Increased Lipocalin 2 level may have important role in thrombotic events in patients with polycythemia vera and essential thrombocythemia.

The aim of our study was to evaluate the possible effect of lipocalin 2 on thrombotic events in polycythemia vera and essential thrombocythemia patients. The samples of 89 patients were collected and RNA based method was used to evaluate the relative gene expression level of lipocalin 2. 74 samples were available for evaluation. Drawing a cut off point at level 30 relative expression rate, 13 patients with elevated lipocalin 2 expression had thrombotic events during the course of their disease. Based on these data high lipocalin 2 expression level seems to have strong positive predictive value on thrombotic events in patients with polycythemia vera and essential thrombocythemia. Lipocalin 2 may be useful in thrombotic risk stratification in these patients.

Development and evaluation of a secondary reference panel for BCR-ABL1 quantification on the International Scale.

Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently use a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR(1)-MR(4)), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that is traceable to and faithfully replicates the WHO panel, with an additional MR(4.5) level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample inputs were used, >60% demonstrated satisfactory IS accuracy, precision and/or MR(4.5) sensitivity, and 58% obtained IS conversion factors from the secondary reference concordant with their current values. Correlation analysis indicated no significant alterations in %BCR-ABL1 results caused by different assay configurations. More assays achieved good precision and/or sensitivity than IS accuracy, indicating the need for better IS calibration mechanisms.

Early Versus Delayed Autologous Stem Cell Transplantation and Interferon Maintenance in Multiple Myeloma: Single-Center Experience of 18 Years.

BACKGROUND: Autologous stem cell transplantation (ASCT) has become the mainstay of 1st-line treatment in younger patients with multiple myeloma (MM), but statistical confirmation of its superiority over other therapies, especially in the era of novel agents, is still lacking. METHODS: We reviewed the results of all 548 myeloma ASCTs performed in our institute over the past 18 years. RESULTS: More than one-half of the patients had access to novel agents before their transplantations. Although the age of the transplanted patients increased significantly over the years, treatment-related mortality (TRM) was remarkably low, especially in 1st-line transplanted patients (100-day TRM, 0.3%). The median overall survival (OS) of the entire cohort was 98.4 months. Patients transplanted within 12 months from the start of their therapy had significantly better responses than those having delayed ASCT (complete response rate, 58.1% vs 46.8%; P = .016) and significant post-ASCT progression-free survival (PFS) benefit (30.2 [26.1-34.3] mo vs 23.3 [16.8-29.8] mo; P = .036), but we found no significant overall survival difference. The results were similar in patients treated with or without novel agents before ASCT. During a period of time, interferon maintenance was our standard approach to post-ASCT maintenance. Our analysis showed not only a significant PFS advantage with interferon, but also a highly significant overall survival benefit (150.4 [105.1-195.8] mo vs 86.1 [72.5-99.7] mo; P = .003). CONCLUSIONS: Our findings demonstrate that delayed ASCT can be feasible in selected patients.

The potential role of HLA-DRB1*11 in the development and outcome of haematopoietic stem cell transplantation-associated thrombotic microangiopathy.

Transplantation-associated thrombotic microangiopathy (TA-TMA) is a serious complication of allogeneic haematopoietic stem cell transplantation (allo-HSCT) with high mortality rate. We retrospectively studied the frequency, clinical and genetic associations and prognostic effect of TA-TMA, in a total of 425 consecutive adult patients, who underwent allo-HSCT for a malignant haematological condition between 2007 and 2013 at our single centre. TA-TMA developed in 19% of the patients. Unrelated donor type (P<0.001), acute GvHD grades II-IV (P<0.001), myeloablative conditioning regimens (P=0.003), tacrolimus-based GvHD prophylaxis (P=0.003), CMV infection (P=0.003) and carriership for HLA-DRB1*11 (P=0.034) were associated with the development of TA-TMA. Survival was adversely affected by the presence of TA-TMA (P<0.001). Among patients with TA-TMA, the outcome of HLA-DRB1*11 carriers was significantly better compared with non-carriers (P=0.003). As a new finding, our observations suggest that the presence of HLA-DRB1*11 antigen contributes to the development of TA-TMA and affects the outcome.

Low occurrence of the HLA-C*04:09N allele in a large Hungarian cohort.

The presence of null alleles may affect the outcome of stem cell transplantation. HLA-C*04:09N was defined as 'common' with a frequency of 2-5/1000 in Caucasians, and its presence is routinely tested as part of haplotypes HLA-A*02:01/A*23:01-B*44:03-DRB1*07:01-DQB1*02:01. We aimed to investigate HLA-C*04:09N in a representative Hungarian cohort. HLA-typing data of 7345 unrelated persons were analyzed. The presence of HLA-C*04:09N was excluded in 157 chromosomes with either serology typing or with an allele-specific polymerase chain reaction for HLA-C*04:09N. HLA-C*04:09N was identified in a single chromosome with HLA-A*02, B*44, C*04, DRB1*07 resulting in a HLA-C*04:09N allele frequency of 0.0068% (1/14,690). This is approximately a 10- to 40-fold lower frequency compared with the previous data. Our results emphasize the need of precise local population-specific HLA-data, allowing appropriate modifications of local HLA-typing protocols.

A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR.

Serial quantification of BCR-ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR-ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 10(6), 1.08±0.11 × 10(5), 1.03±0.10 × 10(4), 1.02±0.09 × 10(3), 1.04±0.10 × 10(2) and 10.0±1.5 copies/µl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR-ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR-ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).

ATG16L1 and IL23 receptor (IL23R) genes are associated with disease susceptibility in Hungarian CD patients.

BACKGROUND: North American and European genome-wide association scans have identified ATG16L1 and IL23R as novel inflammatory bowel disease (IBD) susceptibility genes and subsequent reports confirmed these findings in large independent populations. The aims of this study were to investigate the association and examine genotype-phenotype relationships in a Hungarian IBD cohort. METHODS: 415 unrelated IBD patients (CD: 266, age: 35.2+/-12.1 years, duration: 8.7+/-7.5 years and UC: 149, age: 44.4+/-15.4 years, duration: 10.7+/-8.9 years) and 149 healthy subjects were investigated. IL23R Arg381Gln (R381Q, rs11209026) and ATG16L1 Thr300Ala (T300A, rs2241880) polymorphisms were tested using LightCycler allele discrimination method. Detailed clinical phenotypes were determined by reviewing the medical charts. RESULTS: The association between IL23R rs11209026, ATG16L1 rs2241880 and CD was confirmed (OR(IL23R381Q): 0.38, 95% CI: 0.16-0.87; OR(ATG16L1300AA): 1.86, 95% CI: 1.04-3.40). No difference was found between patients with UC and either controls or CD. In CD, IL23R 381Gln heterozygosity was associated with inflammatory disease (70% vs. 34%, p=0.037), while disease restricted to the colon was more prevalent in patients with the ATG16L1 300Ala/Ala homozygosity (33.3% vs. 21.1%, p=0.036). In addition, carriage of the variant alleles did not predict response to steroids, infliximab or need for surgery. CONCLUSIONS: We confirmed that ATG16L1 and IL23R are susceptibility loci for CD in Hungarian CD patients. Further studies are needed to confirm the reported phenotype-genotype associations found in this study.

Synergistic interaction of ABCB1 and ABCG2 polymorphisms predicts the prevalence of toxic encephalopathy during anticancer chemotherapy.

Polymorphisms of the ABCB1 (MDR1) and ABCG2 (BCRP) genes were reported to alter the expression and function of these drug transporters. Both proteins are present at the main pharmacokinetic barriers including the blood-brain barrier. Data from 291 children with acute lymphoblastic leukaemia were analysed in this retrospective study. ABCB1 3435T>C, 2677G>T/A, 1236C>T and ABCG2 421C>A, 34G>A genotypes were determined. Encephalopathy episodes were more frequent among those with ABCB1 3435TT genotype than in the 3435CC/CT group (odds ratio (OR) 3.5; P=0.03). Patients with the ABCG2 421A allele tended to have more complications than wild type homozygotes (OR=2.0; P=0.25). The rate of the adverse effect was similar in those harbouring no or only one of the predisposing genotypes, that is, either ABCB1 3435TT or ABCG2 421AA/AC. However, significantly more children suffered encephalopathy in the group with both predisposing genotypes (OR=12.3; P=0.005). In conclusion, these variations exert synergistic effect in predisposing patients to toxic neurological complications of chemotherapy.

Reduction of Bcr-Abl function leads to erythroid differentiation of K562 cells via downregulation of ERK.

The chimeric bcr-abl gene encodes a constitutively active tyrosine kinase that leads to abnormal transduction of growth and survival signals leading to chronic myeloid leukemia (CML). According to our previous observations, in vitro differentiation of several erythroid cell lines is accompanied by the downregulation of extracellular signal-regulated kinases (ERK)1/2 mitogen-activated protein kinase (MAPK) activities. In this work we investigated whether ERKs have a decisive role in either the erythroid differentiation process or apoptosis of bcr-abl+ K562 cells by means of direct (MEK1/2 inhibitor UO126) and indirect (reduced Bcr-Abl function) inhibition of their activities. We found that both Gleevec and UO126 induced hemoglobin expression. Gleevec treatment reduced the phosphorylation of Bcr-Abl, ERK and STAT-5 for up to 24 h, decreased Bcl-XL levels, and induced caspase-3-dependent apoptosis. In contrast, UO126 treatment resulted in only a transient decrease of ERK activity and did not induce cell death. For studying the effect of reduced Bcr-Abl function on erythroid differentiation at the level of the bcr-abl transcript, we applied the siRNA approach. Stable degradation of bcr-abl mRNA was achieved by using a retroviral vector with enhanced green fluorescent protein (EGFP) reporter. Despite a high (>90%) transduction efficiency we detected only a transient decrease in Bcr-Abl protein and in phosphorylated ERK1/2 levels. This transient change in Bcr-Abl signaling was sufficient to induce hemoglobin expression without significant cell death. These results suggest that by transiently reducing Bcr-Abl function it is possible to overcome the differentiation blockade without evoking apoptosis in CML cells and that reduced ERK activity may have a crucial role in this process.

Homogeneous assay based on 52 primer sets to scan for mutations of the ABCA1 gene and its application in genetic analysis of a new patient with familial high-density lipoprotein deficiency syndrome.

Familial high-density lipoprotein (HDL)-deficiency syndromes are caused by mutations of the ABCA1 gene, coding for the ATP-binding cassette transporter 1. We have developed a homogeneous assay based on 52 primer sets to amplify all 50 ABCA1 exons and approximately 1 kb of its promoter. The assay allows for convenient amplification of the gene from genomic DNA and easy mutational analysis through automatic sequencing. It obviates the need to use mRNA preparations, which were difficult to handle and posed a risk to miss splice junction or promoter mutations. The application of the test to the molecular analysis of a new patient with familial HDL-deficiency (Tangier disease) led to a discovery of two novel ABCA1 mutations: C2665del and C4457T.

Genotype screening for hereditary hemochromatosis among voluntary blood donors in Hungary.

Hereditary hemochromatosis (HH) is a common genetic disorder. Although it is inherited in an autosomal recessive manner, heterozygous individuals are believed to be protected against iron deficiency. Screening to estimate the prevalence of HH was frequently performed among blood donors, not considering that carriers of the HH gene mutations may be present in higher proportion in this population. To examine the allele frequencies of the HH gene (HFE) point mutations, C282Y and H63D genotyping was carried out in 996 consecutive, first-time, and regular Hungarian blood donors by PCR-RFLP techniques. Iron parameters of the first-time donors and the identified C282Y heterozygotes and age, gender, and number of previous blood donation-matched wild-type donors were also determined. We were not able to demonstrate a significant increase in the frequency of C282Y and H63D alleles among regular blood donors, compared to first-time blood donors. However, there was a trend of higher C282Y allele frequency among women with higher number of previous blood donations (2.2 +/- 1.5% in female blood donors with 0-8 previous blood donations compared to 4.8 +/- 2.3% in women with more than 8 previous blood donations, P = 0.06). No detectable phenotypic differences were observed in serum iron, ferritin, and transferrin saturation values between C282Y wild-type and heterozygous groups. However, the single identified C282Y homozygous male (age 21) showed definite signs of iron overload. Our observations suggest that the protective effect of C282Y heterozygosity against iron deficiency may be less significant than other environmental (e.g., iron-rich diet) or genetic factors.

A haemophilia A and B molecular genetic diagnostic programme in Hungary: a highly informative and cost-effective strategy.

Our aim was to set up a protocol in order to provide carrier and prenatal diagnosis to Hungarian haemophilia A (HA) and B (HB) patients and their relatives. For HA, a combination of direct mutation detection and some indirect marker analyses were used: the detection of the inversion mutation and analysis of three polymorphisms, BclI, IVS13 (CA)n and P39(CA)n. In severe cases, direct mutation detection was performed first. In inversion-negative severe cases and in moderate and mild cases, indirect methods were used. For carrier and prenatal diagnosis in HB, four polymorphisms, DdeI, TaqI, XmnI, and HhaI were examined. Our DNA bank contains samples from 50 HA families (34 severe, 15 moderate and one mild) and seven HB families from different parts of the country. In 100% of the HA cases either the gene inversion and/or at least one of the polymorphisms was found to be informative for carrier or prenatal diagnosis. In the HB cases, an informative marker was found in 95% of the cases (19 of 20). We conclude that these strategies are sufficient to make genetic diagnosis available to almost all HA and HB families in the region. This approach is highly informative and cost-effective, so it can be very useful in countries where direct sequencing of genes for factor VIII and IX is not available for routine diagnosis.

[A new method of molecular testing in the differential diagnosis of hereditary hemochromatosis]. / Uj, molekuláris genetikai módszer az öröklödö haemochromatosis differenciáldiagnosztikájában.

Hereditary hemochromatosis is an autosomal, recessive disorder of the iron metabolism. The hemochromatosis gene (HFE) was previously located on chromosome 6 and recently identified by positional cloning. A point mutation, C282Y, was found to be present in the HFE gene in homozygous form in 64 to 100% of patients with established hemochromatosis. The relationship of a second polymorphic variant of the HFE gene, H63D to the formation of iron overload is debated. Although hemochromatosis is one of the most common inherited disorders among Caucasians, in the absence of specific signs it is rarely diagnosed. In order to obtain comparable epidemiological data for Hungary, we tested 1271 and 277 randomly selected, unrelated, healthy subjects for C282Y and H63D respectively. In addition C282Y testing was carried out in 58 patients suffering from liver cirrhosis, and in 191 individuals with suspected hemochromatosis. For C282Y and H63D mutation analyses polymerase chain reaction technique followed by Rsa I and Bcl I restriction enzyme digestion was used. We developed an alternative method for the detection of C282Y based on an amplification-generated Kpn I restriction site. The allele frequencies were 3.8% and 12.3% for C282Y and H63D respectively in the normal Hungarian population. There was no significant difference in C282Y allele frequencies between liver disease patients (1.7%) and the normal population. We identified 15 homozygous and 25 heterozygous individuals among 191 individuals with suspected hemochromatosis. The C282Y and the H63D allele frequencies in the normal Hungarian population were found to be similar to the allele frequencies observed in other European populations, indicating that there is a large number of individuals susceptible for iron overload in Hungary (1:700). Mutation analysis is a novel, non-invasive method in the diagnostics of hereditary hemochromatosis, which increasingly becomes part of the routine clinical work.