A comprehensive microarray-based DNA methylation study of 367 hematological neoplasms

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

Deregulation of the telomerase reverse transcriptase (TERT) gene by chromosomal translocations in B-cell malignancies

Sequence variants at the TERT-CLPTM1L locus in chromosome 5p have been recently associated with disposition for various cancers. Here we show that this locus including the gene encoding the telomerase reverse-transcriptase TERT at 5p13.33 is rarely but recurrently targeted by somatic chromosomal translocations to IGH and non-IG loci in B-cell neoplasms, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, mantle cell lymphoma and splenic marginal zone lymphoma. In addition, cases with genomic amplification of TERT locus were identified. Tumors bearing chromosomal aberrations involving TERT showed higher TERT transcriptional expression and increased telomerase activity. These data suggest that deregulation of TERT gene by chromosomal abnormalities leading to increased telomerase activity might contribute to B-cell lymphomagenesis

GeneChip analyses point to novel pathogenetic mechanisms in mantle cell lymphoma

The translocation t(11;14)(q13;q32) is the genetic hallmark of mantle cell lymphoma (MCL) but is not sufficient for inducing lymphomagenesis. Here we performed genome-wide 100K GeneChip Mapping in 26 t(11;14)-positive MCL and six MCL cell lines. Partial uniparental disomy (pUPD) was shown to be a recurrent chromosomal event not only in MCL cell lines but also in primary MCL. Remarkably, pUPD affected recurrent targets of deletion like 11q, 13q and 17p. Moreover, we identified 12 novel regions of recurrent gain and loss as well as 12 high-level amplifications and eight homozygously deleted regions hitherto undescribed in MCL. Interestingly, GeneChip analyses identified different genes, encoding proteins involved in microtubule dynamics, such as MAP2, MAP6 and TP53, as targets for chromosomal aberration in MCL. Further investigation, including mutation analyses, fluorescence in situ hybridisation as well as epigenetic and expression studies, revealed additional aberrations frequently affecting these genes. In total, 19 of 20 MCL cases, which were subjected to genetic and epigenetic analyses, and five of six MCL cell lines harboured at least one aberration in MAP2, MAP6 or TP53. These findings provide evidence that alterations of microtubule dynamics might be one of the critical events in MCL lymphomagenesis contributing to chromosomal instability

Characterization of 8p21.3 chromosomal deletions in B-cell lymphoma: TRAIL-R1 and TRAIL-R2 as candidate dosage-dependent tumor suppressor genes

Deletions of chromosome 8p are a recurrent event in B-cell non-Hodgkin lymphoma (B-NHL), suggesting the presence of a tumor suppressor gene. We have characterized these deletions using comparative genomic hybridization to microarrays, fluorescence in situ hybridization (FISH) mapping, DNA sequencing, and functional studies. A minimal deleted region (MDR) of 600 kb was defined in chromosome 8p21.3, with one mantle cell lymphoma cell line (Z138) exhibiting monoallelic deletion of 650 kb. The MDR extended from bacterial artificial chromosome (BAC) clones RP11-382J24 and RP11-109B10 and included the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor gene loci. Sequence analysis of the individual expressed genes within the MDR and DNA sequencing of the entire MDR in Z138 did not reveal any mutation. Gene expression analysis and quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) showed down-regulation of TRAIL-R1 and TRAIL-R2 receptor genes as a consistent event in B-NHL with 8p21.3 loss. Epigenetic inactivation was excluded via promoter methylation analysis. In vitro studies showed that TRAIL-induced apoptosis was dependent on TRAIL-R1 and/or -R2 dosage in most tumors. Resistance to apoptosis of cell lines with 8p21.3 deletion was reversed by restoration of TRAIL-R1 or TRAIL-R2 expression by gene transfection. Our data suggest that TRAIL-R1 and TRAIL-R2 act as dosage-dependent tumor suppressor genes whose monoallelic deletion can impair TRAIL-induced apoptosis in B-cell lymphoma

A cyclin-D1 interaction with BAX underlies its oncogenic role and potential as a therapeutic target in mantle cell lymphoma

The chromosomal translocation t(11;14)(q13;q32) leading to cyclin-D1 overexpression plays an essential role in the development of mantle cell lymphoma (MCL), an aggressive tumor that remains incurable with current treatment strategies. Cyclin-D1 has been postulated as an effective therapeutic target, but the evaluation of this target has been hampered by our incomplete understanding of its oncogenic functions and by the lack of valid MCL murine models. To address these issues, we generated a cyclin-D1-driven mouse model in which cyclin-D1 expression can be regulated externally. These mice developed cyclin-D1-expressing lymphomas capable of recapitulating features of human MCL. We found that cyclin-D1 inactivation was not sufficient to induce lymphoma regression in vivo; however, using a combination of in vitro and in vivo assays, we identified a novel prosurvival cyclin-D1 function in MCL cells. Specifically, we found that cyclin-D1, besides increasing cell proliferation through deregulation of the cell cycle at the G(1)-S transition, sequestrates the proapoptotic protein BAX in the cytoplasm, thereby favoring BCL2's antiapoptotic function. Accordingly, cyclin-D1 inhibition sensitized the lymphoma cells to apoptosis through BAX release. Thus, genetic or pharmacologic targeting of cyclin-D1 combined with a proapoptotic BH3 mimetic synergistically killed the cyclin-D1-expressing murine lymphomas, human MCL cell lines, and primary lymphoma cells. Our study identifies a role of cyclin-D1 in deregulating apoptosis in MCL cells, and highlights the potential benefit of simultaneously targeting cyclin-D1 and survival pathways in patients with MCL. This effective combination therapy also might be exploited in other cyclin-D1-expressing tumors

A Comprehensive Microarray-Based DNA Methylation Study of 367 Hematological Neoplasms

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

Deregulation of the telomerase reverse transcriptase (TERT) gene by chromosomal translocations in B-cell malignancies

Sequence variants at the TERT-CLPTM1L locus in chromosome 5p have been recently associated with disposition for various cancers. Here we show that this locus including the gene encoding the telomerase reverse-transcriptase TERT at 5p13.33 is rarely but recurrently targeted by somatic chromosomal translocations to IGH and non-IG loci in B-cell neoplasms, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, mantle cell lymphoma and splenic marginal zone lymphoma. In addition, cases with genomic amplification of TERT locus were identified. Tumors bearing chromosomal aberrations involving TERT showed higher TERT transcriptional expression and increased telomerase activity. These data suggest that deregulation of TERT gene by chromosomal abnormalities leading to increased telomerase activity might contribute to B-cell lymphomagenesis. Introduction Chromosomal translocations to the immunoglobulin heavy chain (IGH) or light chain (IGL or IGK) loci, and less frequently to non-IG loci represent well known mechanisms of oncogene activation in B-cell neoplasms. 1,2 Many of the genes deregulated by chromosomal translocations in these neoplasms are involved in important cellular processes like cell-cycle control (CCND1, CCND3, BCL6, and CDK6), apoptosis (BCL2), proliferation (MYC), or signal transduction (BCL3 and MALT1). Methods Patient samples The features of B-cell malignancies showing aberrations in the TERT region (5p1) are listed in supplemental Table 1 (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). The study was performed as part of the "Molecular Mechanisms in Malignant Lymphoma" Network Project for which approval from the Institutional Review Board of the Medical Faculity of the Christian-Albrechts-University Kiel has been obtained. FISH Fluorescence in situ hybridization (FISH) was performed on fixed cells from bone marrow, peripheral blood, or lymph node cell suspensions as described previously. 7 Generation of FISH probes as well as FISH procedures are described in supplemental Methods. A list of FISH probes used is shown in supplemental Array CGH to custom-designed arrays The microarrays were designed using the eArray software from Agilent (https://earray.chem.agilent.com/earray/) and the 4 ϫ 44k format. The experimental procedures were performed according to the manufacturer's instructions. Arrays were scanned with the GenePix4000B Scanner (Axon Instruments) and log ratios obtained with the comparative genomic hybridization (CGH) Analytics Version 3.5.14 software (Agilent). All microarray data are available to be viewed at ArrayExpress under accession number E-MEXP-2675 (http://www.ebi.ac.uk/miamexpress/). Quantitative reverse transcription PCR RNA was extracted from tumor cell-containing samples and control tissue, using the RNeasy Mini Kit (QIAGEN) and transcribed into cDNA with the QuantiTect Rev. Transcription Kit (QIAGEN). TERT transcripts were amplified and detected as described elsewhere. 8 TRAP assay The PCR-based telomeric repeat amplification protocol (TRAP) assay was performed as described previously. 9,10 Protein from tumor cell containing samples and control tissue was isolated according to Kim et al. 11 Additional information about materials and methods is available in supplemental Methods. Results and discussion By FISH screening of B-cell neoplasms for translocations affecting the IGH locus we observed a cytogenetically cryptic translocation t(5;14)(p15;q32) involving the IGH locus in 3 cases of chronic lymphocytic leukemia (CLL) and one case of precursor B-cell acute lymphoblastic leukemia (ALL; The IGH locus harbors strong transcriptional enhancers, which can activate oncogenes by translocation on both der(14) and der(5) chromosomes 12 (supplemental To investigate the frequency of rearrangements in the TERT-CLPTM1L region we screened additional cases by FISH (supplemental None of the additional 6 cases with translocations detected by FISH showed juxtaposition to any of the 3 IG loci. Cytogenetically, in 4 of the 6 variant translocations (ie, non-IG), 7p11, 9q31ϳ33, 10q25 and 19p13 were identified as partners of 5p15. In a splenic marginal zone lymphoma (SMZL, case 5) with t(5;7)(p15.33;p11), FISH and tiling array CGH again confirmed a breakpoint centromeric to TERT with loss of CLPTM1L similar to that in the t(5;14)-positive CLL mentioned above ( The inactivation of CLPTM1L by deletion and disruption in 3/10 cases with break at the TERT-CLPTM1L region and also the fact that in precursor B-cell ALL with IGH translocation oncogene activation has always been assigned to the der14 chromosome 13 (supplemental To investigate the effect of the identified chromosomal aberrations on TERT expression, we performed qRT-PCR in cases with t(5;14)/TERT-IGH juxtaposition (n ϭ 3; 1318 NAGEL et al BLOOD, 26 AUGUST 2010 ⅐ VOLUME 116, NUMBER 8 We next assessed whether the up-regulation of TERT mRNA was also associated with increased telomerase activity. Using the TRAP assay, cases of CLL (cases 1 and 6) and MCL (cases 8 and 9) with chromosomal breakpoints at the TERT-CLPTM1L locus showed markedly higher telomerase activity compared with appropriate disease and tissue controls ( Taken together, we show that the TERT-CLPTM1L region in 5p15 is recurrently targeted by chromosomal translocations in B-cell neoplasms. Based on the finding that CLPTM1L is recurrently lost or disrupted by the mentioned aberrations, it does not seem to contribute to the process of lymphomagenesis. Interestingly, germ line sequence variants in the TERT-CLPTM1L region were recently shown to be associated with predisposition for several cancer types. 14-17 All 6 additional translocations and both amplifications identified herein were of somatic origin as cells without the aberration were always present. The pattern of IGH translocations as well as the increased TERT expression and telomerase activation in cases with 5p15 translocations strongly suggest TERT to be the candidate gene involved in lymphomagenesis. Telomerase activity, which is clearly detectable in up to 90% of human tumors, including hematologic neoplasias, but not in most normal somatic cells, is in part explained by changes in chromatin structure or amplification of the TERT locus

Defining the prognosis of early stage chronic lymphocytic leukaemia patients

Approximately 70% of chronic lymphocytic leukaemia (CLL) patients present with early stage disease, therefore defining which patients will progress and require treatment is a major clinical challenge. Here, we present the largest study of prognostic markers ever carried out in Binet stage A patients (n = 1154) with a median follow-up of 8 years. We assessed the prognostic impact of lymphocyte doubling time (LDT), immunoglobulin gene (IGHV) mutation status, CD38 expression, ZAP-70 expression and fluorescence in situ hybridization (FISH) cytogenetics with regards to time to first treatment (TTFT) and overall survival (OS). Univariate analysis revealed LDT as the most prognostic parameter for TTFT, with IGHV mutation status most prognostic for OS. CD38 expression, ZAP-70 expression and FISH were also prognostic variables; combinations of these markers increased prognostic power in concordant cases. Multivariate analysis revealed that only LDT, IGHV mutation status, CD38 and age at diagnosis were independent prognostic variables for TTFT and OS. Therefore, IGHV mutation status and CD38 expression have independent prognostic value in early stage CLL and should be performed as part of the routine diagnostic workup. ZAP-70 expression and FISH were not independent prognostic markers in early stage disease and can be omitted at diagnosis but FISH analysis should be undertaken at disease progression to direct treatment strategy

Deregulation of the telomerase reverse transcriptase (TERT) gene by chromosomal translocations in B-cell malignancies

Sequence variants at the TERT-CLPTM1L locus in chromosome 5p have been recently associated with disposition for various cancers. Here we show that this locus including the gene encoding the telomerase reverse-transcriptase TERT at 5p13.33 is rarely but recurrently targeted by somatic chromosomal translocations to IGH and non-IG loci in B-cell neoplasms, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, mantle cell lymphoma and splenic marginal zone lymphoma. In addition, cases with genomic amplification of TERT locus were identified. Tumors bearing chromosomal aberrations involving TERT showed higher TERT transcriptional expression and increased telomerase activity. These data suggest that deregulation of TERT gene by chromosomal abnormalities leading to increased telomerase activity might contribute to B-cell lymphomagenesis