Background: The genomic landscape of pediatric acute lymphoblastic leukemia (ALL) is heterogeneous with distinct copy number aberrations (CNAs) being detectable in vast majority of the patients. A subset of these alterations provides prognostic and/or predictive information; therefore, various CNA-based patient classifiers were introduced in the past. Aims: We applied a next-generation sequencing (NGS) based method to comprehensively screen for recurrent, disease-relevant CNAs in a cohort of Hungarian patients, allowing us to establish novel patient risk stratification approaches. Methods: Diagnostic bone marrow samples from 261 children with B-ALL and 22 matching samples drawn from 21 patients at first or second relapse were investigated by digital multiplex ligation-dependent probe amplification (digitalMLPA) using the ALL-specific D007 probemix. DigitalMLPA libraries were sequenced on an NGS platform. Whole chromosome gains and losses, as well as subchromosomal CNAs were simultaneously profiled. Survival rates were estimated using the Kaplan-Meier method and compared by log-rank tests in R version 4.1.2. Results: In total, 1,400 CNAs including numerical chromosomal aberrations and subchromosomal CNAs were detected in 93.5% of the diagnostic samples. On average, 5.36 CNAs were observed per patient with a mean of 2.45 subchromosomal alterations. Subtype-defining aberrations were identified in 36.0% of the patients with hyperdiploidy and iAMP21 detected in 32.2% and 3.8% of the cases, respectively. Numerous CNAs in disease-relevant genes responsible for cell cycle control, lymphoid development, signaling, or tumor suppression were identified. Considering all affected exons and an upstream region, 10 distinct patterns of IKZF1 deletion were observed. Unbiased co-segregation analysis revealed 15 positive and 3 negative correlations between the various CNAs, e.g. common co-occurrence of iAMP21 and CDKN2A/B deletion, and negative correlation between ETV6 and BTG1 deletions. Comparative analysis of diagnostic and matching relapse samples revealed characteristic temporal changes of copy number profiles with additional aberrations (65%), as well as both emerging and disappearing CNAs (30%) detected at relapse as compared with diagnosis. One patient did not show CNAs in the analyzed samples (5%). Prognostic subgroups determined based on cytogenetic findings were combined (i) with IKZF1del/IKZF1plus status and (ii) with CNA subgroups defined by a comprehensive digitalMLPA profiling. The combined genetic classification methods identified 3 and 4 patient subgroups, respectively, with significantly different 5-year progression-free survival rates. Summary/Conclusion: Comprehensive and highly optimized CNA profiling with digitalMLPA revealed subtype-defining gross-chromosomal changes and additional disease-relevant subchromosomal CNAs in a large cohort of Hungarian patients treated with ALL IC BFM protocols. Comparative scrutiny of matching diagnostic and relapse samples from 21 patients unveiled two different patterns of temporal evolution. Two novel risk stratification approaches have been established by combining cytogenetic data with digitalMLPA-based copy number profiling, with one of those laying more emphasis on CNA data than seen in previously introduced classifications, and the other one combining cytogenetic data with IKAROS status for the first time. DigitalMLPA offers a fast, reliable and standardizable DNA copy number analysis which is easily implementable in the diagnostic workflow of pediatric ALL.

We describe here a novel c.137 + 5G > A intronic mutation in the SH2D1A gene of the signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) in association with Epstein-Barr virus (EBV)-induced fatal infectious mononucleosis (FIM) in an 8-year-old male patient and his 3-year-old step brother. The mother and the maternal grandmother of the boys are healthy and heterozygous for this sequence variant. Genetic sequencing of blood-cell-derived cDNA in the younger patient revealed a 22 bp deletion in the SH2D1A cDNA. Immunoblot and flow cytometry analysis performed in this younger patient showed the lack of SAP protein expression in peripheral blood lymphocytes. These data suggest that the novel c.137 + 5G > A mutation results in loss of function of SAP protein and leads to typical X-linked lymphoproliferative disease phenotype. We propose that intron 1 and the c.137 + 5G may be the most frequent intronic hot spot for SH2D1A splicing mutation.

CYP3A4 has an important role in the metabolisms of many drugs used in acute lymphoblastic leukemia (ALL) therapy; still, there are practically no publications about the role of CYP3A4 polymorphisms in ALL pharmacogenomics. We genotyped eight common single-nucleotide polymorphisms (SNPs) in the CYP3A4 and CYP3A5 genes in 511 children with ALL and investigated whether they influenced the survival of the patients. We involved additional 127 SNPs in 34 candidate genes and searched for interactions with respect to the survival rates. Significant association between the survival rates and the common rs2246709 SNP in the CYP3A4 gene was observed. The gender of the patients and the rs1076991 in the MTHFD1 gene strongly influenced this effect. We calculated new risk assessments involving the gender-rs2246709 interaction and showed that they significantly outperformed the earlier risk-group assessments at every time point. If this finding is confirmed in other populations, it can have a considerable prognostic significance.

Publisher SummaryFolate is one of several biological active vitamers, which mediates the transfer of one carbon in numerous reactions including the synthesis of purine nucleotides, amino acids, camitine, creatine, lipids, hormones, and also serves as a cofactor for proteins involved in the control of circadian rhythm. Adults should get at least 400 μg/d. Women who may become pregnant should use a supplement with this amount. Its requirements during pregnancy and breastfeeding are slightly higher. Inadequate intakes predispose to anemia and abnormal white blood cells, atherosclerosis, thrombosis, cancer, and to birth of children with severe defects. People with certain common genetic dispositions are most sensitive to low intakes. While folate in itself is not likely to cause harm at intake levels of several times the recommended amount, harmful effects in people with vitamin B12 deficiency are possible, and intakes over 1000 μg should be avoided. Folate absorption is most active in the jejunum. In healthy people, about 85% of a moderate dose of pure folic acid is absorbed and absorption of folate from non-fortified foods is only about 50%. Losses of folate occur mostly through metabolic inactivation and with urine. Significant amounts of folate are secreted into bile and largely recovered again by intestinal absorption, although some folate is lost with the feces.

Anthracyclines are potent cytostatic drugs, the correct dosage being critical to avoid possible cardiac side effects. ABCC1 [ATP-binding cassette, sub-family C, member 1; also denoted as MRP1 (multidrug resistance-associated protein 1)] is expressed in the heart and takes part in the detoxification and protection of cells from the toxic effects of xenobiotics, including anthracyclines. Our objective was to search for associations between LV (left ventricular) function and single-nucleotide polymorphisms of the ABCC1 gene in children receiving anthracycline chemotherapy. Data of 235 paediatric patients with acute lymphoblastic leukaemia was analysed. Patients were followed-up by echocardiography (median follow-up 6.3 years). Nine polymorphisms in the ABCC1 gene were genotyped. The ABCC1 rs3743527TT genotype and rs3743527TT-rs246221TC/TT genotype combination were associated with lower LVFS (left ventricular fractional shortening) after chemotherapy. The results suggest that genetic variants in the ABCC1 gene influence anthracycline-induced LV dysfunction.

INTRODUCTIONNew prognostic factors discovered in chronic lymphocytic leukemia have recently got into the center of clinical interest. While the predictive value of cytogenetical abnormalities, immunoglobulin heavy chain gene mutation status, CD38 and ZAP70 expression is already well known, the significance of multi-drug resistance in chronic lymphocytic leukemia is not well characterized.AIMSThe goal of this study was to characterize the multidrug resistance features in 82 patients with chronic lymphocytic leukemia at the genetical, expression- and functional level and to compare it with the patient's clinical behavior (survival and response to therapy).METHODSLight Cycler Real Time PCR based "Single Nucleotide Polymorphism" analysis of the MDR1 gene, as a biological predictor of the expression level of P-glycoprotein was tested in 66 patients with chronic lymphocytic leukemia. P-glycoprotein expression and MDR-function was detected in 82 cases by flow cytometry (by use of anti-P-glycoprotein monoclonal antibody and calcein-verapamil functional test). Response to therapy was analyzed by statistical Fisher-test in the treated 35 patients. The survival analysis (Log-rank test) was performed on the whole population ( n = 82).RESULTSNo significant correlation was found between the three levels of multidrug resistance (genetics, phenotype, function) in our patients with chronic lymphocytic leukemia. P-glycoprotein positive cases (n = 9) were predominantly non-responders (8/9, 89%). There must be, however, other mechanisms causing non-response (total non-responders: 13/35 treated cases). Most of P-glycoprotein negative CLL patients (n = 26) responded well (21/26, 80%) to chemotherapy (responders: 22/35 treated CLL) (p < 0,001). The tendency was the same in the average expected survival rate between P-glycoprotein positive and negative patients (84 vs 203 months) but the difference was not significant (p = 0,106).CONCLUSIONSThis study proved the clinical prognostic significance of P-glycoprotein expression of leukaemic cells predicting the chemotherapy response and partially estimating the general survival of patients suffering from chronic lymphocytic leukemia.