Andrea Sipos

Distinct clinical characteristics of myeloproliferative neoplasms with calreticulin mutations

Somatic insertions/deletions in the calreticulin gene have recently been discovered to be causative alterations in myeloproliferative neoplasms. A combination of qualitative and quantitative allele-specific polymerase chain reaction, fragment-sizing, high resolution melting and Sanger-sequencing was applied for the detection of three driver mutations (in Janus kinase 2, calreticulin and myeloproliferative leukemia virus oncogene genes) in 289 cases of essential thrombocythemia and 99 cases of primary myelofibrosis. In essential thrombocythemia, 154 (53%) Janus kinase 2 V617F, 96 (33%) calreticulin, 9 (3%) myeloproliferative leukemia virus oncogene gene mutation-positive and 30 triple-negative (11%) cases were identified, while in primary myelofibrosis 56 (57%) Janus kinase 2 V617F, 25 (25%) calreticulin, 7 (7%) myeloproliferative leukemia virus oncogene gene mutation-positive and 11 (11%) triple-negative cases were identified. Patients positive for the calreticulin mutation were younger and had higher platelet counts compared to Janus kinase 2 mutation-positive counterparts. Calreticulin mutation-positive patients with essential thrombocythemia showed a lower risk of developing venous thrombosis, but no difference in overall survival. Calreticulin mutation-positive patients with primary myelofibrosis had a better overall survival compared to that of the Janus kinase 2 mutation-positive (P=0.04) or triple-negative cases (P=0.01). Type 2 calreticulin mutation occurred more frequently in essential thrombocythemia than in primary myelofibrosis (P=0.049). In essential thrombocythemia, the calreticulin mutational load was higher than the Janus kinase 2 mutational load (P<0.001), and increased gradually in advanced stages. Calreticulin mutational load influenced blood counts even at the time point of diagnosis in essential thrombocythemia. We confirm that calreticulin mutation is associated with distinct clinical characteristics and explored relationships between mutation type, load and clinical outcome.

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.

Remarkably Reduced Transplant-Related Complications by Dibromomannitol Non-Myeloablative Conditioning before Allogeneic Bone Marrow Transplantation in Chronic Myeloid Leukemia

A non-myeloablative conditioning protocol containing dibromomannitol (DBM/cytosine arabinoside/cyclophosphamide) has been applied to 36 chronic myeloid leukemia (CML) patients followed by bone marrow transplantation (BMT) from sibling donors. Risk factors include: accelerated phase (10 patients), older age (17 patients over >40 years) and long interval between diagnosis and BMT (27 months on average). Severe mucositis did not occur. Venoocclusive liver disease was absent. Infectious complications were rare. Although grade II–IV acute graft-versus-host disease (GVHD) was present in 9 (25%) cases, there were only 2 serious (III–IV) ones. Chronic GVHD occurred in 25 (69%) cases, preceded by acute GVHD in 9 of the 25 affected patients. Early hematological relapse, 7–29 weeks after BMT, developed in 6 patients (17.6%). No relapse was noted in the completely chimeric patients, however molecular genetic residual disease was observed in 6 patients, in most of them after transient short-term mixed chimeric state. Overall actual survival rate is 83.3% for the 36 cases, and leukemia-free survival is 72.2% for the 34 engrafted patients.

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.

Immunological importance of chimerism in transplantation: new conditioning protocol in BMT and the development of chimeric state

Chimerism is an exceptional immunogenetic state, characterized by the survival and collaboration of cell populations originated from two different individuals. The prerequisits to induce chimerism are immuno-suppression, myeloablation, or severe immunodeficiency of the recipients on the one side and donor originated immuno-hematopoietic cells in the graft on the other. The pathologic or special immunogenetic conditions to establish chimerism are combined with bone marrow transplantation, transfusion, and various kinds of solid organ grafting. Different types of chimerism are known including complete, mixed and mosaic, or split chimerism. There are various methods used to detect the type of chimera state, depending on the immunogenetic differences between the donor and recipient. The induction of complete or mixed chimerism is first determinated by the effect of myeloablative therapy. The chimera state seems to be one of the leading factors to influence the course of the post-transplant period, the frequency and severity of GVHD, and the rate of relapse. However, the most important contribution of the chimeric state is in development of graft versus leukemia effect. A new conditioning protocol (DBM/Ara-C/Cy) for allogeneic BMT in CML patients and its consequence on chimera state and GVL effect is demonstrated.

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.

Homogeneous Immunoglobulins following Allogeneic Bone Marrow Transplantation

Homogeneous immunoglobulins are frequently detected in the serum of patients undergoing allogeneic bone marrow transplantation (BMT). The aim of the present study was to further characterize the incidence of this phenomenon and its correlations with laboratory and clinical data. Serum samples were gathered from 29 patients undergoing allogeneic or syngeneic BMT for chronic myeloid leukemia (CML), and serial protein (IgG, IgA and IgM) quantification, electrophoresis and immunofixation were performed. Transient mono- or oligoclonal gammopathies were observed in 23 out of 29 patients between days 20 and 1,750 following transplantation. The presence of homogeneous immunoglobulins was not correlated with the following clinical parameters: graft-versus-host disease, bacterial sepsis, Epstein-Barr virus or cytomegalovirus infection or invasive fungal infection. Therefore, the development of mono- or oligoclonal immunoglobulins may represent a complex disorder of B cell regeneration which may be caused by an intrinsic B cell defect, or a failure in the regenerating T cell system, or both, manifesting itself in a restricted antibody diversity after allogeneic BMT.