Hairy cell leukemia (HCL) is a well defined clinico-pathological entity whose underlying genetic lesion is still obscure.
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.
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.
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).
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.
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.
Acute promyelocytic leukemia; Elderly patients; Early death; Treatment
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.
microarray analysis; gene expression profiling; acute leukaemia; diagnosis
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.
CMPD; BCR/ABL; Molecular marker; Cytomorphology
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.
classification; eosinophilic leukemia; FIP1L1-PDGFRA; hypereosinophilia; hypereosinophilic syndromes; targeted therapy
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.
AML; Normal karyotype; SNP array; Old age
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.
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.
► 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
eosinophil disorders; hypereosinophilic syndrome (HES); global consensus; classification
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.
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.
tumor suppressor gene; cancer; phosphorylation
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.
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.
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.
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.
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.
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.
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.
microarray; gene expression profiling; leukaemia; standardization; diagnostics
Elevated levels of nucleosomal DNA fragments can be detected in plasma and sera of patients with malignant diseases.
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.
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).
Our results indicate that nucleosomal DNA fragments are valuable markers for the early prediction of therapeutic efficacy in patients with acute myeloid leukemia.
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.
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.
data management; DNA microarray; medical diagnostics; clinical database; gene expression
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.