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Find video protocols related to scientific articles indexed in Pubmed.
Limited duration of complete remission on ruxolitinib in myeloid neoplasms with PCM1-JAK2 and BCR-JAK2 fusion genes.
Ann. Hematol.
PUBLISHED: 09-11-2014
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Rearrangements of chromosome band 9p24 are known to be associated with JAK2 fusion genes, e.g., t(8;9)(p22;p24) with a PCM1-JAK2 and t(9;22)(p24;q11) with a BCR-JAK2 fusion gene, respectively. In association with myeloid neoplasms, the clinical course is aggressive, and in absence of effective conventional treatment options, long-term remission is usually only observed after allogeneic stem cell transplantation (ASCT). With the discovery of inhibitors of the JAK2 tyrosine kinase and based on encouraging in vitro and in vivo data, we treated two male patients with myeloid neoplasms and a PCM1-JAK2 or a BCR-JAK2 fusion gene, respectively, with the JAK1/JAK2 inhibitor ruxolitinib. After 12 months of treatment, both patients achieved a complete clinical, hematologic, and cytogenetic response. Non-hematologic toxicity was only grade 1 while no hematologic toxicity was observed. However, remission in both patients was only short-term, with relapse occurring after 18 and 24 months, respectively, making ASCT indispensable in both cases. This data highlight (1) the ongoing importance of cytogenetic analysis for the diagnostic work-up of myeloid neoplasms as it may guide targeted therapy and (2) remission under ruxolitinib may only be short-termed in JAK2 fusion genes but it may be an important bridging therapy prior to ASCT.
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Analysis for loss of heterozygosity on chromosome arm 13q by STR analysis or SNP sequencing can replace analysis of FLT3-ITD to detect patients with prognostically adverse AML.
Genes Chromosomes Cancer
PUBLISHED: 09-03-2014
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We aimed at developing novel assays for Loss of Heterozygosity (LOH) detection on 13q as a substitute for FLT3-ITD analysis to identify acute myeloid leukemia (AML) patients with high risk of shorter survival. To this aim, we first analyzed a selected cohort of 185 patients with (n?=?138) or without (n?=?47) FLT3-ITD by short tandem repeat (STR) analysis for 13q LOH. In 46 of 138 FLT3-ITD positive cases, a FLT3-ITD/FLT3wt ratio of ?1 was measured indicating LOH. Applying analysis with a combination of five different STR markers, a threshold of an STR allelic ratio of >65% allows the identification of LOH in 40/46 (87%) samples. Survival analysis revealed significantly inferior outcome in patients with LOH as detected by STR analysis (event free survival (EFS): no LOH: 13.3 months versus LOH: 4.5 months, p?
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Distinct characteristics of e13a2 versus e14a2 BCR-ABL1 driven chronic myeloid leukemia under first-line therapy with imatinib.
Haematologica
PUBLISHED: 05-16-2014
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The vast majority of chronic myeloid leukemia patients express a BCR-ABL1 fusion gene mRNA encoding a 210 kDa tyrosine kinase which promotes leukemic transformation. A possible differential impact of the corresponding BCR-ABL1 transcript variants e13a2 ("b2a2") and e14a2 ("b3a2") on disease phenotype and outcome is still a subject of debate. A total of 1105 newly diagnosed imatinib-treated patients were analyzed according to transcript type at diagnosis (e13a2, n=451; e14a2, n=496; e13a2+e14a2, n=158). No differences regarding age, sex, or Euro risk score were observed. A significant difference was found between e13a2 and e14a2 when comparing white blood cells (88 vs. 65 × 10(9)/L, respectively; P<0.001) and platelets (296 vs. 430 × 10(9)/L, respectively; P<0.001) at diagnosis, indicating a distinct disease phenotype. No significant difference was observed regarding other hematologic features, including spleen size and hematologic adverse events, during imatinib-based therapies. Cumulative molecular response was inferior in e13a2 patients (P=0.002 for major molecular response; P<0.001 for MR4). No difference was observed with regard to cytogenetic response and overall survival. In conclusion, e13a2 and e14a2 chronic myeloid leukemia seem to represent distinct biological entities. However, clinical outcome under imatinib treatment was comparable and no risk prediction can be made according to e13a2 versus e14a2 BCR-ABL1 transcript type at diagnosis. (clinicaltrials.gov identifier:00055874).
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TP53 mutations occur in 15.7% of ALL and are associated with MYC-rearrangement, low hypodiploidy, and a poor prognosis.
Blood
PUBLISHED: 05-14-2014
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TP53 is the most extensively studied gene in cancer. However, data on frequency and the prognostic impact of TP53 mutations in acute lymphoblastic leukemia (ALL) remain scarce. Thus, we aimed at identifying the mutation frequency of TP53, its association with cytogenetic subgroups, and its impact on survival in a large cohort of 625 patients with ALL. Our data revealed an overall mutation incidence of 15.7%, which increases with age. Correlation with cytogenetic subgroups showed that mutations were most frequent in ALL with low hypodiploidy or MYC-rearrangements. Furthermore, for a large number of patients, both TP53 alleles were altered, either by 2 TP53 mutations (12%) or by a TP53 mutation and a TP53 deletion in the second allele (39%). A high TP53 mutation load was correlated to low hypodiploidy, high hyperdiploidy, and a complex karyotype. Moreover, a higher mutation load was found in B-lineage ALL compared with T-lineage ALL. Similar to other cancers, the median overall survival was significantly shorter in patients with TP53 mutation compared with patients with wild-type TP53. This effect was especially pronounced when both TP53 alleles were affected, either by 2 TP53 mutations or by both a mutation and an accompanying TP53 deletion.
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Equivalence of BCR-ABL transcript levels with complete cytogenetic remission in patients with chronic myeloid leukemia in chronic phase.
J. Cancer Res. Clin. Oncol.
PUBLISHED: 04-29-2014
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Chronic myeloid leukemia (CML) patients are monitored by both cytogenetic and molecular assessments, although present guidelines appear to switch from cytogenetic to molecular criteria. Due to the increasing use of molecular measurements, it was the aim of this work to identify a BCR-ABL level according to the international scale (BCR-ABL(IS)) as an equivalent substitute for complete cytogenetic remission (CCyR).
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Identification and functional characterization of imatinib-sensitive DTD1-PDGFRB and CCDC88C-PDGFRB fusion genes in eosinophilia-associated myeloid/lymphoid neoplasms.
Genes Chromosomes Cancer
PUBLISHED: 04-29-2014
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Eosinophilia-associated myeloid neoplasms with rearrangement of chromosome bands 5q31-33 are frequently associated with PDGFRB fusion genes, which are exquisitely sensitive to treatment with imatinib. In search for novel fusion partners of PDGFRB, we analyzed three cases with translocation t(5;20)(q33;p11), t(5;14)(q33;q32), and t(5;17;14)(q33;q11;q32) by 5?-rapid amplification of cDNA ends polymerase chain reaction (5?-RACE-PCR) and DNA-based long-distance inverse PCR (LDI-PCR) with primers derived from PDGFRB. LDI-PCR revealed a fusion between CCDC88C exon 25 and PDGFRB exon 11 in the case with t(5;17;14)(q33;q11;q32) while 5?-RACE-PCR identified fusions between CCDC88C exon 10 and PDGFRB exon 12 and between DTD1 exon 4 and PDGFRB exon 12 in the cases with t(5;14)(q33;q32) and t(5;20)(q33;p11), respectively. The PDGFRB tyrosine-kinase domain is predicted to be retained in all three fusion proteins. The partner proteins contained coiled-coil domains or other domains, which putatively lead to constitutive activation of the PDGFRB fusion protein. In vitro functional analyses confirmed transforming activity and imatinib-sensitivity of the fusion proteins. All three patients achieved rapid and durable complete hematologic remissions on imatinib.
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Myelodysplastic cells in patients reprogram mesenchymal stromal cells to establish a transplantable stem cell niche disease unit.
Cell Stem Cell
PUBLISHED: 02-26-2014
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Myelodysplastic syndromes (MDSs) are a heterogeneous group of myeloid neoplasms with defects in hematopoietic stem and progenitor cells (HSPCs) and possibly the HSPC niche. Here, we show that patient-derived mesenchymal stromal cells (MDS MSCs) display a disturbed differentiation program and are essential for the propagation of MDS-initiating Lin(-)CD34(+)CD38(-) stem cells in orthotopic xenografts. Overproduction of niche factors such as CDH2 (N-Cadherin), IGFBP2, VEGFA, and LIF is associated with the ability of MDS MSCs to enhance MDS expansion. These factors represent putative therapeutic targets in order to disrupt critical hematopoietic-stromal interactions in MDS. Finally, healthy MSCs adopt MDS MSC-like molecular features when exposed to hematopoietic MDS cells, indicative of an instructive remodeling of the microenvironment. Therefore, this patient-derived xenograft model provides functional and molecular evidence that MDS is a complex disease that involves both the hematopoietic and stromal compartments. The resulting deregulated expression of niche factors may well also be a feature of other hematopoietic malignancies.
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Acute lymphoblastic leukemia with low hypodiploid/near triploid karyotype is a specific clinical entity and exhibits a very high TP53 mutation frequency of 93%.
Genes Chromosomes Cancer
PUBLISHED: 02-26-2014
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B lymphoblastic leukemia/lymphoma (ALL) are subdivided by the WHO classification into five subgroups defined by specific translocations and two further subgroups defined by the number of chromosomes. The hypodiploid subgroup is heterogeneous and comprises ALL with a chromosome number of <46. To characterize a specific subset with low hypodiploid karyotype, we performed chromosome banding analysis, FISH, array comparative genomic hybridization, and mutational analyses of FBXW7, NOTCH1, KRAS, NRAS, TP53, and IKZF1 in 29 cases. We observed a nonrandom pattern of chromosome losses, including chromosomes 3, 7, 13, 15, 16, and 17. A deletion encompassing the CDKN2A/B locus was the only recurrent structural abnormality. A duplication of the low hypodiploid karyotype occurred frequently, resulting in a near triploid karyotype based on the definition by merely counting chromosomes but in fact was a very low tetraploid chromosome set. Mutational analyses revealed no mutations in IKZF1, FBXW7, NOTCH1, and KRAS and only one mutation in NRAS. However, we discovered a high frequency of TP53 mutations in 93% (27/29) of cases. In 26/27 cases with TP53 mutation, the second TP53 allele was lost due to monosomy 17. Median overall survival was short (18.5 months), which might be related to the high frequency of TP53 alterations. Therefore, ALL with low hypodiploidy is characterized by a typical pattern of chromosome losses and a remarkably high TP53 mutation frequency. Our data suggest the introduction of a novel WHO entity within the B lymphoblastic leukemia/lymphoma group showing low hypodiploid/very low tetraploid karyotype and concomitant TP53 mutation.
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Constitutional and somatic rearrangement of chromosome 21 in acute lymphoblastic leukaemia.
Nature
PUBLISHED: 01-30-2014
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Changes in gene dosage are a major driver of cancer, known to be caused by a finite, but increasingly well annotated, repertoire of mutational mechanisms. This can potentially generate correlated copy-number alterations across hundreds of linked genes, as exemplified by the 2% of childhood acute lymphoblastic leukaemia (ALL) with recurrent amplification of megabase regions of chromosome 21 (iAMP21). We used genomic, cytogenetic and transcriptional analysis, coupled with novel bioinformatic approaches, to reconstruct the evolution of iAMP21 ALL. Here we show that individuals born with the rare constitutional Robertsonian translocation between chromosomes 15 and 21, rob(15;21)(q10;q10)c, have approximately 2,700-fold increased risk of developing iAMP21 ALL compared to the general population. In such cases, amplification is initiated by a chromothripsis event involving both sister chromatids of the Robertsonian chromosome, a novel mechanism for cancer predisposition. In sporadic iAMP21, breakage-fusion-bridge cycles are typically the initiating event, often followed by chromothripsis. In both sporadic and rob(15;21)c-associated iAMP21, the final stages frequently involve duplications of the entire abnormal chromosome. The end-product is a derivative of chromosome 21 or the rob(15;21)c chromosome with gene dosage optimized for leukaemic potential, showing constrained copy-number levels over multiple linked genes. Thus, dicentric chromosomes may be an important precipitant of chromothripsis, as we show rob(15;21)c to be constitutionally dicentric and breakage-fusion-bridge cycles generate dicentric chromosomes somatically. Furthermore, our data illustrate that several cancer-specific mutational processes, applied sequentially, can coordinate to fashion copy-number profiles over large genomic scales, incrementally refining the fitness benefits of aggregated gene dosage changes.
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Association of the type of 5q loss with complex karyotype, clonal evolution, TP53 mutation status, and prognosis in acute myeloid leukemia and myelodysplastic syndrome.
Genes Chromosomes Cancer
PUBLISHED: 01-13-2014
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We analyzed 1,200 patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) harboring a 5q deletion in order to clarify whether the type of 5q loss is associated with other biological markers and prognosis. We investigated all patients by chromosome banding analysis, FISH with a probe for EGR1 (5q31) and, if necessary, to resolve complex karyotypes with 24-color-FISH. Moreover, 420 patients were analyzed for mutations in the TP53 gene. The patient cohort was subdivided based on type of 5q loss: Patients with interstitial deletions and patients with 5q loss due to unbalanced rearrangements or monosomy 5. Loss of the long arm of chromosome 5 due to an unbalanced rearrangement occurred more often in AML (286/627; 45.6%) than MDS (188/573; 32.8%; P?
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Relapse kinetics in acute myeloid leukaemias with MLL translocations or partial tandem duplications within the MLL gene.
Br. J. Haematol.
PUBLISHED: 01-10-2014
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Correct action upon re-emergence of minimal residual disease in acute myeloid leukaemia (AML) patients has not yet been established. The applicability of demethylating agents and use of allogeneic stem cell transplantation will be dependent on pre-relapse AML growth rates. We here delineate molecular growth kinetics of AML harbouring MLL partial tandem duplication (MLL-PTD; 37 cases) compared to those harbouring MLL translocations (43 cases). The kinetics of MLL-PTD relapses was both significantly slower than those of MLL translocation positive ones (median doubling time: MLL-PTD: 24 d, MLL-translocations: 12 d, P = 0·015, Wilcoxon rank sum test), and displayed greater variation depending on additional mutations. Thus, MLL-PTD+ cases with additional RUNX1 mutations or FLT3-internal tandem duplication relapsed significantly faster than cases without one of those two mutations (Wilcoxon rank sum test, P = 0·042). As rapid relapses occurred in all MLL subgroups, frequent sampling are necessary to obtain acceptable relapse detection rates and times from molecular relapse to haematological relapse (blood sampling every second month: MLL-PTD: 75%/50 d; MLL translocations: 85%/25 d). In conclusion, in this cohort relapse kinetics is heavily dependent on AML subtype as well as additional genetic aberrations, with possibly great consequences for the rational choice of pre-emptive therapies.
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Amplification of EVI1 on cytogenetically cryptic double minutes as new mechanism for increased expression of EVI1.
Cancer Genet
PUBLISHED: 01-07-2014
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In acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), increased expression of EVI1 (ecotropic virus integration site 1) was found to be associated with adverse prognosis. Although increased expression of the EVI1 gene is mainly caused by chromosomal rearrangements involving chromosome band 3q26, where EVI1 is located, it can also be identified in cases without these rearrangements. The mechanisms that cause increased EVI1 expression in the absence of 3q26 rearrangements remain unclear. Here, we present four cases with increased EVI1 expression due to EVI1 amplification on cytogenetically cryptic double minutes (dmin). The dmin are small acentric chromosome structures and were observed in about 1% of AML and MDS. We investigated the four cases by conventional cytogenetics, fluorescence in situ hybridization, and array comparative genomic hybridization. Furthermore, EVI1 expression was measured by quantitative reverse transcriptase-PCR. By conventional chromosome analysis, the EVI1 dmin cannot be detected, due to the small size of the amplicons of 0.49-0.78 Mbp. Median % EVI1/ABL expression was 88.9% and therefore comparable to the median % EVI1/ABL expression of patients with EVI1 rearrangements. In conclusion, EVI1 amplification on cytogenetically cryptic dmin causes increased expression of EVI1 and is a new mechanism that causes increased EVI1 expression without a 3q26 rearrangement.
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Investigation of 305 patients with myelodysplastic syndromes and 20q deletion for associated cytogenetic and molecular genetic lesions and their prognostic impact.
Br. J. Haematol.
PUBLISHED: 10-11-2013
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In patients with myelodysplastic syndromes (MDS), sole 20q deletion [del(20q)] is a recurrent favourable abnormality. We studied additional molecular and cytogenetic lesions and their prognostic impact in 305 MDS patients with del(20q) (229 males/76 females; 29-90 years). All patients were investigated by cytomorphology and chromosome banding analysis (CBA), subsets by fluorescence in situ hybridization, molecular mutation screening, and array comparative genomic hybridization (aCGH). By aCGH (n = 30), the minimal common deleted region (CDR) was flanked by PTPRT (20q13·11) and EYA2 (20q13·12). 210 (68·9%) patients had early MDS without blast increase, 95 (31·1%) advanced MDS with blast increase (5-19%). Additional chromosomal abnormalities (ACAs) were detected in 88/305 (28·9%) patients. Patients with advanced MDS more frequently had ACAs (P = 0·003) and had a higher mean number of ACAs (P = 0·020) and of molecular mutations (P = 0·060). Spliceosome mutations were frequent (U2AF1: n = 31/155; 20·0%; SRSF2: n = 31/159; 19·5%; SF3B1mut: n = 8/159; 5·0%). ASXL1mut (25/153; 16·3%) were associated with advanced MDS (P = 0·001). Presence of ?3 ACAs (P = 0·003) and ASXL1mut (P = 0·002) were associated with worse 2-year survival. In conclusion, the cytogenetic subgroup of MDS with del(20q) has a good prognosis but may be further subclassified by additional cytogenetic and molecular lesions. U2AF1mut is overrepresented in MDS with del(20q), and ASXL1mut is prognostically adverse.
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Flow cytometric identification of 76 patients with biclonal disease among 5523 patients with chronic lymphocytic leukaemia (B-CLL) and its genetic characterization.
Br. J. Haematol.
PUBLISHED: 05-23-2013
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Multiparameter flow cytometry (MFC) identifies rare cases of biclonal disease in chronic lymphocytic leukaemia (CLL). By MFC, we identified 76 patients with biclonal disease in a cohort of 5523 CLL patients (1·4%). Fluorescence in situ hybridization and chromosome banding analysis revealed five and six cases, respectively, with two different cytogenetic aberrations due to clonal evolution. Two different B-cell receptor rearrangements and IGHV subtypes were more frequent in biclonal than in monoclonal CLL by MFC (37·1% vs. 2·7%; P < 0·001). Patients with biclonal CLL by MFC showed a trend to a shorter time to treatment than monoclonal CLL (P = 0·080).
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Cyclin D1 (CCND1) messenger RNA expression as assessed by real-time PCR contributes to diagnosis and follow-up control in patients with mantle cell lymphoma.
Exp. Hematol.
PUBLISHED: 04-26-2013
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Molecular diagnosis of mantle cell lymphoma (MCL) can be difficult because the t(11;14)/IGH@-CCND1 is extremely heterogeneous at the DNA level. Aiming to establish a reliable molecular tool that could be easily implemented in routine diagnostics, we developed a new real-time polymerase chain reaction (PCR) assay for CCND1 expression measurement and evaluated 451 cases: 142 MCL, 76 chronic lymphocytic leukemia, 20 hairy cell leukemia, 13 hairy cell leukemia-variant, 20 splenic marginal zone lymphoma, 91 other mature B-cell neoplasms, 29 other hematologic neoplasms, and 60 healthy individuals. Sensitivity of the real-time PCR assay was up to 10(-4). In t(11;14)/IGH@-CCND1 positive lymphoma samples (n = 150), median %CCND1/ABL1 expression level was 178.2 (range: 1.5-4, 152.0). Normalized by t(11;14)/IGH@-CCND1 positive cells as determined by fluorescence in situ hybridization IGH@-CCND1 positive samples showed a median %CCND1/ABL1 of 445.8 (range: 17.9-4,848.5). A normalized %CCND1/ABL1 expression of at least 17.0 was chosen as threshold for CCND1 positivity. For unnormalized samples, the positive detection rate of t(11;14)/IGH@-CCND1 by CCND1 expression was 87.3%. Healthy individuals had low %CCND1/ABL1 (median, 1.1; range, 0.0-7.8). The negative predictive value for exclusion of a t(11;14)/IGH@-CCND1 by CCND1 expression was 95.3% by the above threshold. %CCND1/ABL1 was higher in MCL than in the remaining B-cell lymphomas (mean ± SD, 392.9 ± 685.3 vs. 46.0 ± 305.0; p < 0.001). In 66 follow-up samples, CCND1 showed 2.5-3.5 log reduction after chemotherapy and increase at relapse. CCND1 expression could serve as adjunct to other techniques in diagnosis and follow-up of B-cell lymphomas.
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Recurrent mutations in multiple components of the cohesin complex in myeloid neoplasms.
Nat. Genet.
PUBLISHED: 02-15-2013
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Cohesin is a multimeric protein complex that is involved in the cohesion of sister chromatids, post-replicative DNA repair and transcriptional regulation. Here we report recurrent mutations and deletions involving multiple components of the cohesin complex, including STAG2, RAD21, SMC1A and SMC3, in different myeloid neoplasms. These mutations and deletions were mostly mutually exclusive and occurred in 12.1% (19/157) of acute myeloid leukemia, 8.0% (18/224) of myelodysplastic syndromes, 10.2% (9/88) of chronic myelomonocytic leukemia, 6.3% (4/64) of chronic myelogenous leukemia and 1.3% (1/77) of classical myeloproliferative neoplasms. Cohesin-mutated leukemic cells showed reduced amounts of chromatin-bound cohesin components, suggesting a substantial loss of cohesin binding sites on chromatin. The growth of leukemic cell lines harboring a mutation in RAD21 (Kasumi-1 cells) or having severely reduced expression of RAD21 and STAG2 (MOLM-13 cells) was suppressed by forced expression of wild-type RAD21 and wild-type RAD21 and STAG2, respectively. These findings suggest a role for compromised cohesin functions in myeloid leukemogenesis.
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Robustness of amplicon deep sequencing underlines its utility in clinical applications.
J Mol Diagn
PUBLISHED: 02-08-2013
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We investigated the robustness of amplicon deep sequencing to study its utility in routine clinical applications offering patient-specific individualized assays for molecular disease characterization and monitoring. Amplicons were designed targeting RUNX1, CEBPA, CBL, NRAS, KRAS, DNMT3A, EZH2, and TP53 using different PCR amplification strategies and Roche GS FLX Titanium and Illumina MiSeq sequencing platforms. Thirty-three patients with leukemia were selected as an exemplary cohort representing heterogeneous cancer specimens. Both standard two-primer amplification and four-primer microfluidics PCRs yielded highly linear characteristics in detecting molecular alterations in series of dilution experiments. By fitting a linear mixed-effects model to the logarithmized data, a slope ? of -1.000 (95% CI, ±0.046) was obtained for two-primer assays and of -0.998 (95% CI, ±0.105) was obtained for four-primer assays, which represented a near-perfect decrease of the mutation load. Furthermore, data are presented on technical precision, limit of detection, and occurrence of small subclones in TP53- and RUNX1-mutated patients to identify clonal disease progression and residual disease. We demonstrate that, depending on the local sequence context for each amplicon, the limit of detection of the assay cannot be lower than a range of 0.25% to 3.5%. In conclusion, amplicon deep sequencing enabled the assessment of mutations in a highly robust manner and across a broad range of relative frequencies of mutations. This assay detects residual disease or identifies mutations with predictive relevance to direct treatment strategies.
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Frequency and prognostic impact of CEBPA proximal, distal and core promoter methylation in normal karyotype AML: a study on 623 cases.
PLoS ONE
PUBLISHED: 02-01-2013
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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.
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Hematologic malignancies with PCM1-JAK2 gene fusion share characteristics with myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, and FGFR1.
Ann. Hematol.
PUBLISHED: 01-30-2013
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The translocation t(8;9)(p22;p24) is a rare event that results in the fusion of JAK2 to PCM1 and thus leads to the activation of the Janus Kinase 2. In 2008, the WHO introduced a new entity called "Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB or FGFR1", which are characterized by the formation of a fusion gene encoding an aberrant tyrosine kinase. These disorders share characteristics with myeloproliferative neoplasms and typically show an eosinophilia. We here now report on 6 new cases with PCM1-JAK2 fusion. These patients show characteristics with respect to epidemiology, clinical presentation, and genetic changes that are very similar to patients with rearrangements of PDGFRA, PDGFRB, or FGFR1. Our data suggests the integration of cases with JAK2-PCM1 fusion in the respective WHO category of myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1.
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The molecular profile of adult T-cell acute lymphoblastic leukemia: mutations in RUNX1 and DNMT3A are associated with poor prognosis in T-ALL.
Genes Chromosomes Cancer
PUBLISHED: 01-23-2013
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T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive and heterogeneous disease. The diagnosis is predominantly based on immunophenotyping. In addition to known cytogenetic abnormalities molecular mutations were recently identified. Here, 90 adult T-ALL cases were investigated for mutations in NOTCH1, FBXW7, PHF6, CDKN2A, DNMT3A, FLT3, PTEN, and RUNX1 using 454 next-generation amplicon sequencing and melting curve analyses. These data were further complemented by FISH, chromosome banding, array CGH, and CDKN2B promoter methylation analyses. NOTCH1 was the most frequently mutated gene with a 71.1% frequency followed by FBXW7 (18.9%), PHF6 (39.5%), DNMT3A (17.8%), RUNX1 (15.5%), PTEN (10.0%), CDKN2A (4.4%), FLT3-ITD (2.2%), and FLT3-TKD (1.1%). In total, 84/90 (93.3%) cases harbored at least one mutation. Combining these data with CDKN2A/B deletions and CDKN2B methylation status, we detected minimum one aberration in 89/90 (98.9%) patients. Survival analyses revealed the subtype as defined by the immunophenotype as the strongest independent prognostic factor. When restricting the survival analysis to the early T-ALL subtype, a strong association of RUNX1 (P = 0.027) and DNMT3A (P = 0.005) mutations with shorter overall survival was observed. In conclusion, RUNX1 and DNMT3A are frequently mutated in T-ALL and are associated with poor prognosis in early T-ALL.
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Protein kinase c-?-dependent activation of NF-?B in stromal cells is indispensable for the survival of chronic lymphocytic leukemia B cells in vivo.
Cancer Cell
PUBLISHED: 01-19-2013
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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)-?II and the subsequent activation of NF-?B in bone marrow stromal cells are prerequisites to support the survival of malignant B cells. PKC-? knockout mice are insusceptible to CLL transplantations, underscoring the in vivo significance of the PKC-?II-NF-?B signaling pathway in the tumor microenvironment. Upregulated stromal PKC-?II 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.
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CEBPA double-mutated acute myeloid leukaemia harbours concomitant molecular mutations in 76·8% of cases with TET2 and GATA2 alterations impacting prognosis.
Br. J. Haematol.
PUBLISHED: 01-07-2013
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Acute myeloid leukaemia (AML) with CEBPA mutations is listed as a provisional entity in the current World Health Organization classification. A difference in clinical outcome between single- (sm) and double-mutated (dm) cases has been reported, whereupon CEBPAdm cases were shown to be associated with better overall survival (OS). The occurrence and prognostic impact of concomitant molecular mutations in addition to CEBPAdm has not been assessed until now with exception of GATA2 mutations. Here, we investigated a cohort of 95 AML CEBPAdm cases for concomitant mutations. TET2 was found to be most frequently mutated (34·0%) gene, followed by GATA2 (21·0%), WT1 (13·7%), DNMT3A (9·6%), ASXL1 (9·5%), NRAS (8·4%), KRAS (3·2%), IDH1/2 (6·3%), FLT3-internal tandem duplication (6·3%), FLT3-tyrosine kinase domain (2·1%), NPM1 (2·1%), and RUNX1 (1/94). Patients harbouring additional mutations in the TET2 gene showed significantly worse OS than TET2 wild-type cases (P = 0·035), whereas GATA2-mutated patients showed improved OS (P = 0·032). Serial analyses were performed for 39 CEBPAdm cases with concomitant mutations. Here, we observed that CEBPA mutations present the primary pathogenetic event in the majority of cases (76·9%). Further, a distinct gene expression profile (GEP) was confirmed for CEBPAdm versus CEBPAsm or CEBPA wild-type cases while no significant changes in GEP were observed related to additional mutations within the CEBPAdm AML.
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Multiparameter flow cytometry reveals myelodysplasia-related aberrant antigen expression in myelodysplastic/myeloproliferative neoplasms.
Cytometry B Clin Cytom
PUBLISHED: 01-02-2013
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Within the myelodysplastic/myeloproliferative neoplasm (MDS/MPN) category of the WHO (2008), only chronic myelomonocytic leukemia was so far evaluated by multiparameter flow cytometry (MFC).
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Prognostic value of monosomal karyotype in comparison to complex aberrant karyotype in acute myeloid leukemia: a study on 824 cases with aberrant karyotype.
Blood
PUBLISHED: 12-29-2011
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In acute myeloid leukemia (AML) the subset with complex karyotype (CK) is traditionally regarded as the worst prognostic group. However, ? 3, ? 4, or ? 5 abnormalities have been variably used for its definition. Recently, monosomal karyotype (MSK) was suggested to indicate an even inferior outcome. We tested which definition fits best to identify the most unfavorable subgroup. After excluding patients with t(15;17)/PML-RARA, t(8;21)/RUNX1-RUNX1T1, inv (16)/t(16;16)/CBFB-MYH11, and normal karyotype, 824 patients with AML with cytogenetic abnormalities were analyzed. Patients with MSK or CK defined as ? 3, ? 4, or ? 5 abnormalities showed an inferior overall survival compared with the respective remaining patients not fulfilling these criteria (for all, P < .001). Hazard ratios were 1.93, 1.68, 1.94, and 1.92. CK ? 4 as a single parameter identified the largest proportion of patients with very poor risk. However, combining CK ? 4 and MSK detected an even larger number of patients with very unfavorable outcome (261 of 824; 31.7%).
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Impact of additional cytogenetic aberrations at diagnosis on prognosis of CML: long-term observation of 1151 patients from the randomized CML Study IV.
Blood
PUBLISHED: 10-28-2011
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The prognostic relevance of additional cytogenetic findings at diagnosis of chronic myeloid leukemia (CML) is unclear. The impact of additional cytogenetic findings at diagnosis on time to complete cytogenetic (CCR) and major molecular remission (MMR) and progression-free (PFS) and overall survival (OS) was analyzed using data from 1151 Philadelphia chromosome-positive (Ph(+)) CML patients randomized to the German CML Study IV. At diagnosis, 1003 of 1151 patients (87%) had standard t(9;22)(q34;q11) only, 69 patients (6.0%) had variant t(v;22), and 79 (6.9%) additional cytogenetic aberrations (ACAs). Of these, 38 patients (3.3%) lacked the Y chromosome (-Y) and 41 patients (3.6%) had ACAs except -Y; 16 of these (1.4%) were major route (second Philadelphia [Ph] chromosome, trisomy 8, isochromosome 17q, or trisomy 19) and 25 minor route (all other) ACAs. After a median observation time of 5.3 years for patients with t(9;22), t(v;22), -Y, minor- and major-route ACAs, the 5-year PFS was 90%, 81%, 88%, 96%, and 50%, and the 5-year OS was 92%, 87%, 91%, 96%, and 53%, respectively. In patients with major-route ACAs, the times to CCR and MMR were longer and PFS and OS were shorter (P < .001) than in patients with standard t(9;22). We conclude that major-route ACAs at diagnosis are associated with a negative impact on survival and signify progression to the accelerated phase and blast crisis.
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TET2 deletions are a recurrent but rare phenomenon in myeloid malignancies and are frequently accompanied by TET2 mutations on the remaining allele.
Br. J. Haematol.
PUBLISHED: 10-24-2011
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TET2 mutations have been identified in 10-25% of patients with myeloid malignancies, but TET2 gene copy number alterations remain to be evaluated. We performed interphase and metaphase fluorescence in situ hybridization (FISH), using newly designed probes that span TET2, in 893 patients with acute myeloid leukaemia (AML) and chronic myeloid malignancies and validated the new assay by single nucleotide polymorphism arrays. Next-generation sequencing for molecular TET2 mutations was performed in 37/50 del(TET2) patients. We identified TET2 deletions in 50/893 patients (26 males, 24 females; 44-87 years) resulting in a 5.6% frequency [22/425 AML (5.2%), 15/217 chronic myelomonocytic leukaemia (CMML; 6.9%), 9/188 myelodysplastic syndromes (4.8%), 4/63 myeloproliferative neoplasms (6.3%)]. Cytogenetic alterations (mostly complex) were detected in 35/50 (70.0%) of del(TET2) cases. TET2 deletions were cytogenetically cryptic in 25/50 (50.0%). Sequencing detected TET2mut in 19/37 (51.4%) del(TET2) cases investigated, predominantly CMML (10/14; 71.4%). In de novo AML with intermediate cytogenetics, presence of any TET2 alteration was related to worse median overall survival (347 d vs. not reached; P = 0.052) and event-free survival (164 vs. 457 d; P = 0.024). JAK2(V617F) was found in 6/18 (33.3%) del(TET2) cases analysed. Thus, TET2 haploinsufficiency recurrently occurs in myeloid malignancies, frequently combined with TET2 mutations on the remaining allele. The prognostic impact of del(TET2) in myeloid malignancies should be further evaluated.
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Whole-exome sequencing identifies somatic mutations of BCOR in acute myeloid leukemia with normal karyotype.
Blood
PUBLISHED: 10-19-2011
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Among acute myeloid leukemia (AML) patients with a normal karyotype (CN-AML), NPM1 and CEBPA mutations define World Health Organization 2008 provisional entities accounting for approximately 60% of patients, but the remaining 40% are molecularly poorly characterized. Using whole-exome sequencing of one CN-AML patient lacking mutations in NPM1, CEBPA, FLT3-ITD, IDH1, and MLL-PTD, we newly identified a clonal somatic mutation in BCOR (BCL6 corepressor), a gene located on chromosome Xp11.4. Further analyses of 553 AML patients showed that BCOR mutations occurred in 3.8% of unselected CN-AML patients and represented a substantial fraction (17.1%) of CN-AML patients showing the same genotype as the AML index patient subjected to whole-exome sequencing. BCOR somatic mutations were: (1) disruptive events similar to the germline BCOR mutations causing the oculo-facio-cardio-dental genetic syndrome; (2) associated with decreased BCOR mRNA levels, absence of full-length BCOR, and absent or low expression of a truncated BCOR protein; (3) virtually mutually exclusive with NPM1 mutations; and (4) frequently associated with DNMT3A mutations, suggesting cooperativity among these genetic alterations. Finally, BCOR mutations tended to be associated with an inferior outcome in a cohort of 422 CN-AML patients (25.6% vs 56.7% overall survival at 2 years; P = .032). Our results for the first time implicate BCOR in CN-AML pathogenesis.
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Frequency and prognostic impact of the aberrant CD8 expression in 5,523 patients with chronic lymphocytic leukemia.
Cytometry B Clin Cytom
PUBLISHED: 09-30-2011
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In patients with chronic lymphocytic leukemia (CLL), aberrant expression of the T-lineage antigen CD8 was reported in low frequencies. The clinical impact of this phenomenon remains in discussion.
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Gene expression of BAALC, CDKN1B, ERG, and MN1 adds independent prognostic information to cytogenetics and molecular mutations in adult acute myeloid leukemia.
Genes Chromosomes Cancer
PUBLISHED: 09-27-2011
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Expression of BAALC, ERG, and MN1 is associated with outcome in normal karyotype acute myeloid leukemia (AML). In this study, the expression of these markers and of EVI1 and CDKN1B was determined using oligonucleotide microarrays in 286 AML comprising all cytogenetic groups. Higher expression of each gene was associated with an inferior outcome: CDKN1B, median overall survival (mOS): 14.9 months vs. not reached (nr), P = 0.005, median event-free survival (mEFS): 9.7 vs. 31.0 months, P = 0.013; BAALC, no impact on OS, mEFS: 6.2 vs. 13.0 months, P = 0.03; ERG: mOS: 12.5 months vs. nr, P = 0.002, mEFS: 8.1 vs. 15.7 months, P = 0.001; MN1: mOS: 12.3 months vs. nr, P = 0.004, mEFS: 8.1 vs. 16.7 months, P = 0.001. A multivariate analysis revealed an independent impact on OS for CDKN1B, ERG, and MN1 expression. A novel score based on BAALC, CDKN1B, ERG, and MN1 expression had an impact on OS and EFS independent of cytogenetics and age. A score taking into account gene expression and karyotype allowed the separation of four prognostic groups with significant differences in OS and EFS (OS at 2 years: 90.4%, 56.4%, 34.0%, 12.6%; mEFS: n.r., 18.1 months, 8.7 months, 2.5 months). The impact on outcome of this score was independent of NPM1mut/FLT3-ITD- status, MLL-PTD, and age.
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Update on developments in the diagnosis and prognostic evaluation of patients with myelodysplastic syndromes (MDS): consensus statements and report from an expert workshop.
Leuk. Res.
PUBLISHED: 09-15-2011
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Several new treatments for myelodysplastic syndromes (MDS) have recently become available, or are in development. Patients who could benefit from active treatment must be effectively identified and followed up. Therefore, guidelines for the diagnosis and prognostic evaluation of MDS need to be kept up to date with technological and scientific advances. An expert workshop was convened to review currently available and emerging diagnostic technologies and developments in prognostic classification systems, to ensure appropriate management of individual patients. The panel also provided suggestions to ensure adherence to guidelines and highlighted the mandatory requirement for cytogenetic evaluation in patients with MDS.
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ETV6 rearrangements are recurrent in myeloid malignancies and are frequently associated with other genetic events.
Genes Chromosomes Cancer
PUBLISHED: 08-29-2011
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ETV6 (TEL) rearrangements are favorable in pediatric acute lymphoblastic leukemia but are less well characterized in myeloid malignancies. We investigated 9,550 patients with myeloid disorders for ETV6 rearrangements by chromosome banding analysis and interphase fluorescence in situ hybridization. ETV6 rearrangements were identified in 51 of 9,550 (0.5%) patients (range, 19.2-85.3 years). Frequencies were in detail: acute myeloid leukemia (AML): 40 of 3,798, 1.1%; myelodysplastic syndromes (MDS): 6 of 3,375, 0.2%; myeloproliferative neoplasms (MPNs): 5 of 1,720, 0.3%; MDS/MPN: 0 of 210; and chronic myelomonocytic leukemia: 0 of 447. Thirty-three different partner bands of ETV6 were identified, and most were recurrent: 3q26 (n = 10), 5q33 (n = 4), 17q11 (n = 3), 22q12 (n = 3), 5q31 (n = 2), and 2q31 (n = 2). Additional chromosomal abnormalities were identified in 29 of 51 (57%) ETV6 rearranged cases. In AML, ETV6 rearrangements were frequently associated with NPM1 (9/39, 23%) and RUNX1 mutations (6/31, 19%). The FAB M0 subtype was more frequent in ETV6 rearranged de novo AML than other AML (P < 0.001); expression of CD7 and CD34 by immunophenotyping was higher in ETV6 rearranged AML compared with other subgroups. Survival of 29 ETV6 rearranged de novo AML was compared with 818 AML from other cytogenetic subgroups. Median overall and event-free survival of ETV6 rearranged cases was similar to the intermediate-risk cohort (26.3 vs. 62.2 months and 14.0 vs. 15.4 months) defined according to Medical Research Council criteria. Our study confirms the variety of ETV6 rearrangements in AML, MDS, and MPNs often in association with other genetic events. Prognosis of ETV6 rearranged de novo AML seems to be intermediate, which should be independently confirmed.
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Clinical impact of FLT3 mutation load in acute promyelocytic leukemia with t(15;17)/PML-RARA.
Haematologica
PUBLISHED: 08-22-2011
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Combined treatment with all-trans-retinoic acid and chemotherapy is extremely efficient in patients with acute promyelocytic leukemia with t(15;17)/PML-RARA, but up to 15% of patients relapse.
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Prognostic relevance of RUNX1 mutations in T-cell acute lymphoblastic leukemia.
Haematologica
PUBLISHED: 08-09-2011
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The runt-related transcription factor 1, RUNX1, is crucial in the development of myeloid and lymphoid cell lineages and has been reported to be mutated in myeloid malignancies in approximately 30% of cases. In this study, the mutational status of RUNX1 was investigated in 128 acute lymphoblastic leukemia patients. We detected a mutation rate of 18.3% (13 of 71) in patients with T-cell acute lymphoblastic leukemia, 3.8% (2 of 52) in patients with B-cell acute lymphoblastic leukemia and no mutation (0 of 5) in patients with natural killer cell leukemia, respectively. In T-cell acute lymphoblastic leukemia patients, RUNX1 mutations were significantly associated with higher age (P=0.017) and lower white blood cell count (P=0.038). Moreover, an inferior outcome was observed in the subgroup of early T-cell acute lymphoblastic leukemia patients carrying RUNX1 mutations for overall survival (P=0.043). In conclusion, RUNX1 mutations are an important novel biomarker for a comprehensive characterization of T-cell acute lymphoblastic leukemia with poor prognostic impact and have implications for use also in monitoring disease.
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Response of ETV6-FLT3-positive myeloid/lymphoid neoplasm with eosinophilia to inhibitors of FMS-like tyrosine kinase 3.
Blood
PUBLISHED: 06-24-2011
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Imatinib-resistant tyrosine kinase (TK) fusions involving FGFR1, JAK2, or FLT3 are rare but recurrent in patients with eosinophilia-associated neoplasms. We report here 2 male patients with ETV6-FLT3(+) myeloid/lymphoid neoplasms with eosinophilia who were treated with the multitargeted TK inhibitors sunitinib and sorafenib. Patient 1 achieved rapid complete hematologic response and complete cytogenetic response after 3 months of taking sunitinib. A secondary blast phase caused by clonal evolution was diagnosed after 6 months. He achieved a second complete hematologic response after taking sorafenib but relapsed 2 months later. An N841K point mutation within the TK domain of FLT3, previously reported in acute myeloid leukemia and potentially conferring resistance to sorafenib, was subsequently identified. Patient 2 was heavily pretreated according to the initial diagnosis of T-lymphoblastic lymphoma and died in sunitinib-induced pancytopenia. This report highlights the importance of a careful diagnostic workup for eosinophilia-associated neoplasms to evaluate the possibility of TK inhibitor therapy.
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Frequent pathway mutations of splicing machinery in myelodysplasia.
Nature
PUBLISHED: 06-07-2011
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Myelodysplastic syndromes and related disorders (myelodysplasia) are a heterogeneous group of myeloid neoplasms showing deregulated blood cell production with evidence of myeloid dysplasia and a predisposition to acute myeloid leukaemia, whose pathogenesis is only incompletely understood. Here we report whole-exome sequencing of 29 myelodysplasia specimens, which unexpectedly revealed novel pathway mutations involving multiple components of the RNA splicing machinery, including U2AF35, ZRSR2, SRSF2 and SF3B1. In a large series analysis, these splicing pathway mutations were frequent (?45 to ?85%) in, and highly specific to, myeloid neoplasms showing features of myelodysplasia. Conspicuously, most of the mutations, which occurred in a mutually exclusive manner, affected genes involved in the 3-splice site recognition during pre-mRNA processing, inducing abnormal RNA splicing and compromised haematopoiesis. Our results provide the first evidence indicating that genetic alterations of the major splicing components could be involved in human pathogenesis, also implicating a novel therapeutic possibility for myelodysplasia.
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Comparison of genetic and clinical aspects in patients with acute myeloid leukemia and myelodysplastic syndromes all with more than 50% of bone marrow erythropoietic cells.
Haematologica
PUBLISHED: 05-23-2011
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The World Health Organization separates acute erythroid leukemia (erythropoiesis in ?50% of nucleated bone marrow cells; ?20% myeloblasts of non-erythroid cells) from other entities with increased erythropoiesis - acute myeloid leukemia with myelodysplasia-related changes (?20% myeloblasts of all nucleated cells) or myelodysplastic syndromes - and subdivides acute erythroid leukemia into erythroleukemia and pure erythroid leukemia subtypes. We aimed to investigate the biological/genetic justification for the different categories of myeloid malignancies with increased erythropoiesis (?50% of bone marrow cells).
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Cytogenetic methods in chronic lymphocytic leukemia.
Methods Mol. Biol.
PUBLISHED: 03-25-2011
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In chronic lymphocytic leukemia of the B-lineage (B-CLL), cytogenetic alterations are highly relevant for prognosis and therapeutic decisions. With conventional techniques, chromosome banding analysis in CLL has been hampered by the low quality of metaphases and low rates of cytogenetic alterations due to a low in vitro proliferation rate of CLL cells. Thus, interphase fluorescence in situ hybridization (FISH) has become the standard technique for cytogenetic analysis in CLL. However, interphase FISH is not able to provide an overview on the whole karyotype. In order to improve chromosome banding analysis in CLL, specific stimulation techniques have been developed. These either use CD40 ligand or oligonucleotides (e.g., CpG) and IL-2 in combination. With the respective techniques, metaphase cultivation is successful in >90% of CLL cases and aberrant karyotypes can be detected in nearly 90% of CLL cases. This has allowed the detection of new clinically relevant subgroups (e.g., complex aberrant karyotype cases) and a more differentiated picture of distinct cytogenetic subtypes, e.g., cases with a 13q deletion. Efforts should continue to define the value of chromosomal banding in CLL focusing as well on the interaction with already established techniques such as interphase FISH or immunophenotyping.
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CDKN1B, encoding the cyclin-dependent kinase inhibitor 1B (p27), is located in the minimally deleted region of 12p abnormalities in myeloid malignancies and its low expression is a favorable prognostic marker in acute myeloid leukemia.
Haematologica
PUBLISHED: 03-21-2011
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Alterations of the short arm of chromosome 12 (12p) occur in various hematologic malignancies and ETV6 and CDKN1B, which are located on 12p, have been implicated as leukemogenic genes of interest.
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Amount of bone marrow blasts is strongly correlated to NPM1 and FLT3-ITD mutation rate in AML with normal karyotype.
Leuk. Res.
PUBLISHED: 03-09-2011
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FLT3-ITDs are linked to higher leukocytes/blasts in acute myeloid leukemia. To evaluate the effect of NPM1 mutations, we correlated NPM1mut status with morphology in 805 adult normal karyotype AML. NPM1mut were found in 391/805 (48.6%), FLT3-ITD in 219/805 (27.2%). Frequencies of FLT3-ITD and NPM1mut cases were continuously increasing by blast decades: NPM1mut from 38/123 (30.9%) in 20-29% blast decade to 70/103 (68.0%) in 90-100% decade (p<0.001), FLT3-ITDs from 15/123 (12.2%) to 58/103 (56.3%) (p<0.001). Mean WBC count was highest in NPM1-mut/FLT3-ITD-positive and lowest in NPM1-wildtype/FLT3-ITD-negative patients (p<0.0001); similar for BM blasts. Therefore, FLT3-ITD and NPM1mut might synergistically stimulate blast proliferation.
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Strategy for robust detection of insertions, deletions, and point mutations in CEBPA, a GC-rich content gene, using 454 next-generation deep-sequencing technology.
J Mol Diagn
PUBLISHED: 03-01-2011
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CEBPA mutations are of prognostic relevance in acute myeloid leukemia (AML) and are currently detected using a combination of denaturing high-performance liquid chromatography (DHPLC), gene scan/fragment length analysis, and direct Sanger sequencing. Next-generation deep pyrosequencing, principally, allows for the highly sensitive detection of molecular mutations. However, standard 454 chemistry laboratory procedures lack efficient amplification of guanine-cytosine (GC)-rich amplicons during the emulsion PCR (emPCR) steps allowing direct massively parallel clonal amplification of PCR products. To solve this problem, we investigated six distinct emPCR conditions. The coding sequence of CEBPA was subdivided into four overlapping amplicons: GC content for amplicon 1, 74%; amplicon 2, 76%; amplicon 3, 77%; and amplicon 4, 69%. A new emPCR condition, improving the standard titanium assay, presents a robust solution to sequence amplicons with a GC content of up to 77%. Moreover, this assay was subsequently tested on a larger independent cohort of 23 AML patients. For each patient, a median of 737 reads was generated (coverage range, 397-fold to 1194-fold) and therefore allowed a robust detection of insertions, deletions, and point mutations. In conclusion, next-generation amplicon sequencing enables the highly sensitive detection of molecular mutations and is a feasible assay for routine assessment of GC-rich content amplicons.
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Several lymphoma-specific genetic events in parallel can be found in mature B-cell neoplasms.
Genes Chromosomes Cancer
PUBLISHED: 02-23-2011
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The mature B-cell neoplasms frequently result from translocations involving the IGH gene localized on 14q32. In rare instances, combinations of different IGH rearrangements were described, and even coincidence of three genetic lymphoma-specific events has been rarely reported. We here present eight patients with triple hit lymphoma, and two with four different lymphoma specific events detected by chromosome banding analysis and interphase FISH (seven males, three females, 47-82 years). All showed bone marrow involvement (Burkitts lymphoma: n = 5, diffuse large B-cell lymphoma: n = 2; features intermediate between BL/DLBCL: n = 1; secondary aggressive B-cell lymphoma: n = 1; follicular lymphoma: n = 1). Eight patients had triple hit lymphoma combining IGH-BCL2/t(14;18)(q32;q21), MYC/8q24, and BCL6/3q27 rearrangements. Two showed an additional IGH-CCND1/t(11;14)(q13;q32) as fourth genetic event (according to research of the Mitelman database, the first reports on four genetic hits in lymphomas). All cases had complex aberrant karyotypes (median, 11 cytogenetic alterations per patient). Interphase FISH revealed a high rate of CDKN2A deletions (five of nine cases investigated; 56%), being homozygous in two of these cases. At the time of study, four of nine patients with follow-up data were alive (44%), one of these in remission. The two patients with four genetic hits died 6 and 9 days from diagnosis. Additional cases of lymphomas with three or four genetic hits should be collected to evaluate whether their clinical course differs from dual hit lymphomas.
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Subclones with the t(9;22)/BCR-ABL1 rearrangement occur in AML and seem to cooperate with distinct genetic alterations.
Br. J. Haematol.
PUBLISHED: 01-31-2011
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In AML, cooperation of mutations suppressing differentiation (class-II-mutations) with class-I-mutations increasing cell proliferation is frequent. In rare cases of myeloid malignancies, the BCR-ABL1 fusion was reported to cooperate as class-I-mutation with class-II-mutations, but most cases had to be classified as blast phase of chronic myeloid leukaemia (CML). We identified five cases of Philadelphia positive subclones in AML occurring in coincidence with other genetic lesions: 1:220 patients with inv(16)/CBFB-MYH11 (0·5%), 2:272 AML cases with t(8;21)/RUNX1-RUNX1T1 (0·7%), 1:1029 NPM1-mutated AML (0·1%), and one patient with s-AML following MDS with a 5q-deletion. Four patients had m-BCR (e1a2) BCR-ABL1 transcripts; one case only had an M-BCR (b3a2) breakpoint. These cases allow some interesting conclusions: The BCR-ABL1 rearrangement apparently can cooperate with the NPM1 mutation similar to other class-I-mutations. The identification of Philadelphia positive subclones in <1% of patients with CBF-leukaemias fits well with previous observations that most CBF-AML are accompanied by activating mutations in genes enhancing proliferation. Since we observed the occurrence of the Philadelphia positive subclones at diagnosis, at relapse, or throughout the disease, the time point of the emergence of Philadelphia subclones seems variable in AML. Clinical research should further concentrate on Philadelphia positive subclones in AML to assess the clinical impact.
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Acute monoblastic/monocytic leukemia and chronic myelomonocytic leukemia share common immunophenotypic features but differ in the extent of aberrantly expressed antigens and amount of granulocytic cells.
Leuk. Lymphoma
PUBLISHED: 01-12-2011
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Differentiation between acute monoblastic/monocytic leukemia (AMoL) and chronic myelomonocytic leukemia (CMML) can be difficult. Therefore, we compared immunophenotypes between 27 cases of AMoL and 138 cases of CMML. Monocytopoietic cells showed aberrant coexpression of CD56 in all cases of AMoL vs. 81.9% of CMML (p?=?0.015). No other aberrantly expressed antigen was found in AMoL, while in CMML CD2 coexpression was found in 21.7% (p?=?0.005), lack of CD13 in 10.9%, and of HLA-DR in 4.3% (NS). Cytomorphology identified higher blast percentages and lower percentages of monocytes and granulocytic cells in AMoL (p?<0.001). Multiparameter flow-cytometry (MFC) found higher percentages of blasts (17.6?±?25.2 vs. 4.1?±?3.2, p?<0.001) and monocytopoietic cells (29.1?±?27.5 vs. 19.9?±?12.2, p?=?0.012) in AMoL and more granulocytic cells in CMML (52.4?±?19.2 vs. 26.0?±?22.4, p?<0.001). The mean ratio of monocytic:granulocytic cells was higher in AMoL (5.0 vs. 0.8; p?<0.001). It can be concluded that AMoL and CMML differ in aberrantly expressed antigens and the amount of granulocytic cells.
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RUNX1 mutations are frequent in de novo AML with noncomplex karyotype and confer an unfavorable prognosis.
Blood
PUBLISHED: 12-09-2010
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Analyses of 164 RUNX1 mutations (RUNX1mut) in 147 of 449 patients (32.7%) with normal karyotype or noncomplex chromosomal imbalances were performed. RUNX1mut were most frequent in acute myeloid leukemia French-American-British classification M0 (65.2%) followed by M2 (32.4%) and M1 (30.2%). Considering cytogenetics, RUNX1mut were most frequent in cases with +13 (27 of 30, 90%), whereas frequencies were similar in other cytogenetic groups (26%-36%). The molecular genetic markers most frequently associated with RUNX1mut were partial tandem duplication in the MLL gene (19.7%), internal tandem duplication in the FLT3 gene (FLT3-ITD; 16.3%), and NRAS mutations (9.5%). Patients with RUNX1mut had shorter overall and event-free survival compared with RUNX1 wild-type cases (median, 378 days vs not reached, P = .003; and median, 285 vs 450 days, P = .003, respectively). In addition, it was shown that the adverse effect of RUNX1 was independent of the adverse effect of FLT3-ITD as well as of the high frequency of prognostically favorable NPM1mut and CEBPAmut in the RUNX1wt group. No effect of the type or localization of the individual RUNX1 mutations was observed. Multivariate analysis showed independent prognostic relevance for overall survival for RUNX1mut (P = .029), FLT3-ITD (P = .003), age (P < .001), and white blood cell count (P < .002).
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Complete remission and early death after intensive chemotherapy in patients aged 60 years or older with acute myeloid leukaemia: a web-based application for prediction of outcomes.
Lancet
PUBLISHED: 12-03-2010
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About 50% of patients (age ?60 years) who have acute myeloid leukaemia and are otherwise medically healthy (ie, able to undergo intensive chemotherapy) achieve a complete remission (CR) after intensive chemotherapy, but with a substantially increased risk of early death (ED) compared with younger patients. We verified the association of standard clinical and laboratory variables with CR and ED and developed a web-based application for risk assessment of intensive chemotherapy in these patients.
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The European LeukemiaNet: achievements and perspectives.
Haematologica
PUBLISHED: 11-03-2010
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The only way to cure leukemia is by cooperative research. To optimize research, the European LeukemiaNet integrates 105 national leukemia trial groups and networks, 105 interdisciplinary partner groups and about 1,000 leukemia specialists from 175 institutions. They care for tens of thousands of leukemia patients in 33 countries across Europe. Their ultimate goal is to cure leukemia. Since its inception in 2002, the European LeukemiaNet has steadily expanded and has unified leukemia research across Europe. The European LeukemiaNet grew from two major roots: 1) the German Competence Network on Acute and Chronic Leukemias; and 2) the collaboration of European Investigators on Chronic Myeloid Leukemia. The European LeukemiaNet has improved leukemia research and management across Europe. Its concept has led to funding by the European Commission as a network of excellence. Other sources (European Science Foundation; European LeukemiaNet-Foundation) will take over when the support of the European Commission ends.
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IDH1 mutations are detected in 6.6% of 1414 AML patients and are associated with intermediate risk karyotype and unfavorable prognosis in adults younger than 60 years and unmutated NPM1 status.
Blood
PUBLISHED: 08-30-2010
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Mutations in the IDH1 gene at position R132 coding for the enzyme cytosolic isocitrate dehydrogenase are known in glioma and have recently been detected also in acute myeloid leukemia (AML). These mutations result in an accumulation of ?-ketoglutarate to R (2)-2-hydroxyglutarate (2HG). To further clarify the role of this mutation in AML, we have analyzed IDH1R132 in 1414 AML patients. We detected IDH1R132 mutations in 93 of 1414 patients (6.6%) with a clear prevalence in intermediate risk karyotype group (10.4%, P < .001). Although IDH1R132 mutations can incidentally occur together with all other molecular markers, there were strong associations with NPM1 mutations (14.2% vs 5.4% in NPM1wt, P < .001) and MLL-PTD (18.2% vs 7.0% in MLLwt, P = .020). IDH1-mutated cases more often had AML without maturation/French-American-British M1 (P < .001), an immature immunophenotype, and female sex (8.7% vs 4.7% in male, P = .003) compared with IDH1wt cases. Prognosis was adversely affected by IDH1 mutations with trend for shorter overall survival (P = .110), a shorter event-free survival (P < .003) and a higher cumulative risk for relapse (P = .001). IDH1 mutations were of independent prognostic relevance for event-free survival (P = .039) especially in the age group < 60 years (P = .028). In conclusion, these data show that IDH1R132 may significantly add information regarding characterization and prognostication in AML.
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The role of multiparameter flow cytometry for disease monitoring in AML.
Best Pract Res Clin Haematol
PUBLISHED: 08-12-2010
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Monitoring of the minimal residual disease (MRD) load has become essential for the choice of post-induction strategies in patients with acute myeloid leukemia (AML). Quantitative real-time and nested PCR guarantee highest sensitivities, but suitable markers for follow-up are available for ?60% of patients only: e.g. for those with reciprocal gene rearrangements or those with NPM1 mutations. On the other hand, for most AML patients, multiparameter flow cytometry (MFC) represents a good option for MRD monitoring. In virtually all AML patients, leukemia-associated immunophenotypes (LAIPs) are detectable with MFC. These can be targeted with high sensitivity ranging up to 10(-4) during the course of disease. Numerous studies demonstrated the prognostic power of the MRD levels determined by MFC at post-induction as well as post-consolidation time points in adults and children considered to be in hematologic remission of AML. The post-consolidation MRD status seems to have more prognostic power than post-induction levels. Thus, MFC can significantly contribute to risk assessment of patients with AML during and after treatment and allows clinicians to consider alternative strategies (e.g. allogeneic hematopoietic stem cell transplantation) earlier. Clinical studies need to focus on a standardization of these approaches to facilitate the translation of MFC-based MRD assessment into therapeutic decisions in patients with AML.
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Biological and clinical characterization of recurrent 14q deletions in CLL and other mature B-cell neoplasms.
Br. J. Haematol.
PUBLISHED: 07-22-2010
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14q-deletions have been repeatedly described in mature B-cell neoplasms, but not yet characterized in a larger cohort. Based on chromosome banding analysis, the present study identified 47 del(14q) cases in 3054 mature B-cell neoplasms (1·5%) (chronic lymphocytic leukaemia [CLL]: 1·9%; CLL/prolymphocytic leukaemia [PL]: 9·0%; others: 0·2%). Interphase fluorescence in situ hybridization was performed with probes for 14q22.1, 14q24.1, 14q32.33, and IGH@ (14q32.3). The del(14q) had heterogeneous size but showed a breakpoint cluster at the centromeric site in 14q24.1 (62% of cases). At the telomeric side, the most frequent breakpoint was within the IGH@ locus (14q32.3) between IGH@ 3-flanking and IGHV (IgVH) probes (45%). In 16 cases (34%), breakpoints occurred within 14q24.1 and 14q32.3. Eighty-one percent of del(14q) cases showed 1-3 additional cytogenetic alterations (in 45%, +12), and 56% were IGHV-unmutated. In all cases (16/16) with breakpoints in 14q24.1 and 14q32.3, a B-CLL immunophenotype was found. Clinical follow-up in 32 del(14q) patients was compared to 383 CLL and CLL/PL patients without del(14q). While 3-year-overall survival did not differ significantly, time to treatment was significantly shorter in the del(14q) cohort (21·0 months vs. 80·1 months, P = 0·015). In conclusion, the del(14q) is a rare recurrent alteration in diverse mature B-cell neoplasms, shows variable size but distinct clustering of breakpoints, and is associated with short time to treatment.
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Next-generation sequencing technology reveals a characteristic pattern of molecular mutations in 72.8% of chronic myelomonocytic leukemia by detecting frequent alterations in TET2, CBL, RAS, and RUNX1.
J. Clin. Oncol.
PUBLISHED: 07-19-2010
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Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic malignancy that is characterized by features of both a myeloproliferative neoplasm and a myelodysplastic syndrome. Thus far, data on a comprehensive cytogenetic or molecular genetic characterization are limited.
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A copy number repeat polymorphism in the transactivation domain of the CEPBA gene is possibly associated with a protective effect against acquired CEBPA mutations: an analysis in 1135 patients with AML and 187 healthy controls.
Exp. Hematol.
PUBLISHED: 07-10-2010
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CEBPA-mutated normal karyotype acute myeloid leukemia (AML) has recently been included as a provisional entity in the World Health Organization classification. The CEBPA mutations are heterogeneous, including missense/nonsense base exchanges, frameshift mutations, and insertions/deletions being distributed throughout the gene. One of the genetic alterations within CEBPA (c.1175_1180dup; p.P194_H195dup) was later suggested to represent an inherited polymorphism. As this is not a simple single nucleotide polymorphism, but a six-base pair insertion that leads to a two amino acid elongation of the protein, functional implications cannot be excluded.
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Associations between imatinib resistance conferring mutations and Philadelphia positive clonal cytogenetic evolution in CML.
Genes Chromosomes Cancer
PUBLISHED: 07-08-2010
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In patients with chronic myeloid leukemia (CML), resistance against imatinib is associated with mutations in the kinase domain (KD) of the BCR-ABL1 fusion gene and/or with additional chromosomal abnormalities (ACAs) secondary to the Philadelphia chromosome. To evaluate associations between these molecular and cytogenetic events, we correlated cytogenetic data with results of KD mutation analysis in 205 CML patients with acquired resistance to imatinib (100 females, 105 males, 17.9-90.3 years). In 79/205 patients (38.5%), at least one KD mutation was detected; in 13/79 (16.5%) even two different mutations were observed. A second KD mutation was frequent in cases with G250E (4/9), E255V (1/3), T315I (5/18), F317L (2/7), F359C/V (4/7), or H396R (2/3), but was never detected in cases with V299L (n = 3) or Y253H (n = 4). With respect to cytogenetics, ACAs were identified in 76/205 patients (37.1%), in 29 (36.7%) together with KD mutations. ACAs were frequent in cases with E255V (2/3), T315I (8/18), F317L (4/7), F359C/V (4/7), or H396R (2/3), but rare in those with M351T (1/9). Therefore, some KD mutations (e.g., T315I) might be associated with higher genetic instability paving the way to additional cytogenetic and molecular genetic events.
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Multilineage dysplasia (MLD) in acute myeloid leukemia (AML) correlates with MDS-related cytogenetic abnormalities and a prior history of MDS or MDS/MPN but has no independent prognostic relevance: a comparison of 408 cases classified as "AML not otherwis
Blood
PUBLISHED: 06-25-2010
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The World Health Organization classification of acute myeloid leukemia (AML) is hierarchically structured and integrates genetics, data on patients history, and multilineage dysplasia (MLD). The category "AML with myelodysplastic syndrome (MDS)-related changes" (AML-MRC) is separated from "AML not otherwise specified" (AML-NOS) by presence of MLD, MDS-related cytogenetics, or history of MDS or MDS/myeloproliferative neoplasm (MPN). We analyzed 408 adult patients categorized as AML-MRC or AML-NOS. Three-year event-free survival (EFS; median, 13.8 vs 16.0 months) and 3-year overall survival (OS; 45.8% vs 53.9%) did not differ significantly between patients with MLD versus without. However, MLD correlated with preexisting MDS (P < .001) and MDS-related cytogenetics (P = .035). Patients with MLD as sole AML-MRC criterion (AML-MLD-sole; n = 90) had less frequently FLT3 internal tandem duplication (P = .032) and lower median age than AML-NOS (n = 232). Contrarily, patients with AML-NOS combined with AML-MLD-sole (n = 323) had better 3-year EFS (16.9 vs 10.7 months; P = .005) and 3-year OS (55.8% vs 32.5%; P = .001) than patients with history of MDS or MDS/MPN or MDS-related cytogenetics (n = 85). Gene expression analysis showed distinct clusters for AML-MLD-sole combined with AML-NOS versus AML with MDS-related cytogenetics or MDS history. Thus, MLD alone showed no independent clinical effect, whereas cytogenetics and MDS history were prognostically relevant.
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Clinical utility of multiparameter flow cytometry in the diagnosis of 1013 patients with suspected myelodysplastic syndrome: correlation to cytomorphology, cytogenetics, and clinical data.
Cancer
PUBLISHED: 06-24-2010
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The diagnosis and classification of myelodysplastic syndromes (MDS) is based on cytomorphology (CM) and cytogenetics (CG). Multiparameter flow cytometry (MFC) may add important diagnostic information.
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Toward a comprehensive prognostic scoring system in chronic lymphocytic leukemia based on a combination of genetic parameters.
Genes Chromosomes Cancer
PUBLISHED: 06-17-2010
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Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with a variable clinical course. The aim of this study was to evaluate whether a combination of genetic parameters can improve prediction of outcome irrespective of clinical stage. The prognostic impact of chromosome banding analysis (CBA) in addition to FISH and IgVH mutation status was evaluated. In total, 482 patients were analyzed, but evaluation of prognostic factors was restricted to 399 untreated cases. The prognostic significance of age, white blood cell (WBC) count, IgVH status, and TP53 and ATM deletions was confirmed. In addition, a prognostic impact of translocations involving the IGH@ locus (t(IgH)) and of a complex aberrant karyotype was found. On the basis of these results, we propose a scoring system for overall survival (OS) based on: age >or=65 years, WBC >or=20 x 10(9)/l, unmutated IgVH status, TP53 deletion, t(IgH), and the number of chromosome aberrations observed with CBA. Three risk groups showed considerable differences in OS (94.5% vs. 64.3% vs. 41.1% surviving at 5 years, P < 0.0001). Time to treatment (TTT) can be predicted best by unmutated IgVH status, ATM deletion, t(IgH), and number of chromosome aberrations. Four distinct subgroups were separated with median TTT of 110.7 months, 39.8 months, 19.5 months, and 3.8 months, respectively (P < 0.0001). In conclusion, cytogenetic data from CBA add prognostic information. The proposed scoring systems for OS and TTT based on a combination of genetic markers improve the separation of prognostic subgroups in CLL already early in the course of the disease.
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Genomic profiling of adult acute lymphoblastic leukemia by single nucleotide polymorphism oligonucleotide microarray and comparison to pediatric acute lymphoblastic leukemia.
Haematologica
PUBLISHED: 04-30-2010
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Differences in survival have been reported between pediatric and adult acute lymphoblastic leukemia. The inferior prognosis in adult acute lymphoblastic leukemia is not fully understood but could be attributed, in part, to differences in genomic alterations found in adult as compared to in pediatric acute lymphoblastic leukemia.
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Detection of centrosome aberrations in disease-unrelated cells from patients with tumor treated with tyrosine kinase inhibitors.
Eur. J. Haematol.
PUBLISHED: 04-16-2010
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Tyrosine kinase inhibitors (TKIs) target various pathways associated with proliferation of aberrant clones in malignant diseases. Despite good response and acceptable tolerability, little is known concerning long-term toxicity. Furthermore, the influence of these inhibitors on disease-unrelated cells is not investigated yet.
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Comparison of cytogenetic clonal evolution patterns following allogeneic hematopoietic transplantation versus conventional treatment in patients at relapse of AML.
Biol. Blood Marrow Transplant.
PUBLISHED: 03-21-2010
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Relapse of acute myelogenous leukemia has been associated with clonal cytogenetic evolution, but no study focused specifically on relapse after allogeneic hematopoietic stem cell transplantation (HSCT). We compared karyotypes in 160 patients at both diagnosis and relapse either after allo-HSCT (n = 26) or standard chemotherapy (n = 134) using chromosome banding analysis combined with fluorescein in situ hybridization. There were 71 females and 89 males (19.7-80.6 years). At diagnosis, aberrant karyotypes were more frequent in the HSCT than in the chemotherapy cohort (16 of 26; 61.5% versus 63 of 134; 47.0%). This was most obvious in patients with unfavorable cytogenetics (8 of 26; 30.8% versus 19 of 134; 14.2%; P = .032). Differences in the karyotypes between diagnosis and relapse were more frequent in the allo-cohort (14 of 26; 53.8% versus 49 of 134; 36.6%) than in the conventional cohort (n.s.), mainly because of newly emerging cytogenetic alterations. Appearance of ? 3 new clonal alterations was more frequent in the allo-cohort (6 of 12; 50.0% with clonal evolution versus 5 of 41; 12.2%, P = .005). The mean number of cytogenetic alterations per patient was increasing from 2.0 at diagnosis to 4.0 at relapse in the allo-cohort, in the conventionally treated patients from 0.9 to 1.3 (both P < .001). Thus, higher frequencies of clonal evolution and increasing cytogenetic complexity were observed in the stem cell recipients probably related to the more unfavorable cytogenetic profiles already depicted at diagnosis.
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Detection of a t(4;14)(p16;q32) in two cases of lymphoma showing both the immunophenotype of chronic lymphocytic leukemia.
Cancer Genet. Cytogenet.
PUBLISHED: 03-15-2010
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Reciprocal IGH/14q32 translocations are detectable in 55-70% of patients with plasma cell myeloma; e.g., the adverse t(4;14)(p16;q32) fusing the IGH and FGFR3 genes (immunoglobulin heavy chain/fibroblast growth factor receptor 3). In a minority of patients with B-lineage chronic lymphocytic leukemia (CLL), reciprocal IGH/14q32 translocations have been reported as well. We describe the occurrence of a t(4;14)(p16;q32) in two lymphoma patients showing the immunophenotype of B-CLL, which, to our knowledge, is the first report on such an association. The first patient, a 72-year-old female, showed mature lymphocyte infiltration of the bone marrow and marked splenomegaly. Immunophenotyping revealed aberrant CD5 expression and light-chain lambda restriction on mature B-lymphocytes corresponding to a B-CLL. Interphase fluorescence in situ hybridization (FISH) plus chromosome banding revealed a t(4;14)(p16;q32) in addition to an unbalanced der(16)t(8;16)(q23;p13) and a del(8)(p11). The second patient, a male of 65 years, showed marked peripheral leukocytosis. Immunophenotyping revealed the phenotype of CLL/PLL (chronic lymphocytic leukemia/prolymphocytic leukemia). Again, FISH together with karyotyping revealed a t(4;14)(p16;q32). These two cases with an t(4;14), but the immunophenotype of B-CLL, demonstrate the genetic variability of B-cell lymphomas and the potential of specific metaphase cultivation techniques using oligonucleotides to increase our insights in the genetic pathways of these heterogeneous disorders.
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Multilineage dysplasia has no impact on biologic, clinicopathologic, and prognostic features of AML with mutated nucleophosmin (NPM1).
Blood
PUBLISHED: 03-04-2010
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NPM1-mutated acute myeloid leukemia (AML) is a provisional entity in the 2008 World Health Organization (WHO) classification of myeloid neoplasms. The significance of multilineage dysplasia (MLD) in NPM1-mutated AML is unclear. Thus, in the 2008 WHO classification, NPM1-mutated AML with MLD is classified as AML with myelodysplasia (MD)-related changes (MRCs). We evaluated morphologically 318 NPM1-mutated AML patients and found MLD in 23.3%. Except for a male predominance and a lower fms-related tyrosine kinase 3-internal tandem duplication (FLT3-ITD) incidence in the MLD(+) group, no differences were observed in age, sex, cytogenetics, and FLT3--tyrosine kinase domain between NPM1-mutated AML with and without MLD. NPM1-mutated AML with and without MLD showed overlapping immunophenotype (CD34 negativity) and gene expression profile (CD34 down-regulation, HOX genes up-regulation). Moreover, overall and event-free survival did not differ among NPM1-mutated AML patients independently of whether they were MLD(+) or MLD(-), the NPM1-mutated/FLT3-ITD negative genotype showing the better prognosis. Lack of MLD impact on survival was confirmed by multivariate analysis that highlighted FLT3-ITD as the only significant prognostic parameter in NPM1-mutated AML. Our findings indicate that NPM1 mutations rather than MLD dictate the distinctive features of NPM1-mutated AML. Thus, irrespective of MLD, NPM1-mutated AML represents one disease entity clearly distinct from AML with MRCs.
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Correlation of cytomorphology, immunophenotyping, and interphase fluorescence in situ hybridization in 381 patients with monoclonal gammopathy of undetermined significance and 301 patients with plasma cell myeloma.
Cancer Genet. Cytogenet.
PUBLISHED: 02-19-2010
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To further clarify the transformation from monoclonal gammopathy of undetermined significance (MGUS) to plasma cell myeloma (PCM), we compared interphase fluorescence in situ hybridization (FISH) patterns in 381 MGUS and 301 PCM patients. According to the World Health Organization and the International Myeloma Working Group, a threshold of 10% of bone marrow plasma cells separated MGUS from PCM. After magnetic activated cell sorting for CD138(+) cells, FISH succeeded in 272 of 301 (90.4%) PCM, but in only 302 of 381 (79.3%) MGUS cases (P < 0.001). Cytogenetic alterations were more frequent in PCM (237 of 272; 87.1%) than MGUS (169 of 302; 56.0%; P = 0.0002). PCM showed a median of two cytogenetic alterations (range, 0-9) and MGUS one (range, 0-6). Considering only cases with a yield of plasma cells allowing five or more FISH probes, del(13)(q14) was found in 99 of 251 (39.3%) PCM but in only 59 of 267 (22.1%) MGUS (P = 0.0001), del(17p) in 15 PCM (6.0%) and in 6 MGUS (2.2%) patients (P = 0.029). A t(4;14)/IGH-FGFR3 was detected in 28 PCM (11.1%) and 5 MGUS (1.9%; P < 0.001). The t(11;14)/IGH-CCND1 and the t(14;16)/IGH-MAF showed no significant differences. Cytomorphology detected higher numbers of plasma cells than multiparameter flow cytometry (median ratio 4.25). This study underlines the genetic heterogeneity of MGUS similar to PCM. Genetic analysis might contribute to more diversified monitoring strategies for MGUS patients.
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Atypical mRNA fusions in PML-RARA positive, RARA-PML negative acute promyelocytic leukemia.
Genes Chromosomes Cancer
PUBLISHED: 02-16-2010
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Reciprocal RARA-PML transcripts are not detected in approximately 25% of patients with PML-RARA positive acute promyelocytic leukemia (APL), but the reasons for this are poorly understood. We studied 21 PML-RARA positive/RARA-PML negative cases by bubble PCR and multiplex long template PCR to identify the genomic breakpoints. Additional RT-PCR analysis was performed based on the DNA findings. Three cases were found to have complex rearrangements involving a third locus: the first had a PML-CDC6-RARA forward DNA fusion and expressed a chimeric PML-CDC6-RARA mRNA in addition to a PML-RARA. The other two had HERC1-PML and NT_009714.17-PML genomic fusion sequences at their respective reciprocal breakpoints. Six patients were falsely classified as RARA-PML negative due to deletions on chromosome 15 and/or 17, or alternative splicing leading to atypical RARA-PML fusion transcripts, which were not identified by conventional RT-PCR assays. This study demonstrates that the frequency of RARA-PML expression has been underestimated and highlights remarkable complexity at chromosomal breakpoint regions in APL even in cases with an apparently simple balanced t(15;17)(q24;q12).
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Screening for diverse PDGFRA or PDGFRB fusion genes is facilitated by generic quantitative reverse transcriptase polymerase chain reaction analysis.
Haematologica
PUBLISHED: 01-27-2010
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Rapid identification of diverse fusion genes with involvement of PDGFRA or PDGFRB in eosinophilia-associated myeloproliferative neoplasms is essential for adequate clinical management but is complicated by the multitude and heterogeneity of partner genes and breakpoints.
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The idic(X)(q13) in myeloid malignancies: breakpoint clustering in segmental duplications and association with TET2 mutations.
Hum. Mol. Genet.
PUBLISHED: 01-21-2010
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Myelodysplastic syndromes and acute myeloid leukemia with an isodicentric X chromosome [idic(X)(q13)] occur in elderly women and frequently display ringed sideroblasts. Because of the rarity of idic(X)(q13), little is known about its formation, whether a fusion gene is generated, and patterns of additional aberrations. We here present an SNP array study of 14 idic(X)-positive myeloid malignancies, collected through an international collaborative effort. The breakpoints clustered in two regions of segmental duplications and were not in a gene, making dosage effects from the concurrent gain of Xpter-q13 and loss of Xq13-qter, rather than a fusion gene, the most likely pathogenetic outcome. Methylation analysis revealed involvement of the inactive X chromosomes in five cases and of the active in two. The ABCB7 gene, mutated in X-linked sideroblastic anemia and spinocerebellar ataxia, is in the deleted region, suggesting that loss of this gene underlies the frequent presence of ringed sideroblasts. Additional genetic abnormalities were present in 12/14 (86%), including partial uniparental disomies for 9p (one case) and 4q (two cases) associated with homozygous mutations of JAK2 and TET2, respectively. In total, TET2 mutations were seen in 4/11 (36%) analyzed cases, thus constituting a common secondary event in idic(X)-positive malignancies.
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Mutations of the TET2 and CBL genes: novel molecular markers in myeloid malignancies.
Ann. Hematol.
PUBLISHED: 01-02-2010
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Despite recent progress in molecular research in myeloid malignancies, in subsets of patients with myelodysplastic syndrome (MDS) so far no underlying mutation was identified. In the myeloproliferative neoplasms (MPNs), the JAK2V617F alone cannot explain the phenotypic heterogeneity. In acute myeloid leukemia (AML), clinical variability exists within distinct subgroups. Thus, the search for novel molecular markers continues. Recently, mutations of the tet oncogene family member 2 (TET2) and Casitas B-cell lymphoma (CBL) genes became the focus of interest. With diverse genetic methods, TET2 on chromosome 4q24 was identified as candidate tumor suppressor gene. Sequencing studies revealed heterogeneous mutations in 10-25% of patients with acute myeloid leukemia (AML), MDS, and MPNs, while the frequency might be higher in chronic myelomonocytic leukemia (CMML). The prognostic impact is being explored. The CBL gene is involved in the degradation of tyrosine kinases. In rare cases of human AML (<2%), CBL mutants were identified, with a higher frequency in core binding factor leukemias. Presence of these mutations was suggested to be involved in aberrant FLT3 expression. In the MPNs, a 2-8% frequency of CBL mutations was reported. These novel mutations deepened insights in the mechanisms of leukemogenesis, might contribute to the identification of new therapeutic targets, and improve diagnostics in the myeloid malignancies.
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SNP array analysis of tyrosine kinase inhibitor-resistant chronic myeloid leukemia identifies heterogeneous secondary genomic alterations.
Blood
PUBLISHED: 12-02-2009
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To elucidate whether tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia is associated with characteristic genomic alterations, we analyzed DNA samples from 45 TKI-resistant chronic myeloid leukemia patients with 250K single nucleotide polymorphism arrays. From 20 patients, matched serial samples of pretreatment and TKI resistance time points were available. Eleven of the 45 TKI-resistant patients had mutations of BCR-ABL1, including 2 T315I mutations. Besides known TKI resistance-associated genomic lesions, such as duplication of the BCR-ABL1 gene (n = 8) and trisomy 8 (n = 3), recurrent submicroscopic alterations, including acquired uniparental disomy, were detectable on chromosomes 1, 8, 9, 17, 19, and 22. On chromosome 22, newly acquired and recurrent deletions of the IGLC1 locus were detected in 3 patients, who had previously presented with lymphoid or myeloid blast crisis. This may support a hypothesis of TKI-induced selection of subclones differentiating into immature B-cell progenitors as a mechanism of disease progression and evasion of TKI sensitivity.
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