancer.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression and their deregulation is involved in tumor development. Epigenetic gene silencing in cancer by DNA methylation contributes to the silencing of tumor-suppressor genes, including miRNAs. We have recently shown that the promoter of miR-708 is aberrantly methylated in chronic lymphocytic leukemia (CLL). To characterize the molecular signaling networks that are influenced by miR-708, we performed a luciferase-based screen evaluating the effects of ectopic miR-708 expression on leukemia-relevant signaling pathways. We found that miR-708 strongly repressed NF-κB signaling, a pathway known to be deregulated in CLL. Among the predicted miR-708 targets was IKKβ (inhibitor of kappa light polypeptide gene enhancer in B cells, kinase-β/IKBKB), a key kinase facilitating NF-κB signaling. We validated the interaction of miR-708 with the 3'-untranslated region of IKKβ and found that miR-708 overexpression represses endogenous IKKβ. Phosphorylation of the IKKβ target IκBα and expression of known NF-κB target genes were impaired by miR-708. Furthermore, we identified an enhancer region downstream of the miR-708 promoter that displays a distinct DNA methylation status in CLL. High enhancer methylation is significantly correlated with lower miR-708 expression and is predominantly found in patients with poor prognosis and shorter time to treatment. These results demonstrate that miR-708 regulates the NF-κB pathway by targeting IKKβ, and that methylation of a key enhancer region contributes to its suppression in CLL.

Despite some considerable progress in the therapy for chronic lymphocytic leukaemia (CLL) owing to fludarabine-based regimens and rituximab, no curative treatment is available so far. We conducted an explorative phase II study in patients with CLL, prolymphocytic leukaemia (PLL) and leukaemic lymphoplasmacytic lymphoma (LL) with the combination of fludarabine, epirubicin and rituximab (FER) to improve the complete remission (CR) rate and progression-free survival (PFS). Fludarabine 25 mg/m(2) was administered i.v. on days 1-5 and epirubicin 25 mg/m(2) i.v. on days 4 and 5, and rituximab was added at a dose of 375 mg/m(2) i.v. day 1 in the first cycle and at a dose of 500 mg/m(2) in all consecutive cycles. Patients exhibiting responsive disease after FER were eligible to receive maintenance therapy of up to 12 cycles of rituximab 375 mg/m(2) bimonthly. Forty-four patients (38 CLL, 4 PLL and 2 LL) with a median age of 65 yrs (43-84 yrs) were evaluable. Seventeen patients with CLL had stage Binet C, 14 Binet B and seven symptomatic or rapid progressive stage Binet A. Cytogenetic features showed normal karyotype in nine cases, an isolated deletion (del) 13q in 12 patients, trisomy 12 in 7, del 11 in two and del 17p in 4. Half of the patients (48%) had mutated IgVH genes. Treatment with FER achieved an overall response rate of 95%, including 63% CRs and 32% PRs. Haematological toxicity was considerable. After a median follow-up period of 34 months (range: 8-84 months), median PFS was 61 months and overall survival was yet not reached. All patients with PLL and LL achieved CR. The data support the high efficacy of the combination of rituximab with chemotherapy (FE) and are suggestive of possible benefit with rituximab maintenance therapy for PFS and DFS.

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 (PPARα), 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.

Background: The role of Daxx, in particular its ability to promote or hinder apoptosis, still remains controversial. In order to elucidate the functional relevance of Daxx in the extrinsic signaling of malignant lymphocytes Jurkat T-cells were stably transfected with a Daxx-expressing vector or with the respective Daxx-negative control vector. Results: Assessing first the impact of Daxx expression on the rate of proliferation we demonstrate that overexpression of Daxx alone is not sufficient to alter proliferation in neoplastic lymphocytes. Nevertheless, expression of Daxx down-regulates anti-apoptotic Bcl-2 and up-regulates pro-apoptotic BID. In addition, Daxx-overexpressing Jurkat cells exhibit a decreased expression of the pro-caspase-8, -10, -9 and -3 and a concomitant increase of the inhibitors of apoptosis proteins survivin, XIAP, cIAP-1 and -2. We further demonstrate, that upon incubation with various chemotherapeutic agents these Daxx-induced molecular alterations sensitize Jurkat T-cells to the apoptosis-inducing effects of specific chemotherapeutic agents. Conclusions: We here outline the molecular changes elicited by Daxx on major components of the apoptotic cascade of malignant lymphocytes and demonstrate the capacity of Daxx to sensitize these cells to the apoptosis-inducing effect of various chemotherapeutic agents.

The mutational status of immunoglobulin variable region genes (Ig VH) is a well established prognostic parameter in chronic lymphocytic leukemia (CLL). Recently, a subset of genes with a characteristic expression profile correlating with the mutational status of B-CLLs has been identified. One of the overexpressed genes in the prognostically unfavorable group of CLL patients with unmutated Ig VH genes encodes for the protein tyrosine kinase ZAP-70, which is physiologically involved in T-cell signaling. Since ZAP-70 has been described to be prognostically relevant in CLL, we analyzed the possible relationship of its expression to the mutational status of Ig VH genes as well as to other prognostic factors in CLL and indolent lymphomas. The mutational status of Ig VH genes was analyzed by seminested PCR, direct sequencing and comparison with the sequences of the EMBL databases in 60 samples of patients with B-CLL and 18 samples of patients with indolent B-cell malignancies. ZAP-70 protein expression was assessed in all samples by immunoblotting and for semiquantitative analysis the ratio of ZAP-70 to tubulin expression was calculated. ZAP-70 protein was found to be expressed in all investigated B-cell malignancies. Expression levels varied within a wide range in each entity. The highest mean level of ZAP-70 expression was observed in unmutated B-CLLs, however, with broad expression variability. High levels of ZAP-70 expression correlated with higher stage Binet B or C and with unmutated Ig VH genes. Overall survival rates estimated by Kaplan-Meier curves did not differ among patients with high or low ZAP-70 expression. We conclude that ZAP-70 is associated with the mutational status of Ig VH genes, but this expression pattern is not present in all individual cases. Furthermore, high levels of ZAP-70 correlated with Binet stages B or C indicating an involvement of ZAP-70 in mechanisms promoting growth of B-CLL cells.

The X-linked inhibitor of apoptosis (XIAP) and cellular inhibitor of apoptosis-1 (cIAP-1) are emerging as versatile proteins in programmed cell death with a scope of possible functions reaching far beyond their well known inhibitory effects on caspases. We previously demonstrated that the ability of drugs to modify expression and cleavage of the IAPs are crucial for the synergistic effects achieved by the combinations of different cytotoxic drugs employed to treat malignant lymphomas. In order to more clearly assess the underlying molecular mechanisms, we here evaluated the consequences of drug-induced apoptosis on the localization and aggregation of XIAP and cIAP-1. The influence of drug-induced apoptosis on localization of IAPs was investigated using immunofluorescence microscopy as well as western blot analysis. Apoptosis was induced by chemotherapeutic drugs with different modes of action (bendamustine, cladribine, fludarabine, doxorubicin and mitoxantrone) and assessed by flow-cytometry using Annexin V. We demonstrate that XIAP and cIAP-1 are downregulated and/or cleaved in a dose-dependent manner upon treatment with a variety of anti-cancer drugs. Moreover we provide evidence that in the context of drug-induced apoptosis XIAP, its BIR3-RING cleavage product and cIAP-1 undergo an extensive change of subcellular localization. Immunofluorescence microscopy reveals that XIAP, in contrast to cIAP-1, is located in discrete cytosolic protein aggregates and-upon induction of apoptosis with cytotoxic drugs--redistributes into large nuclear inclusions. This translocation of XIAP and its BIR3-RING cleavage product from the cytosol into the nucleus is confirmed by cell fractionation and western blot analyses. Of note, in this experimental setting putative interaction partners of XIAP-such as Apaf-1, caspase-3 and -7--do not co-localize with XIAP. These results imply a new unknown function of XIAP and its BIR3-RING fragment in the nucleus in the context of drug-induced apoptosis. The localization of cIAP-1 in mitochondria and its liberation from these indicate a profoundly different function of this protein despite its similar modular structure to XIAP.

The role of Daxx, in particular, its ability to promote or hinder apoptosis, still remains controversial. In order to elucidate the functional relevance of Daxx in apoptosis signaling of malignant lymphocytes, Jurkat T-cells were stably transfected with a Daxx-expressing vector or with the respective Daxx-negative control vector. We thus demonstrate that ectopic expression of Daxx substantially increases the rate of apoptosis upon incubation with death receptor agonists such as Fas and TRAIL as well as upon incubation with the cytotoxic drug doxorubicin (DOX). Analysis of the molecular changes induced in the extrinsic and intrinsic apoptosis pathways reveals that augmentation of apoptosis by Daxx overexpression is conveyed by distinctly different mechanisms. Although enforced apoptosis caused by ectopic Daxx expression is caspase-dependent in both cases, major differences between Fas/TRAIL-induced apoptosis and doxorubicin-induced apoptosis are observed in expression patterns of X-linked inhibitor of apoptosis (XIAP), p53, Bid, ZIP kinase, and prostate apoptosis response gene 4 (Par-4). Moreover, we could show that addition of a CD95 blocking antibody to the clones treated with doxorubicin was able to increase apoptosis as compared to doxorubicin treatment alone and was accompanied by an enhancement of the mitochondrial branch of apoptosis. In conclusion, we here outline the major molecular mechanisms underlying the apoptosis-promoting effect of Daxx in neoplastic lymphocytes and demonstrate fundamental molecular differences elicited by the overexpression of Daxx in the extrinsic and intrinsic signaling pathways.

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 (atypical CLL, typical CLL, Immunocytoma, Mantle Cell Lymphoma, Prolymphocytic Leukemia (PLL)) 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 significant 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 and cell proliferation signalling including complement lysis inhibitor (clusterin, CLU, SP40), beta-catenin interacting protein (ICAT), peroxisome proliferator activated receptor alpha (PPARα), TNF alpha converting enzyme (ADAM 17 / TACE), homeo box A3 (HOXA), inositol polyphosphate 5 phophatase (PPI 5 PIV, SHIP1), FK 506 binding protein (FKBP 38) and inhibitor of p53 induced apoptosis alpha (NME 6). Clusterin is able to mediate apoptosis via p53 and increases drug-induced cell death when overexpressed as detected in our treated samples. The downregulation of NME 6 during chemotherapeutic treatment may enhance this effect. These results indicate that in vitro chemosensitivity testing and gene expression profiling can successfully be utilised to predict 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.

Background: Aberrant DNA methylation is frequently found in human malignancies including acute myeloid leukemia (AML). While most studies focus on later disease stages, the onset of aberrant DNA methylation events and their dynamics during leukemic progression are largely unknown. Methods: We screened genome-wide for aberrant CpG island methylation in three disease stages of a murine AML model that is driven by hypomorphic expression of the hematopoietic transcription factor PU.1. DNA methylation levels of selected genes were correlated with methylation levels of CD34+ cells and lineage negative, CD127-, c-Kit+, Sca-1+ cells; common myeloid progenitors; granulocyte-macrophage progenitors; and megakaryocyte-erythroid progenitors. Results: We identified 1,184 hypermethylated array probes covering 762 associated genes in the preleukemic stage. During disease progression, the number of hypermethylated genes increased to 5,465 in the late leukemic disease stage. Using publicly available data, we found a significant enrichment of PU.1 binding sites in the preleukemic hypermethylated genes, suggesting that shortage of PU.1 makes PU.1 binding sites in the DNA accessible for aberrant methylation. Many known AML associated genes such as RUNX1 and HIC1 were found among the preleukemic hypermethylated genes. Nine novel hypermethylated genes, FZD5, FZD8, PRDM16, ROBO3, CXCL14, BCOR, ITPKA, HES6 and TAL1, the latter four being potential PU.1 targets, were confirmed to be hypermethylated in human normal karyotype AML patients, underscoring the relevance of the mouse model for human AML. Conclusions: Our study identified early aberrantly methylated genes as potential contributors to onset and progression of AML.