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Összefoglaló. Bevezetés: Az allogén hematopoetikus őssejt-transzplantáció (allo-HSCT) kiemelkedő szerepet tölt be a hematológiai betegségek gyógyításában. A kimérizmusvizsgálat a donor és a recipiens típusú vérképzés jelenlétét különíti el örökletes genetikai markerek segítségével. Az allo-HSCT kimenetele (pl. az engraftment dinamikája vagy a relapsus előrejelzése) jól követhető kimérizmusvizsgálatokkal, akár a betegségspecifikus markerek hiányában is. Célkitűzés: Munkánk során a kimérizmusvizsgálatok terén arany standardnak számító short tandem repeat (STR) és a fluoreszcens in situ hibridizáció (FISH) módszereket hasonlítottuk össze, valamint vizsgáltuk az allo-HSCT kimenetelét a kimérizmuseredményektől függően. Módszer: Az STR és FISH módszerek összehasonlítását 2018-ban gyűjtött, a két különböző vizsgálatra ugyanazon a napon beküldött 210 mintán végeztük. A kimérizmusvizsgálatok klinikai jelentőségét 2015. január és 2018. február között 378 allogén HSCT-n átesett beteg adatainak elemzésével vizsgáltuk. Eredmények: A két vizsgálati módszerrel kapott eredmények 95,2%-ban megegyeztek. A teljes kimérizmust elért betegek (n = 311) összesített túlélése (OS) kedvezőbb volt, mint azoké, akik nem értek el teljes kimérizmust (n = 44; OS 24 hónapban: 59,4% vs. 23,6%; p < 0,001). Következtetések: Vizsgálatunkban a két kimérizmusvizsgálati módszer (STR és FISH) eredményei jól korreláltak egymással, mindkettő alkalmasnak bizonyult a betegek allo-HSCT utáni követésére. A teljes donorkimérizmus elérése szignifikánsan jobb túléléssel társult. A kevert kimérizmus hatékonyan visszafordítható donor lymphocytainfúzió adásával, mely fokozza a graft alloreaktivitását. Summary. Introduction: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) plays a pivotal role in the cure of hematologic malignancies. Chimerism testing can differentiate recipient and donor hematopoiesis by hereditary genetic markers. The outcome of allo-HSCT (e.g. the dynamics of engraftment and the prediction of relapse) can be monitored by chimerism testing even in the case of lacking disease specific markers. Aims: In the current study we compared the gold standard short tandem repeat (STR) and the fluorescent in situ hybridization (FISH) chimerism testing methods; and we examined the outcome of allo-HSCT depending on chimerism results. Methods: The comparison of STR and FISH methods was performed on 210 samples parallelly tested for chimerism by both methods on the same day in 2018. The clinical significance of chimerism testing was investigated by clinical data analysis of 378 patients treated by allo-HSCT between 1 January 2015 and 28 February 2018. Results: Chimerism results tested by the two methods showed 95.2% concordance. Patients with complete donor chimerism (n = 311) had a more favourable overall survival (OS) than patients without complete donor chimerism (n = 44; OS at 24 month years 59.4% vs. 23.6%, respectively, p < 0.001). Conclusion: In our comparison both chimerism testing methods correlated well, and proved to be suitable for chimerism monitoring after allo-HSCT. Achieving complete donor chimerism was associated with better survival. In case of mixed chimerism, donor lymphocyte infusion can be an effective therapeutic option since it increases graft alloreactivity.

Absztrakt: Az izocitrat dehidrogenaz 1 es 2 szomatikus mutaciok az akut myeloid leukemia visszaterő genetikai elteresei, tagabb ertelemben vett epigenetikus hatasmoddal. Az irodalomban ellentmondo ...

Monitoring measurable residual disease (MRD) in acute myeloid leukemia (AML) plays an important role in predicting relapse and outcome. The applicability of the leukemia-initiating nucleophosmin1 (NPM1) gene mutations in MRD detection is well-established, while that of isocitrate dehydrogenase1/2 (IDH1/2) mutations are matter of debate. The aim of this study was to investigate the stability of NPM1 and IDH1/2 mutations at diagnosis and relapse retrospectively in 916 adult AML patients. The prognostic value of MRD was evaluated by droplet digital PCR on the DNA level in a selected subgroup of patients in remission. NPM1 re-emerged at relapse in 91% (72/79), while IDH1/2 in 87% (20/23) of mutation-positive cases at diagnosis. NPM1 mutation did not develop at relapse, on the contrary novel IDH1/2 mutations occurred in 3% (3/93) of previously mutation-negative cases. NPM1 MRD-positivity after induction (n = 116) proved to be an independent, adverse risk factor (MRDpos 24-month OS: 39.3±6.2% versus MRDneg: 58.5±7.5%, p = 0.029; HR: 2.16; 95%CI: 1.25–3.74, p = 0.006). In the favorable subgroup of mutated NPM1 without fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) or with low allelic ratio, NPM1 MRD provides a valuable prognostic biomarker (NPM1 MRDpos versus MRDneg 24-month OS: 42.9±6.7% versus 66.7±8.6%; p = 0.01). IDH1/2 MRD-positivity after induction (n = 62) was also associated with poor survival (MRDpos 24-month OS: 41.3±9.2% versus MRDneg: 62.5±9.0%, p = 0.003; HR 2.81 95%CI 1.09–7.23, p = 0.032). While NPM1 variant allele frequency decreased below 2.5% in remission in all patients, IDH1/2 mutations (typically IDH2 R140Q) persisted in 24% of cases. Our results support that NPM1 MRD even at DNA level is a reliable prognostic factor, while IDH1/2 mutations may represent pre-leukemic, founder or subclonal drivers.

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. IDH1(mut) and IDH2(mut) were mutually exclusive, detected in 8.5% and 7.5% of cases, respectively. IDH1/2(mut) was associated with: older age (p = 0.001), higher average platelet count (p = 0.001), intermediate karyotype (p0.0001), NPM1(mut) (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 IDH1(mut) or IDH2(mut) vs. IDH(neg). IDH1(mut) and IDH2(mut) were associated differently with NPM1(mut); 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 IDH(neg) (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.

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-ITD(48-60bp) showed worse OS and DFS compared to other groups (FLT3-ITD(neg), 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.

Introduction: The incidence of human parvovirus B19 infection is unknown. Aim: A retrospective analysis of clinical and laboratory findings was carried out in patients diagnosed with human parvovirus B19 infection in 2011 in a virologic laboratory of a single centre in Hungary. Methods: Clinical and laboratory data of patients with proven human parvovirus B19 infection were analysed using in- and out-patient files. Results: In 2011, 72 patients proved to have human parvovirus B19 infection with the use of enzyme immunoassay. The clinical diagnoses of these patients were as follows: human parvovirus B19 infection (30.6%), transient aplastic crisis (16.7%), arthritis (8.3%) and acute hepatitis (4.1%). Symptoms of each of the four phases of the infection occurred in various combinations with the exception of the monophase of cheek exanthema. This occurred without the presence of other symptoms in some cases. Leading symptoms and signs were exanthema (in 74.6% of cases), haematological disorders (in 69% of cases), fever (in 54.9% of cases) and arthritis (in 33.8% of cases). Several atypical dermatological symptoms were also observed. Acute arthritis without exanthema was noted in 8 patients. Of the 72 patients with proven human parvovirus B19 infection there were 7 pregnant women, and one of them had hydrops foetalis resulting spontaneous abortion. In 16 patients (22.5%) human parvovirus B19 IgG was undetectable despite an optimal time for testing. Conclusion: The observations of this study may contribute to a better recognition of clinical symptoms of human parvovirus B19 infection. Orv. Hetil., 2012, 153, 1948–1957.

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.

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 hematopoetic stem cells. According to current results, the frequency of Val617Phe activating mutation is around 80% in polycythaemia vera, 35% in essential thrombocythaemia, and 50% in chronic idiopathic myelofibrosis. The diagnoses of polycythaemia vera, essential thrombocythaemia 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 polycytaemia vera, 56,6% (56/99) in essential thrombocythaemia, 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 polycythaemia vera and essential thrombocythaemia 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. Althought 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.

Treatment outcome in patients with acute myeloid leukemia are determined by prognostic factors.Between January 1996 and December 2001 160 patients were treated with newly diagnosed acute myeloid leukemia. Treatment results were analysed according to the age and cytogenetics. Different types of induction and postremission protocols were applied. The median age was 42.2 +/- 12.8 (16-60) years.Complete remission was reached in 113 (70.6%) patients. 25/160 (15.6%) individuals were refractory to treatment, 22/160 (13.8%) patients died within one month. One hundred and ten out of 113 who went into remission were given postremission therapy. Twelve out of 50 relapsed patients achieved a second complete remission. The complete remission rate and cumulative survival of patients below the age of forty years were significantly higher than of those above the age of 40 years. Four fifths of refractory patients as well as nearly all patients with secondary leukemia were older than 40 years. Similarly to studies published in the literature, the expected survival was the best in patients who had a favourable cytogenetics. In contrast, all patients who fell into the unfavourable cytogenetic group died within three years. Intensification of the postremission treatment resulted in an improved survival.Classification of acute myeloid leukemia and careful determination of prognostic factors are necessary at the time of diagnosis. This predicts outcome, as well as provides means for application of individualized therapy.

Chronic myeloproliferative diseases (CMPD) are clonal disorders of pluripotent hematopoietic stem cells. Acquired JAK2 V617F point mutation has recently been identified as disease causing activating genetic abnormality in classic BCR-ABL negative CMPD (80% of polycythemia vera, 35% of essential thrombocythemia and 50% of chronic idiopathic myelofibrosis cases). JAK2 V617F is rare in other myeloid stem cell disorders like acute myeloid leukemia, chronic myelomonocyter leukemia or myelodysplasia with reported frequency of 3–8%. Between January 2001 and December 2005 155 consecutive adult patients [87 females and 68 males, median age of onset was 49±14 (range 18–83) years] were diagnosed with AML in our institute. Peripheral blood or bone marrow samples drawn at the time point of diagnosis were analyzed for the presence of JAK2 V617F by allele-specific PCR. JAK2 V617F mutation was present in 5 patients (3 males and 2 females; 3.2%). 3 of the 5 patients had prior history of CMPD, while 2 patients were diagnosed with de novo AML. The clinical characteristics and laboratory features of JAK2 V617F positive patients are shown in Table 1. FLT3 internal tandem duplication, FLT3 tyrosine kinase domain mutations, AML1-ETO, CBFB-MYH, PML-RARA rearrangements or nucleophosmin mutations were not present at the time point of AML diagnosis. In the case of patient 1, thrombocytosis was present prior the diagnosis of AML, and bone marrow biopsy revealed grade 3 fibrosis at diagnosis of AML, suggesting the presence of an atypical CMPD with the coexistence of del(5q) MDS. Patient 2 had no remarkable disease in his previous medical history. Induction therapy resulted in complete hematological and cytogenetic remission with persistent JAK2 V617F positivity. 10 month later clinical features of CMPD (elevated white blood cell count, left shifted peripheral blood smear, hepatosplenomegaly) appeared. In conclusion, these two cases suggest that acute myeloid leukemia with JAK-2 V617F mutation in fact corresponds to the blastic transformation of a clinically atipical chronic myeloproliferative disorders. Clinical characteristics and laboratory features of JAK2 V617F positive patients Case Sex AML subtype Age of onset (years) FAB subtype Cytogenetic abnormality Therapy Overall Survival (months) 1 F de novo 52 M1 del(5q) DNR+ara-C, HDara-C 23 2 M de novo 65 M4 t(13;17) DNR+ara-C, HDara-C 12 3 F CMPD blastic transformation 65 M4 trisomy (1q) Supportive 18 4 M CMPD blastic transformation 70 M4 not available Supportive 3 5 M CMPD blastic transformation 71 M4 not available Supportive 6

Treatment outcome in patients with AML is determined by a variety of prognostic factors such as WBC, LDH, age, presence of chromosomal aberration. Recently, two acquired genetic alterations, internal tandem duplications (ITD) and Asp835 codon mutations of the fms-like tyrosine kinase 3 (FLT3) gene were reported in AML as prognostic factors. 66 consecutive adult patients (39 females and 27 males) were treated with newly diagnosed AML in our institute between January 2002 and June 2004. The median age of onset was 49±15 (range 17–83) years. The gender distribution was 1.44 (female/male). We analyzed FLT3 ITD and Asp835 mutations at the time point of diagnosis by fluorescent PCR and PCR-RFLP methods. ITD was present in 22.7% (15/66) of the patients and Asp835 mutations were detected in 7.6 % (5/66). Two patients carried both mutations. 13 of 15 ITD-positive patients had myelomonocytic or monocytic leukemia (M4 and M5 in the FAB classification system). There was no difference between the numbers of ITD-positive and negative patients in complete remission (10/15 [66.7%] vs. 31/51 [60.8%]). In the ITD-positive patient group, the relapse rate was increased (5/15 [33.3%] vs. 7/51 [13.7%]) however, this difference was statistically not significant. The age of onset was greater than 40 years in all Asp835 mutation-positive patients and in this group, no relapse occurred within the follow-up time. The remission rate (4/5) of this group was high in spite of the old age of onset. The single Asp835-positive patient also carried the ITD mutation and showed primary resistance. Our results are in agreement and confirm earlier observations for the ITD mutation in a different patient population. In our relatively small patient group the Asp835 mutation seemed to have a beneficial effect.