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1.  BAALC expression: a suitable marker for prognostic risk stratification and detection of residual disease in cytogenetically normal acute myeloid leukemia 
Blood Cancer Journal  2014;4(1):e173-.
High brain and acute leukemia, cytoplasmic (BAALC) expression defines an important risk factor in cytogenetically normal acute myeloid leukemia (CN-AML). The prognostic value of BAALC expression in relation to other molecular prognosticators was analyzed in 326 CN-AML patients (<65 years). At diagnosis, high BAALC expression was associated with prognostically adverse mutations: FLT3 internal tandem duplication (FLT3-ITD) with an FLT3-ITD/FLT3 wild-type (wt) ratio of ⩾0.5 (P=0.001), partial tandem duplications within the MLL gene (MLL-PTD) (P=0.002), RUNX1 mutations (mut) (P<0.001) and WT1mut (P=0.001), while it was negatively associated with NPM1mut (P<0.001). However, high BAALC expression was also associated with prognostically favorable biallelic CEBPA (P=0.001). Survival analysis revealed an independent adverse prognostic impact of high BAALC expression on overall survival (OS) and event-free survival (EFS), and also on OS when eliminating the effect of allogeneic stem cell transplantation (SCT) (OSTXcens). Furthermore, we analyzed BAALC expression in 416 diagnostic and follow-up samples of 66 patients. During follow-up, BAALC expression correlated with mutational load or expression levels, respectively, of other minimal residual disease markers: FLT3-ITD (r=0.650, P<0.001), MLL-PTD (r=0.728, P<0.001), NPM1mut (r=0.599, P<0.001) and RUNX1mut (r=0.889, P<0.001). Moreover, a reduction in BAALC expression after the second cycle of induction chemotherapy was associated with improved EFS. Thus, our data underline the utility of BAALC expression as a marker for prognostic risk stratification and detection of residual disease in CN-AML.
doi:10.1038/bcj.2013.71
PMCID: PMC3913940  PMID: 24413067
BAALC expression; CN-AML; prognosis; MRD
2.  BRAF MUTATIONS IN HAIRY CELL LEUKEMIA 
The New England journal of medicine  2011;364(24):2305-2315.
Background
Hairy cell leukemia (HCL) is a well defined clinico-pathological entity whose underlying genetic lesion is still obscure.
Methods
We searched for HCL-associated mutations by massively parallel sequencing of the whole exome of leukemic and matched normal mononuclear cells purified from the peripheral blood of one patient with HCL.
Results
Whole exome sequencing identified 5 missense somatic clonal mutations that were confirmed at Sanger sequencing, including a heterozygous V600E mutation involving the BRAF gene. Since the BRAF V600E mutation is oncogenic in other tumors, further analyses were focused on this genetic lesion. Sanger sequencing detected mutated BRAF in 46/46 additional HCL patients (47/47 including the index case; 100%). None of the 193 peripheral B-cell lymphomas/leukemias other than HCL that were investigated carried the BRAF V600E mutation, including 36 cases of splenic marginal zone lymphomas and unclassifiable splenic lymphomas/leukemias. Immunohistological and Western blot studies showed that HCL cells express phospho-MEK and phospho-ERK (the downstream targets of the BRAF kinase), indicating a constitutive activation of the RAF-MEK-ERK mitogen-activated protein kinase pathway in HCL. In vitro incubation of BRAF-mutated primary leukemic cells from 5 HCL patients with PLX-4720, a specific inhibitor of active BRAF, led to marked decrease of phosphorylated ERK and MEK.
Conclusions
The BRAF V600E mutation was present in all HCL patients investigated. This finding may have relevant implications for the pathogenesis, diagnosis and targeted therapy of HCL (Funded by the Associazione Italiana Ricerca Cancro and others).
doi:10.1056/NEJMoa1014209
PMCID: PMC3689585  PMID: 21663470
3.  Frequency and Prognostic Impact of CEBPA Proximal, Distal and Core Promoter Methylation in Normal Karyotype AML: A Study on 623 Cases 
PLoS ONE  2013;8(2):e54365.
The clinical impact of aberrant CEBPA promoter methylation (PM) in AML is controversially discussed. The aim of this study was to clarify the significance of aberrant CEBPA PM with regard to clinical features in a cohort of 623 cytogenetically normal (CN) de novo AML. 555 cases had wild-type CEBPA, 68 cases harbored CEBPA mutations. The distal promoter was methylated in 238/623 cases (38.2%), the core promoter in 8 of 326 cases (2.5%), whereas proximal PM was never detected. CEBPA PM and CEBPA mutations were mutually exclusive. CEBPA distal PM positive cases were characterized by reduced CEBPA mRNA expression levels and elevated white blood cell counts. CEBPA distal PM was less frequent in patients with mutations in FLT3, NPM1 and TET2 and more frequent in cases with RUNX1 and IDH2R140 mutations. Overall, no association of methylation to prognosis was seen. However CEBPA distal PM was associated with inferior outcome in cases with low FLT3-ITD ratio or TET2 mutations. A distinct gene expression profile of CEBPA distal PM positive cases compared to CEBPA mutated and CEBPA distal PM negative cases was observed. In conclusion, the presence of aberrant CEBPA PM is associated with distinct biological features but impact on outcome is weak.
doi:10.1371/journal.pone.0054365
PMCID: PMC3562230  PMID: 23383300
4.  Outcome of elderly patients with acute promyelocytic leukemia: results of the German Acute Myeloid Leukemia Cooperative Group 
Annals of Hematology  2012;92(1):41-52.
Despite improvement of prognosis, older age remains a negative prognostic factor in acute promyelocytic leukemia (APL). Reports on disease characteristics and outcome of older patients are conflicting. We therefore analyzed 91 newly diagnosed APL patients aged 60 years or older (30 % of 305 adults with APL) registered by the German AML Cooperative Group (AMLCG) since 1994; 68 patients (75 %) were treated in studies, 23 (25 %) were non-eligible, and 31 % had high-risk APL. Fifty-six patients received induction therapy with all-trans retinoic acid and TAD (6-thioguanine, cytarabine, daunorubicin), and consolidation and maintenance therapy. Treatment intensification with a second induction cycle (high dose cytarabine, mitoxantrone; HAM) was optional (n = 14). Twelve patients were randomized to another therapy not considered in this report. The early death rate was 48 % in non-eligible and 19 % in study patients. With the AMLCG regimen, 7-year overall, event-free and relapse-free survival (RFS) and cumulative incidence of relapse were 45 %, 40 %, 48 %, and 24 %, respectively. In patients treated with TAD–HAM induction, 7-year RFS was superior (83 %; p = 0.006) compared to TAD only, and no relapse was observed. In our registered elderly patients, we see a high rate of non-eligibility for treatment in studies and of high-risk APL. In patients who can undergo a curative approach, intensified chemotherapy is highly effective, but is restricted to a selection of patients. Therefore, new less toxic treatment approaches with broader applicability are needed. Elderly patients might be a particular target group for concepts with arsenic trioxide.
doi:10.1007/s00277-012-1597-9
PMCID: PMC3536950  PMID: 23090499
Acute promyelocytic leukemia; Elderly patients; Early death; Treatment
7.  Gene expression profiling for the diagnosis of acute leukaemia 
British Journal of Cancer  2006;96(4):535-540.
An optimised diagnostic setting in acute leukaemias combines cytomorphology and cytochemistry, multiparameter immunophenotyping, cytogenetics, fluorescence in situ hybridisation, and polymerase chain reaction (PCR)-based assays. This allows classification and definition of biologically defined and prognostically relevant subtypes, and allows directed treatment in some subentities. Over the last years the microarray technology has helped to quantify simultaneously the expression status of ten thousands of genes in single experiments. This novel approach will hopefully become an essential tool for the molecular classification of acute leukaemias in the near future. It can be anticipated that new biologically defined and clinically relevant subtypes of leukaemia will be identified based on their unique gene expression profiles. This method may therefore guide therapeutic decisions and should be investigated in a diagnostic setting in parallel to established standard methods.
doi:10.1038/sj.bjc.6603495
PMCID: PMC2360048  PMID: 17146476
microarray analysis; gene expression profiling; acute leukaemia; diagnosis
8.  The diagnosis of BCR/ABL-negative chronic myeloproliferative diseases (CMPD): a comprehensive approach based on morphology, cytogenetics, and molecular markers 
Annals of Hematology  2007;87(1):1-10.
Recent years showed significant progress in the molecular characterization of the chronic myeloproliferative disorders (CMPD) which are classified according to the WHO classification of 2001 as polycythemia vera (PV), chronic idiopathic myelofibrosis (CIMF), essential thrombocythemia (ET), CMPD/unclassifiable (CMPD-U), chronic neutrophilic leukemia, and chronic eosinophilic leukemia (CEL)/hypereosinophilic syndrome, all to be delineated from BCR/ABL-positive chronic myeloid leukemia (CML). After 2001, the detection of the high frequency of the JAK2V617F mutation in PV, CIMF, and ET, and of the FIP1L1–PDGFRA fusion gene in CEL further added important information in the diagnosis of CMPD. These findings also enhanced the importance of tyrosine kinase mutations in CMPD and paved the way to a more detailed classification and to an improved definition of prognosis using also novel minimal residual disease (MRD) markers. Simultaneously, the broadening of therapeutic strategies in the CMPD, e.g., due to reduced intensity conditioning in allogeneic hematopoietic stem cell transplantation and the introduction of tyrosine kinase inhibitors in CML, in CEL, and in other ABL and PDGRFB rearrangements, increased the demands to diagnostics. Therefore, today, a multimodal diagnostic approach combining cytomorphology, cytogenetics, and individual molecular methods is needed in BCR/ABL-negative CMPD. A stringent diagnostic algorithm for characterization, choice of treatment, and monitoring of MRD will be proposed in this review.
doi:10.1007/s00277-007-0403-6
PMCID: PMC2082654  PMID: 17938925
CMPD; BCR/ABL; Molecular marker; Cytomorphology
9.  Landscape of genetic lesions in 944 patients with myelodysplastic syndromes 
Leukemia  2013;28(2):241-247.
High-throughput DNA sequencing significantly contributed to diagnosis and prognostication in patients with myelodysplastic syndromes (MDS). We determined the biological and prognostic significance of genetic aberrations in MDS. In total, 944 patients with various MDS subtypes were screened for known/putative mutations/deletions in 104 genes using targeted deep sequencing and array-based genomic hybridization. In total, 845/944 patients (89.5%) harbored at least one mutation (median, 3 per patient; range, 0–12). Forty-seven genes were significantly mutated with TET2, SF3B1, ASXL1, SRSF2, DNMT3A, and RUNX1 mutated in >10% of cases. Many mutations were associated with higher risk groups and/or blast elevation. Survival was investigated in 875 patients. By univariate analysis, 25/48 genes (resulting from 47 genes tested significantly plus PRPF8) affected survival (P<0.05). The status of 14 genes combined with conventional factors revealed a novel prognostic model (‘Model-1') separating patients into four risk groups (‘low', ‘intermediate', ‘high', ‘very high risk') with 3-year survival of 95.2, 69.3, 32.8, and 5.3% (P<0.001). Subsequently, a ‘gene-only model' (‘Model-2') was constructed based on 14 genes also yielding four significant risk groups (P<0.001). Both models were reproducible in the validation cohort (n=175 patients; P<0.001 each). Thus, large-scale genetic and molecular profiling of multiple target genes is invaluable for subclassification and prognostication in MDS patients.
doi:10.1038/leu.2013.336
PMCID: PMC3918868  PMID: 24220272
next-generation sequencing; molecular markers; myelodysplastic syndromes; prognostic score
10.  An international study of intrachromosomal amplification of chromosome 21 (iAMP21): cytogenetic characterization and outcome 
Leukemia  2013;28(5):1015-1021.
Intrachromosomal amplification of chromosome 21 (iAMP21) defines a distinct cytogenetic subgroup of childhood B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). To date, fluorescence in situ hybridisation (FISH), with probes specific for the RUNX1 gene, provides the only reliable detection method (five or more RUNX1 signals per cell). Patients with iAMP21 are older (median age 9 years) with a low white cell count. Previously, we demonstrated a high relapse risk when these patients were treated as standard risk. Recent studies have shown improved outcome on intensive therapy. In view of these treatment implications, accurate identification is essential. Here we have studied the cytogenetics and outcome of 530 iAMP21 patients that highlighted the association of specific secondary chromosomal and genetic changes with iAMP21 to assist in diagnosis, including the gain of chromosome X, loss or deletion of chromosome 7, ETV6 and RB1 deletions. These iAMP21 patients when treated as high risk showed the same improved outcome as those in trial-based studies regardless of the backbone chemotherapy regimen given. This study reinforces the importance of intensified treatment to reduce the risk of relapse in iAMP21 patients. This now well-defined patient subgroup should be recognised by World Health Organisation (WHO) as a distinct entity of BCP-ALL.
doi:10.1038/leu.2013.317
PMCID: PMC4283797  PMID: 24166298
iAMP21; genetics; outcome; poor prognosis; BCP-ALL; chromosomal abnormalities
11.  Selective BCL-2 Inhibition by ABT-199 Causes On Target Cell Death in Acute Myeloid Leukemia 
Cancer discovery  2013;4(3):362-375.
B-cell leukemia/lymphoma 2 (BCL-2) prevents commitment to programmed cell death at the mitochondrion. It remains a challenge to identify those tumors that are best treated by inhibition of BCL-2. Here we demonstrate that acute myeloid leukemia (AML) cell lines, primary patient samples, and murine primary xenografts are very sensitive to treatment with the selective BCL-2 antagonist ABT-199. In primary patient cells, the median IC50 was approximately 10 nM, and cell death occurred within 2 h. Our ex vivo sensitivity results compare favorably with those observed for chronic lymphocytic leukemia (CLL), a disease for which ABT-199 has demonstrated consistent activity in clinical trials. Moreover, mitochondrial studies using BH3 profiling demonstrate activity at the mitochondrion that correlates well with cytotoxicity, supporting an on target mitochondrial mechanism of action. Our protein and BH3 profiling studies provide promising tools that can be tested as predictive biomarkers in any clinical trial of ABT-199 in AML.
doi:10.1158/2159-8290.CD-13-0609
PMCID: PMC3975047  PMID: 24346116
AML; apoptosis; BCL-2; targeted therapy; BH3 profiling
12.  Contemporary consensus proposal on criteria and classification of eosinophilic disorders and related syndromes 
Eosinophilia is an important indicator of various neoplastic and nonneoplastic conditions. Depending on the underlying disease and mechanisms, eosinophil infiltration can lead to organ dysfunction, clinical symptoms, or both. During the past 2 decades, several different classifications of eosinophilic disorders and related syndromes have been proposed in various fields of medicine. Although criteria and definitions are, in part, overlapping, no global consensus has been presented to date. The Year 2011 Working Conference on Eosinophil Disorders and Syndromes was organized to update and refine the criteria and definitions for eosinophilic disorders and to merge prior classifications in a contemporary multidisciplinary schema. A panel of experts from the fields of immunology, allergy, hematology, and pathology contributed to this project. The expert group agreed on unifying terminologies and criteria and a classification that delineates various forms of hypereosinophilia, including primary and secondary variants based on specific hematologic and immunologic conditions, and various forms of the hypereosinophilic syndrome. For patients in whom no underlying disease or hypereosinophilic syndrome is found, the term hypereosinophilia of undetermined significance is introduced. The proposed novel criteria, definitions, and terminologies should assist in daily practice, as well as in the preparation and conduct of clinical trials.
doi:10.1016/j.jaci.2012.02.019
PMCID: PMC4091810  PMID: 22460074
Hypereosinophilic syndrome; eosinophilic leukemia; criteria; classification; hypereosinophilia of undetermined significance
13.  Pathogenesis and classification of eosinophil disorders: a review of recent developments in the field 
Expert review of hematology  2012;5(2):157-176.
Eosinophils and their products play an essential role in the pathogenesis of various reactive and neoplastic disorders. Depending on the underlying disease, molecular defect and involved cytokines, hypereosinophilia may develop and may lead to organ damage. In other patients, persistent eosinophilia is accompanied by typical clinical findings, but the causative role and impact of eosinophilia remain uncertain. For patients with eosinophil-mediated organ pathology, early therapeutic intervention with agents reducing eosinophil counts can be effective in limiting or preventing irreversible organ damage. Therefore, it is important to approach eosinophil disorders and related syndromes early by using established criteria, to perform all appropriate staging investigations, and to search for molecular targets of therapy. In this article, we review current concepts in the pathogenesis and evolution of eosinophilia and eosinophil-related organ damage in neoplastic and non-neoplastic conditions. In addition, we discuss classifications of eosinophil disorders and related syndromes as well as diagnostic algorithms and standard treatment for various eosinophil-related disorders.
doi:10.1586/ehm.11.81
PMCID: PMC3625626  PMID: 22475285
classification; eosinophilic leukemia; FIP1L1-PDGFRA; hypereosinophilia; hypereosinophilic syndromes; targeted therapy
14.  Older patients with normal karyotype acute myeloid leukemia have a higher rate of genomic changes compared to young patients as determined by SNP array analysis 
Leukemia Research  2011;36(4):467-473.
Older patients with AML have a worse outcome compared to young patients. To study for potential contributors to their poor prognosis, we compared two NK-AML cohorts, young (< 60 years old) and old (> 60 years old), via high density SNP array analysis. Older patients had more genomic changes (1.83±0.23 vs. 1.16±0.2, p=0.037) and a trend for a higher number of copy number neutral loss of heterozygosity (0.5±0.2 vs. 0.24±0.08, p=0.088) compared to young patients. We speculate that complex genomic changes in NK-AML may be a sign of an increase in genomic instability and an indicator of a worse prognosis.
doi:10.1016/j.leukres.2011.10.013
PMCID: PMC3288295  PMID: 22071139
AML; Normal karyotype; SNP array; Old age
15.  Recurrent SETBP1 mutations in atypical chronic myeloid leukemia 
Nature genetics  2012;45(1):18-24.
Atypical chronic myeloid leukemia (aCML) shares clinical and laboratory features with CML, but it lacks the BCR-ABL1 fusion. We performed exome sequencing of eight aCMLs and identified somatic alterations of SETBP1 (encoding a p.Gly870Ser alteration) in two cases. Targeted resequencing of 70 aCMLs, 574 diverse hematological malignancies and 344 cancer cell lines identified SETBP1 mutations in 24 cases, including 17 of 70 aCMLs (24.3%; 95% confidence interval (CI) = 16–35%). Most mutations (92%) were located between codons 858 and 871 and were identical to changes seen in individuals with Schinzel-Giedion syndrome. Individuals with mutations had higher white blood cell counts (P = 0.008) and worse prognosis (P = 0.01). The p.Gly870Ser alteration abrogated a site for ubiquitination, and cells exogenously expressing this mutant exhibited higher amounts of SETBP1 and SET protein, lower PP2A activity and higher proliferation rates relative to those expressing the wild-type protein. In summary, mutated SETBP1 represents a newly discovered oncogene present in aCML and closely related diseases.
doi:10.1038/ng.2495
PMCID: PMC3588142  PMID: 23222956
16.  Protein Kinase C-β-Dependent Activation of NF-κB in Stromal Cells Is Indispensable for the Survival of Chronic Lymphocytic Leukemia B Cells In Vivo 
Cancer Cell  2013;23(1):77-92.
Summary
Tumor cell survival critically depends on heterotypic communication with benign cells in the microenvironment. Here, we describe a survival signaling pathway activated in stromal cells by contact to B cells from patients with chronic lymphocytic leukemia (CLL). The expression of protein kinase C (PKC)-βII and the subsequent activation of NF-κB in bone marrow stromal cells are prerequisites to support the survival of malignant B cells. PKC-β knockout mice are insusceptible to CLL transplantations, underscoring the in vivo significance of the PKC-βII-NF-κB signaling pathway in the tumor microenvironment. Upregulated stromal PKC-βII in biopsies from patients with CLL, acute lymphoblastic leukemia, and mantle cell lymphoma suggests that this pathway may commonly be activated in a variety of hematological malignancies.
Highlights
► Malignant B cells induce the expression of PKC-βII in bone marrow stromal cells ► The activation of NF-κB in tumor stromal cells strictly depends on PKC-βII ► The PKC-βII-NF-κB pathway is indispensable for survival of malignant B cells in vivo ► The PKC-βII-NF-κB pathway is activated by ALL and mantle cell lymphoma cells
doi:10.1016/j.ccr.2012.12.003
PMCID: PMC3546417  PMID: 23328482
17.  ICON: Eosinophil Disorders 
doi:10.1097/WOX.0b013e31827f4192
PMCID: PMC3651188  PMID: 23282419
eosinophil disorders; hypereosinophilic syndrome (HES); global consensus; classification
18.  Molecular Diagnostics, Targeted Therapy, and the Indication for Allogeneic Stem Cell Transplantation in Acute Lymphoblastic Leukemia 
Advances in Hematology  2011;2011:154745.
In recent years, the panel of known molecular mutations in acute lymphoblastic leukemia (ALL) has been continuously increased. In Philadelphia-positive ALL, deletions of the IKZF1 gene were identified as prognostically adverse factors. These improved insights in the molecular background and the clinical heterogeneity of distinct cytogenetic subgroups may allow most differentiated therapeutic decisions, for example, with respect to the indication to allogeneic HSCT within genetically defined ALL subtypes. Quantitative real-time PCR allows highly sensitive monitoring of the minimal residual disease (MRD) load, either based on reciprocal gene fusions or immune gene rearrangements. Molecular diagnostics provided the basis for targeted therapy concepts, for example, combining the tyrosine kinase inhibitor imatinib with chemotherapy in patients with Philadelphia-positive ALL. Screening for BCR-ABL1 mutations in Philadelphia-positive ALL allows to identify patients who may benefit from second-generation tyrosine kinase inhibitors or from novel compounds targeting the T315I mutation. Considering the central role of the molecular techniques for the management of patients with ALL, efforts should be made to facilitate and harmonize immunophenotyping, cytogenetics, and molecular mutation screening. Furthermore, the potential of high-throughput sequencing should be evaluated for diagnosis and follow-up of patients with B-lineage ALL.
doi:10.1155/2011/154745
PMCID: PMC3216286  PMID: 22110503
19.  Deletion of the protein tyrosine phosphatase gene PTPN2 in T-cell acute lymphoblastic leukemia 
Nature genetics  2010;42(6):530-535.
PTPN2 (protein tyrosine phosphatase non-receptor type 2, also known as TC-PTP) is a cytosolic tyrosine phosphatase that functions as a negative regulator of a variety of tyrosine kinases and other signaling proteins.1–3 In agreement with its role in the regulation of the immune system, PTPN2 was identified as a susceptibility locus for autoimmune diseases.4,5 In this work, we describe the identification of focal deletions of PTPN2 in human T-cell acute lymphoblastic leukemia (T-ALL). Deletion of PTPN2 was specifically found in T-ALLs with aberrant expression of the TLX1 transcription factor oncogene,6 including four cases also expressing the NUP214-ABL1 tyrosine kinase.7 Knockdown of PTPN2 expression increased the proliferation and cytokine sensitivity of T-ALL cells. In addition, PTPN2 was identified as a negative regulator of NUP214-ABL1 kinase activity. Our study provides genetic and functional evidence for a tumor suppressor role of PTPN2, and suggests that expression levels of PTPN2 may modulate response to treatment.
doi:10.1038/ng.587
PMCID: PMC2957655  PMID: 20473312
tumor suppressor gene; cancer; phosphorylation
20.  Quantitative comparison of microarray experiments with published leukemia related gene expression signatures 
BMC Bioinformatics  2009;10:422.
Background
Multiple gene expression signatures derived from microarray experiments have been published in the field of leukemia research. A comparison of these signatures with results from new experiments is useful for verification as well as for interpretation of the results obtained. Currently, the percentage of overlapping genes is frequently used to compare published gene signatures against a signature derived from a new experiment. However, it has been shown that the percentage of overlapping genes is of limited use for comparing two experiments due to the variability of gene signatures caused by different array platforms or assay-specific influencing parameters. Here, we present a robust approach for a systematic and quantitative comparison of published gene expression signatures with an exemplary query dataset.
Results
A database storing 138 leukemia-related published gene signatures was designed. Each gene signature was manually annotated with terms according to a leukemia-specific taxonomy. Two analysis steps are implemented to compare a new microarray dataset with the results from previous experiments stored and curated in the database. First, the global test method is applied to assess gene signatures and to constitute a ranking among them. In a subsequent analysis step, the focus is shifted from single gene signatures to chromosomal aberrations or molecular mutations as modeled in the taxonomy. Potentially interesting disease characteristics are detected based on the ranking of gene signatures associated with these aberrations stored in the database. Two example analyses are presented. An implementation of the approach is freely available as web-based application.
Conclusions
The presented approach helps researchers to systematically integrate the knowledge derived from numerous microarray experiments into the analysis of a new dataset. By means of example leukemia datasets we demonstrate that this approach detects related experiments as well as related molecular mutations and may help to interpret new microarray data.
doi:10.1186/1471-2105-10-422
PMCID: PMC2803858  PMID: 20003504
21.  A Comprehensive Microarray-Based DNA Methylation Study of 367 Hematological Neoplasms 
PLoS ONE  2009;4(9):e6986.
Background
Alterations in the DNA methylation pattern are a hallmark of leukemias and lymphomas. However, most epigenetic studies in hematologic neoplasms (HNs) have focused either on the analysis of few candidate genes or many genes and few HN entities, and comprehensive studies are required.
Methodology/Principal Findings
Here, we report for the first time a microarray-based DNA methylation study of 767 genes in 367 HNs diagnosed with 16 of the most representative B-cell (n = 203), T-cell (n = 30), and myeloid (n = 134) neoplasias, as well as 37 samples from different cell types of the hematopoietic system. Using appropriate controls of B-, T-, or myeloid cellular origin, we identified a total of 220 genes hypermethylated in at least one HN entity. In general, promoter hypermethylation was more frequent in lymphoid malignancies than in myeloid malignancies, being germinal center mature B-cell lymphomas as well as B and T precursor lymphoid neoplasias those entities with highest frequency of gene-associated DNA hypermethylation. We also observed a significant correlation between the number of hypermethylated and hypomethylated genes in several mature B-cell neoplasias, but not in precursor B- and T-cell leukemias. Most of the genes becoming hypermethylated contained promoters with high CpG content, and a significant fraction of them are targets of the polycomb repressor complex. Interestingly, T-cell prolymphocytic leukemias show low levels of DNA hypermethylation and a comparatively large number of hypomethylated genes, many of them showing an increased gene expression.
Conclusions/Significance
We have characterized the DNA methylation profile of a wide range of different HNs entities. As well as identifying genes showing aberrant DNA methylation in certain HN subtypes, we also detected six genes—DBC1, DIO3, FZD9, HS3ST2, MOS, and MYOD1—that were significantly hypermethylated in B-cell, T-cell, and myeloid malignancies. These might therefore play an important role in the development of different HNs.
doi:10.1371/journal.pone.0006986
PMCID: PMC2737286  PMID: 19750229
22.  An international standardization programme towards the application of gene expression profiling in routine leukaemia diagnostics: the Microarray Innovations in LEukemia study prephase 
British Journal of Haematology  2008;142(5):802-807.
Gene expression profiling has the potential to enhance current methods for the diagnosis of haematological malignancies. Here, we present data on 204 analyses from an international standardization programme that was conducted in 11 laboratories as a prephase to the Microarray Innovations in LEukemia (MILE) study. Each laboratory prepared two cell line samples, together with three replicate leukaemia patient lysates in two distinct stages: (i) a 5-d course of protocol training, and (ii) independent proficiency testing. Unsupervised, supervised, and r2 correlation analyses demonstrated that microarray analysis can be performed with remarkably high intra-laboratory reproducibility and with comparable quality and reliability.
doi:10.1111/j.1365-2141.2008.07261.x
PMCID: PMC2654477  PMID: 18573112
microarray; gene expression profiling; leukaemia; standardization; diagnostics
23.  Early prediction of therapy response in patients with acute myeloid leukemia by nucleosomal DNA fragments 
BMC Cancer  2006;6:143.
Background
Elevated levels of nucleosomal DNA fragments can be detected in plasma and sera of patients with malignant diseases.
Methods
We investigated the course of nucleosomal DNA, thymidine kinase, lactate dehydrogenase and leukocytes in sera of 25 patients with acute myeloid leukemia during the first cycle of induction chemotherapy and tested their power to distinguish between patients with complete remission and those with no remission.
Results
Almost all patients showed strongly decreasing levels of nucleosomal DNA during the first week, in some cases after initial peaks. In overall analysis of variance, DNA levels could clearly distinguish between patients with complete remission, who had higher DNA values, and those with insufficient response (p = 0.017). The area under the curve of DNA values of days 2–4 after start of therapy (AUC 2–4) discriminated between both groups with a sensitivity of 56% at a specificity of 100%. Further, pretherapeutic levels and AUC 2–4 of nucleosomal DNA correlated significantly with blast reduction after 16 days. A tendency to higher levels in patients with complete response was also found for thymidine kinase, lactate dehydrogenase and leukocytes, however the difference did not reach the level of significance (p = 0.542, p = 0.260, and p = 0.144, respectively).
Conclusion
Our results indicate that nucleosomal DNA fragments are valuable markers for the early prediction of therapeutic efficacy in patients with acute myeloid leukemia.
doi:10.1186/1471-2407-6-143
PMCID: PMC1555596  PMID: 16734907
24.  Bioinformatics for Medical Diagnostics: Assessment of Microarray Data in the Context of Clinical Databases 
Motivation
To identify genes suitable for medical diagnostics microarray data is assessed in the context of clinical databases, which store complex information about the patient phenotype. The wealth of data and lack of standards make it difficult to analyse this kind of data.
Results
We present a workflow for exploratory analysis of microarray data together with clinical data consisting of four steps: definition of clinically meaningful research questions in a masterfile, generation of analysis files, selection and characterization of differentially expressed genes, and estimation of classification accuracy. We applied this workflow to large data sets from the field of cardiology and oncology (n~500 patients).
Systematic data management of microarray data and clinical data helps to make results more transparent and comparable.
PMCID: PMC1480146  PMID: 14728164
data management; DNA microarray; medical diagnostics; clinical database; gene expression
25.  Effective mobilisation of peripheral blood progenitor cells with 'Dexa-BEAM' and G-CSF: timing of harvesting and composition of the leukapheresis product. 
British Journal of Cancer  1993;68(5):950-957.
The mini-BEAM regimen (BCNU, etoposide, cytarabine, melphalan) and its modification 'Dexa-BEAM' are effective salvage protocols for relapsed Hodgkin's disease and non-Hodgkin's lymphoma. Since many patients with relapsed lymphoma are eligible for high-dose chemotherapy with autologous stem cell rescue, we were interested in the suitability of these second line regimens for mobilising peripheral blood progenitor cells (PBPC). The kinetics of PBPC were studied in 15 patients treated with Dexa-BEAM and granulocyte colony-stimulating factor (G-CSF). Leukocytes started to rise from < 0.5 nL-1 on day 18 (16-22) after Dexa-BEAM, and exceeded 10 nL-1 on day 20 (18-28). Peripheral blood CFU-GM peaked on day 21 (19-28) and declined slowly thereafter; the median leukocyte count was 18.7 nL-1 (12.2-60) on the day of CFU-GM-peak. The maximum number of CFU-GM circulating in peripheral blood was inversely correlated to the duration of leukopenia after Dexa-BEAM. Measurement of CD34+ cells with the monoclonal antibody 8G12-PE (HPCA-2) predicted the number of CFU-GM precisely in both peripheral blood and leukapheresis products (r = 0.90-0.95). Two to six leukapheresis procedures yielded 6.39 x 10(8) mononuclear cells kg-1 (1.82-13.49) containing 44.4 x 10(4) CFU-GM kg-1 (2.2-213.8). Immunophenotypical analysis revealed that the percentage of CD19+ B cells was very low in all collection products (less than 1%). Nine patients were autografted with PBPC (15.4-213.8 x 10(4) CFU-GM kg-1) after myeloablative chemotherapy and experienced rapid and sustained engraftment (Platelets > 50 nL-1 on day +13 [9-22]). We conclude that PBPC can be mobilised effectively by Dexa-BEAM plus G-CSF. An adequate timing of PBPC collection (when the leukocyte count has exceeded 10 nL-1) and evaluation of the progenitor content of the leukapheresis products with 8G12-PE will allow to minimise the number of leukaphereses.
PMCID: PMC1968738  PMID: 7692921

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