Nóra Meggyesi

Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia

Abstract Mutations of isocitrate dehydrogenase 1 and 2 (IDH1/2) are genetic alterations in acute myeloid leukemia (AML). The aim of our study was to investigate the frequency and prognostic effect of IDH1/2 mutations together followed by an individual analysis of each substitution in a Hungarian cohort consisting of 376 patients with AML. IDH1mut and IDH2mut were mutually exclusive, detected in 8.5% and 7.5% of cases, respectively. IDH1/2mut was associated with: older age (p = 0.001), higher average platelet count (p = 0.001), intermediate karyotype (p < 0.0001), NPM1mut (p = 0.022) and lower mRNA expression level of ABCG2 gene (p = 0.006). Overall survival (OS), remission and relapse rates were not different in IDH1mut or IDH2mut vs. IDHneg. IDH1mut and IDH2mut were associated differently with NPM1mut; co-occurrence was observed in 14.3% of IDH1 R132C vs. 70% of R132H carriers (p = 0.02) and in 47.4% of IDH2 R140Q vs. 0% of R172K carriers (p = 0.02). IDH1 R132H negatively influenced OS compared to IDHneg (p = 0.02) or R132C (p = 0.019). Particular amino acid changes affecting the same IDH1 codon influence the clinical characteristics and treatment outcome in AML.

Additional Chromosome Abnormalities, BCR-ABL Tyrosine Kinase Domain Mutations and Clinical Outcome in Hungarian Tyrosine Kinase Inhibitor-Resistant Chronic Myelogenous Leukemia Patients

Background: Additional chromosome abnormalities (ACAs), mutations of the BCR-ABL tyrosine kinase domain (TKD) and BCR-ABL splice variants may cause resistance to first- and second-generation tyrosine kinase inhibitors (TKIs) in chronic myelogenous leukemia (CML) and Philadelphia-positive (Ph+) acute lymphoid leukemia (ALL). Methods: Karyotyping and BCR-ABL TKD mutation screening were performed in 71 imatinib-resistant CML patients and 6 Ph+ ALL patients. A total of 56 out of these 77 patients received second-generation TKI. Results: ACAs were present in 30 of 65 imatinib-resistant patients (46%). In 27 of 74 imatinib-resistant patients (36%), 15 different BCR-ABL TKD mutations were detected. Mutations were found in 25% of chronic-phase patients (12/47), 33% of accelerated-phase patients (5/15), 71% of blast crisis CML patients (5/7) and 100% of ALL patients. In nilotinib-resistant patients, Y253H, T315I and F359I/V mutations were detected; in dasatinib-resistant patients, L248M, E279K and T315I mutations were detected. T315I was found more frequently in patients on dasatinib than on imatinib therapy. The presence of ACAs predicted shorter survival during first- and second-generation TKI therapy, while TKD mutations only influenced survival during second-generation TKI therapy. Conclusion: For patients with TKI resistance, mutation and ACA screening may play a role in identifying patients with poorer prognosis.

The prognostic impact of germline 46/1 haplotype of Janus kinase 2 in cytogenetically normal acute myeloid leukemia.

Background Prognostic risk stratification according to acquired or inherited genetic alterations has received increasing attention in acute myeloid leukemia in recent years. A germline Janus kinase 2 haplotype designated as the 46/1 haplotype has been reported to be associated with an inherited predisposition to myeloproliferative neoplasms, and also to acute myeloid leukemia with normal karyotype. The aim of this study was to assess the prognostic impact of the 46/1 haplotype on disease characteristics and treatment outcome in acute myeloid leukemia. Design and Methods Janus kinase 2 rs12343867 single nucleotide polymorphism tagging the 46/1 haplotype was genotyped by LightCycler technology applying melting curve analysis with the hybridization probe detection format in 176 patients with acute myeloid leukemia under 60 years diagnosed consecutively and treated with curative intent. Results The morphological subtype of acute myeloid leukemia with maturation was less frequent among 46/1 carriers than among non-carriers (5.6% versus 17.2%, P=0.018, cytogenetically normal subgroup: 4.3% versus 20.6%, P=0.031), while the morphological distribution shifted towards the myelomonocytoid form in 46/1 haplotype carriers (28.1% versus 14.9%, P=0.044, cytogenetically normal subgroup: 34.0% versus 11.8%, P=0.035). In cytogenetically normal cases of acute myeloid leukemia, the 46/1 carriers had a considerably lower remission rate (78.7% versus 94.1%, P=0.064) and more deaths in remission or in aplasia caused by infections (46.8% versus 23.5%, P=0.038), resulting in the 46/1 carriers having shorter disease-free survival and overall survival compared to the 46/1 non-carriers. In multivariate analysis, the 46/1 haplotype was an independent adverse prognostic factor for disease-free survival (P=0.024) and overall survival (P=0.024) in patients with a normal karyotype. Janus kinase 2 46/1 haplotype had no impact on prognosis in the subgroup with abnormal karyotype. Conclusions Janus kinase 2 46/1 haplotype influences morphological distribution, increasing the predisposition towards an acute myelomonocytoid form. It may be a novel, independent unfavorable risk factor in acute myeloid leukemia with a normal karyotype.

Characterization of ABL exon 7 deletion by molecular genetic and bioinformatic methods reveals no association with imatinib resistance in chronic myeloid leukemia

In chronic myeloid leukemia (CML), the best characterized imatinib resistance mechanisms are BCR-ABL tyrosine kinase domain mutations and clonal evolution, but recently alternative splicing of BCR-ABL was also proposed as a mechanism for imatinib resistance. Among recently reported BCR-ABL splice variants, exon 7 deletion (Δexon7) was characterized in this study. The frequency of Δexon7 was investigated in 30 healthy controls and in 76 CML patients at different time points of the disease course by four different molecular genetic methods (direct sequencing, fragment analysis, allele-specific and quantitative PCR). The functionality and viability of the variant protein was tested by bioinformatic prediction. The Δexon7 was abundantly detected with similar frequency in healthy controls, in imatinib naive and resistant CML patients on BCR-ABL and also on the nontranslocated ABL. The detection rate of Δexon7 (varying between 17 and 100%) was highly dependent on the expression levels of BCR-ABL or ABL and the sensitivity of detection method. According to secondary structure prediction by bioinformatic methods, the exon 7 deleted mRNA is a target for nonsense-mediated decay, and the translated protein is likely to be nonfunctional and unstable. Taken together all the above observations, we concluded that Δexon7 is a common splice variant not associating with imatinib resistance.

HFE C282Y Mutation as a Genetic Modifier Influencing Disease Susceptibility for Chronic Myeloproliferative Disease

Iron metabolism has been implicated in carcinogenesis and several studies assessed the potential role of genetic variants of proteins involved in iron metabolism (HFE C282Y, TFR S142G) in different malignancies. Few reports addressed this issue with relation to chronic myeloproliferative disorders (CMPD). The aims of our study were (a) to examine the potential associations of CMPD development with genetic modifiers of iron metabolism in a large cohort of CMPD patients; (b) to examine associations of genetic variants of proteins involved in iron metabolism; and acquired JAK2 V617F mutation with clinical characteristics of CMPD. HFE C282Y was genotyped in 328 CMPD patients and 996 blood donors as controls, HFE H63D, and TFR S142G were tested in CMPD patients and 171 first time blood donors. JAK2 V617F mutation was tested in CMPD patients and in 122 repeated blood donors. Decreased C282Y allele frequency (allele frequency ± 95% confidence interval) was found in the CMPD group (1.8% ± 1.0%) compared with controls (3.4% ± 0.8%; P = 0.048). TFR S142G allele frequency was reduced among V617F-negative CMPD patients (34.8% ±7.6%) compared with controls (47.8% ± 5.4%; P = 0.02). The frequency of JAK2 V617F was 75.9% (249 of 328) in the CMPD group. At presentation, elevated hemoglobin levels were found in V617F-positive patients compared with V617F-negative counterparts (P < 0.000). Vascular complications (26.6% versus 15.2%; P = 0.039) as well as female gender (57.4% versus 41.8%; P = 0.019) were more common in V617F-positive patients. We found that HFE C282Y might be associated with a protective role against CMPD. Because chronic iron deficiency or latent anemia may trigger disease susceptibility for CMPD, HFE C282Y positivity may be a genetic factor influencing this effect. (Cancer Epidemiol Biomarkers Prev 2009;18(3):929–34)

Medium-sized FLT3 internal tandem duplications confer worse prognosis than short and long duplications in a non-elderly acute myeloid leukemia cohort

Abstract Internal tandem duplications (ITDs) of the fms-like tyrosine kinase 3 (FLT3) gene occur in about 25% of patients with adult acute myeloid leukemia (AML). The aim of our study was to investigate the frequency of FLT3-ITD mutations followed by a detailed analysis of the mutational load and size of ITD insertions in a cohort consisting of 324 patients younger than 60 years old and treated with curative intention. FLT3-ITD alone did not influence overall survival (OS) or disease-free survival (DFS). We observed worse OS and DFS for patients with high mutational load indicative for loss of the FLT3 wild type allele (p = 0.010, p = 0.038, respectively). In multivariate analyses, patients with FLT3-ITD48–60bp showed worse OS and DFS compared to other groups (FLT3-ITDneg, FLT3-ITD < 48b, FLT3-ITD > 60bp; p = 0.014, p = 0.019, respectively). Our novel observation suggested that not only high FLT3-ITD load, but also medium-sized ITD insertions (48–60 bp) represented an adverse prognostic subgroup of patients with AML.

A 2-es típusú Janus tirozin kináz Val617Phe aktiváló pontmutáció szerepe és kimutatásának jelentosége myeloproliferatív szindrómában

The Val617Phe point mutation of Janus kinase 2 gene is believed to participate in the pathogenesis of myeloproliferative syndrome characterised by the clonal alteration of hematopoietic stem cells. According to current results, the frequency of Val617Phe activating mutation is around 80% in polycythaemia vera, 35% in essential thrombocythemia, and 50% in chronic idiopathic myelofibrosis. The diagnoses of polycythemia vera, essential thrombocythemia and idiopathic myelofibrosis were so far based on the exclusion of secondary factors as well as bone marrow biopsy histology. The goal of the present work was to establish simple molecular genetic techniques for the routine testing of Janus kinase 2 gene Val617Phe mutation, and to compare the clinical phenotypes of Val617Phe mutation positive and negative myeloproliferative syndromes. We employed the allele specific polymerase chain technique for detection of Val617Phe mutation in 252 patients with myeloproliferative syndrome. We measured Val617Phe frequency as 85,4% (117/137) in polycythemia vera, 56,6% (56/99) in essential thrombocythemia, and 87,5% (14/16) in idiopathic myelofibrosis. We found significantly elevated hemoglobin levels and white blood cell counts (measured at the time of diagnosis) in Val617Phe-positive polycythemia vera and essential thrombocythemia patient groups compared to Val617Phe-negative patients. However, the frequencies of splenomegaly and other complications (thrombosis, bleeding, transformation to acute leukemia) were not significantly different between the mutation-positive and negative groups. In conclusion, the non-invasive mutation analysis of the Janus kinase 2 Val617Phe is suitable for routine laboratory application and helps the differential diagnosis of myeloproliferative syndrome. Although the exact role of Val617Phe mutation testing has not yet been identified on the basis of a broad professional consensus, the testing is suggested in cases of erythrocytoses and thrombocytoses of unknown origin.

Complex molecular genetic algorithm in the diagnosis of myeloproliferative neoplasms

Bevezetes: A Philadelphia-kromoszoma negativ, „klasszikus” myeloproliferativ neoplasiak genetikai hattereben a Janus kinaz 2, a calreticulin- es a trombopoetinreceptor genmutacioit azonositottak. Celkitűzes: A koroki mutacio azonositasa 949, myeloproliferativ neoplasiaban szenvedő betegnel. Modszer: A szerzők minősegi es mennyisegi allelspecifikus polimeraz lancreakcio, fragmensanalizis, nagy felbontasu olvadasigorbe-analizis es Sanger-szekvenalas kombinacioit alkalmaztak. Eredmenyek: 354 polycytaemia veraban szenvedő beteg 98,6%-a (n = 349) hordozta a Janus kinaz 2 gen V617F mutaciojat, 1,4%-uk (n = 5) pedig a 12. exon mutacioit. Esszencialis thrombocythaemiaban (n = 468) a betegek 61,3%-anal (n = 287) V617F-, 25,2%-anal (n = 118) calreticulin- es 2,1%-anal (n = 10) trombopoetinreceptor-genmutaciot talaltak, mig a betegek 11,3%-a (n = 53) a fenti mutaciok egyiket sem hordozta (triplan negativak). Hasonlo eloszlast tapasztaltak primer myelofibrosisban (n = 127): 58,3% (n = 74) V617F-, 23,6% (n = 30) calreticulin-, 6,3% (n = 8) trombopoetinreceptor mutacio pozitiv, 11,8% (n = 15) triplan negativ. Kovetkeztetesek: A calreticulingent erintő, nemregiben felfedezett mutaciok reven a Philadelphia kromoszoma negativ, „klasszikus” myeloproliferativ neoplasiaban szenvedő betegek kozel 90%-anal hatarozhato meg a betegseget okozo genetikai elteres. Orv. Hetil., 2014, 155(52), 2074–2081. n | n Introduction: Mutations in Janus kinase 2, calreticulin and thrombopoietin receptor genes have been identified in the genetic background of Philadelphia chromosome negative, “classic” myeloproliferative neoplasms. Aim: The aim of the authors was to identify driver mutations in a large myeloproliferative cohort of 949 patients. Method: A complex array of molecular techniques (qualitative and quantitative allele-specific polymerase chain reactions, fragment analyzes, high resolution melting and Sanger sequencing) was applied. Results: All 354 patients with polycythemia vera carried Janus kinase 2 mutations (V617F 98.6%, exon 12: 1.4%). In essential thrombocythemia (n = 468), the frequency of V617F was 61.3% (n = 287), that of calreticulin 25.2% (n = 118), and that of thrombopoietin receptor mutations 2.1% (n = 10), while 11.3% (n = 53) were triple-negative. Similar distribution was observed in primary myelofibrosis (n = 127): 58.3% (n = 74) V617F, 23.6% (n = 30) calreticulin, 6.3% (n = 8) thrombopoietin receptor mutation positive and 11.8% (n = 15) triple-negative. Conclusions: The recent discovery of calreticulin gene mutations led to definite molecular diagnostics in around 90% of clonal myeloproliferative cases. Orv. Hetil., 2014, 155(52), 2074–2081.INTRODUCTIONnMutations in Janus kinase 2, calreticulin and thrombopoietin receptor genes have been identified in the genetic background of Philadelphia chromosome negative, classic myeloproliferative neoplasms.nnnAIMnThe aim of the authors was to identify driver mutations in a large myeloproliferative cohort of 949 patients.nnnMETHODnA complex array of molecular techniques (qualitative and quantitative allele-specific polymerase chain reactions, fragment analyzes, high resolution melting and Sanger sequencing) was applied.nnnRESULTSnAll 354 patients with polycythemia vera carried Janus kinase 2 mutations (V617F 98.6%, exon 12: 1.4%). In essential thrombocythemia (n = 468), the frequency of V617F was 61.3% (n = 287), that of calreticulin 25.2% (n = 118), and that of thrombopoietin receptor mutations 2.1% (n = 10), while 11.3% (n = 53) were triple-negative. Similar distribution was observed in primary myelofibrosis (n = 127): 58.3% (n = 74) V617F, 23.6% (n = 30) calreticulin, 6.3% (n = 8) thrombopoietin receptor mutation positive and 11.8% (n = 15) triple-negative.nnnCONCLUSIONSnThe recent discovery of calreticulin gene mutations led to definite molecular diagnostics in around 90% of clonal myeloproliferative cases.

The adverse effect of FOPNL genomic variant is reversed by bortezomib-based treatment protocols in multiple myeloma

Abstract Fibroblast growth factor receptor 1 oncogene partner N-terminal like gene (FOPNL) rs72773978 polymorphism was identified as an adverse prognostic factor in multiple myeloma (MM). We aimed to investigate the associations of rs72773978 with clinical characteristics and treatment outcome in 373 Hungarian MM patients. In our cohort, FOPNL polymorphism showed differential prognostic effect that depended on the treatment applied. Among patients treated with non-proteasome inhibitor (PI)-based therapy, carriership of the minor allele was significantly associated with adverse overall survival (p=.022). In contrast, the adverse effect was overcome by the application of PI-containing treatment (p=.048). Multivariate analyses revealed the independent adverse effect of rs72773978 on survival in the non-PI-treated group (p=.045), but not in PI treatment (OS: p=.093). We confirmed the adverse prognostic effect of rs72773978 associated with non-PI-based treatment regimens. Our results point to the importance of genotypic prognostic information associated with complex clinical background MM.

Proteasome Subunit Beta Type 1 P11A Polymorphism Is a New Prognostic Marker in Multiple Myeloma

Background Proteasome subunit beta type 1 (PSMB1) rs12717 polymorphism, a single nucleotide polymorphism with unknown functional effect, was recently reported to influence response to bortezomib‐based therapy in follicular lymphoma. Patients and Methods We retrospectively analyzed the prognostic impact of this polymorphism in 211 consecutively diagnosed multiple myeloma cases, and performed in vitro experiments to look into its functional consequences. Results On univariate analysis, patients carrying the variant G allele showed significantly shorter progression‐free survival (PFS) with a pattern suggestive of a gene‐dose effect (PFS 26.4, 22.3, and 16.4 months in C/C, C/G, and G/G patients, respectively, P = .002). On multivariate analysis, carrying the G/G genotype was a significant independent risk factor for relapse (hazard ratio [HR] 2.29, P < .001) with a similar trend in C/G carriers (HR 1.33, P = .097) when compared with the major allele carrier C/C cohort. Our subsequent in vitro analyses demonstrated significantly reduced protease activity in proteasomes of individuals with G/G genotype compared with that of C/C carriers, despite that PSMB1 expression and proteasome assembly remained unaltered. Bortezomib exhibited a lower inhibitory capacity on the caspase‐ and trypsin‐like activity of proteasomes from G/G individuals. Conclusion Our results show that carriership of PSMB1 rs12717 minor allele is predictive for suboptimal response with bortezomib treatment, which could be explained by less active proteasomes that are less sensitive to bortezomib, and myeloma cells consequently relying on other escape mechanisms to cope with the abundance of misfolded proteins. Micro‐Abstract We retrospectively analyzed the prognostic impact of proteasome subunit beta type 1 rs12717 polymorphism in 211 consecutively diagnosed multiple myeloma cases. Patients carrying the variant G allele showed significantly shorter progression‐free survival. In proteasomes of individuals with G/G genotype, we found significantly reduced protease activity and lower inhibitory capacity of bortezomib on the caspase‐ and trypsin‐like activity.