Natasa Tosic

Frequent deletions of JARID2 in leukemic transformation of chronic myeloid malignancies

Chronic myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS) have an inherent tendency to progress to acute myeloid leukemia (AML). Using high‐resolution SNP microarrays, we studied a total of 517 MPN and MDS patients in different disease stages, including 77 AML cases with previous history of MPN (N = 46) or MDS (N = 31). Frequent chromosomal deletions of variable sizes were detected, allowing the mapping of putative tumor suppressor genes involved in the leukemic transformation process. We detected frequent deletions on the short arm of chromosome 6 (del6p). The common deleted region on 6p mapped to a 1.1‐Mb region and contained only the JARID2 gene—member of the polycomb repressive complex 2 (PRC2). When we compared the frequency of del6p between chronic and leukemic phase, we observed a strong association of del6p with leukemic transformation (P = 0.0033). Subsequently, analysis of deletion profiles of other PRC2 members revealed frequent losses of genes such as EZH2, AEBP2, and SUZ12; however, the deletions targeting these genes were large. We also identified two patients with homozygous losses of JARID2 and AEBP2. We observed frequent codeletion of AEBP2 and ETV6, and similarly, SUZ12 and NF1. Using next generation exome sequencing of 40 patients, we identified only one somatic mutation in the PRC2 complex member SUZ12. As the frequency of point mutations in PRC2 members was found to be low, deletions were the main type of lesions targeting PRC2 complex members. Our study suggests an essential role of the PRC2 complex in the leukemic transformation of chronic myeloid disorders. Am. J. Hematol. 2012.

Importance of early detection and follow-up of FLT3 mutations in patients with acute myeloid leukemia

Mutations in the fms-like tyrosine kinase 3 (FLT3) gene, such as internal tandem duplication (FLT3/ITD) in the juxtamembrane domain and point mutations in the tyrosine kinase domain, are the most common abnormalities in acute myeloid leukemia (AML). FLT3/ITD and FLT3/D835 mutations were analyzed in 113 Serbian adult AML patients using polymerase chain reaction. Twenty patients were found to be FLT3/ITD positive (17.7%). The mutations occurred most frequently in M5 and M0 subtypes of AML. They were mainly associated with the normal karyotype. All patients harboring FLT3/ITD had a higher number of white blood cells than patients without it (p = 0.027). FLT3/ITD mutations were associated with lower complete remission (CR) rate (χ2 = 5.706; p = 0.017) and shorter overall survival (OS; Log rank = 8.76; p = 0.0031). As for disease-free survival, the difference between FLT3/ITD-positive and FLT3/ITD-negative patients was not statistically significant (Log rank = 0.78; p = 0.3764). In multivariate analysis, the presence of FLT3/ITD mutations was the most significant prognostic factor for both OS and CR rate (p = 0.0287; relative risk = 1.73; 95% CI = 1.06–2.82). However, in the group of patients with the intermediate-risk karyotype, the mere presence of FLT3/ITD was not associated with inferior clinical outcome. FLT3/D835 point mutation was found in four patients (3.5%) only. Follow-up of the FLT3/ITD-positive patients revealed stability of this mutation during the course of the disease. However, changes in the pattern of FLT3/D835 mutations in initial and relapsed AML were observed. Our results indicate an association of FLT3/ITD with the adverse outcome in AML patients treated with standard induction chemotherapy. Because FLT3/ITD mutation is a target for specific therapeutic inhibition, its early detection could be helpful in clinical practice.

Analysis of thiopurine S-methyltransferase polymorphism in the population of Serbia and Montenegro and mercaptopurine therapy tolerance in childhood acute lymphoblastic leukemia

Thiopurine S-methyltransferase (TPMT) is an enzyme that converts thiopurine drugs into inactive metabolites. It is now well established that interindividual variation in sensitivity to thiopurines can be the result of the presence of genetic polymorphisms in the TPMT gene. The aim of this study was to determine the frequency and type of TPMT polymorphisms in the population of Serbia and Montenegro and to assess its relevance in the management of childhood acute lymphoblastic leukemia (ALL). Blood samples from 100 healthy adults and 100 children with ALL were analyzed for common mutations in the TPMT gene using polymerase chain reaction-based assays. The results revealed that allelic frequencies were 0.2% for TPMT*2, 3.2% for TPMT*3A, and 0.5% for TPMT*3B. A rare TPMT*3B allele was detected in 2 families. No TPMT*3C allele was found. The general pattern of TPMT-variant allele distribution as well as their frequencies in the population of Serbia and Montenegro, is similar to those determined for other Slavic and Mediterranean populations. The ability to tolerate 6-mercaptopurine (6-MP) -based maintenance therapy was used as a surrogate marker of hematologic toxicity. In the study of 50 patients with childhood ALL treated according to the BFM-like protocol, it was found that even TPMT-heterozygous patients are at greater risk of thiopurine drug-related leukopenia (mean duration of period when children missed therapy as a result of leukopenia for TPMT-heterozygous patients was 11.3 weeks vs 3.4 weeks for wild-type genotype patients, P < 0.01). In another group of 50 patients, the TPMT genotype was determined prospectively. The therapy protocol was modified considering their TPMT genotype. Administering reduced 6-MP dosages in the initial phase of maintenance allowed TPMT-heterozygous patients to later receive full protocol doses of both 6-MP and nonthiopurine therapy without omitting therapy resulting from myelotoxicity. These results justify performing TPMT genotyping before initiating thiopurine therapy in all children with ALL to minimize consequent toxicity.

Functional analysis of the role of the TPMT gene promoter VNTR polymorphism in TPMT gene transcription

AIMS Thiopurine S-methyltransferase (TPMT) activity is polymorphic, and a trimodal distribution has been demonstrated in Caucasians (low, intermediate and high methylator groups). The TPMT gene promoter contains a variable number of three GC-rich tandem repeats, namely A, B and C, ranging from three to nine in length in a A(n)B(m)C architecture. MATERIALS & METHODS Here, we investigated the influence of number and type of TPMT gene promoter tandem repeats on human TPMT gene transcription in K562 cells transiently transfected with reporter constructs bearing various variable number of tandem repeats (VNTR) and addressed the interaction of transcription factor binding to the VNTRs by electrophoretic mobility shift assays. RESULTS We found that the distribution patterns of VNTR alleles do not significantly differ among acute lymphoblastic leukemia patients, acute myeloid leukemia patients and normal individuals. We also demonstrated that the A repeat has a negative effect in TPMT gene transcription and that a positive regulatory element, identified immediately upstream to the VNTR region of the TPMT gene promoter, is indispensable for TPMT gene transcription. Our electrophoretic mobility shift assay analysis indicated that the Sp1 and Sp3 transcription factors bind to the VNTR repeats. CONCLUSION Overall, our data underline that both the number and type of VNTRs, as well as the upstream regulatory region of the TPMT gene promoter, determine the overall level of TPMT gene transcription. It remains to be seen whether these VNTRs can be employed as pharmacogenetic markers to individualize thiopurine therapy.

Molecular and phenotypic characteristics of patients with phenylketonuria in Serbia and Montenegro

Phenylketonuria (PKU) is the most common inborn error of amino acid metabolism in Caucasians. PKU is caused by mutations in the gene encoding phenylalanine hydroxylase (PAH) enzyme. Here, we report the spectrum and the frequency of mutations in the PAH gene and discuss genotype–phenotype correlation in 34 unrelated patients with PKU from Serbia and Montenegro. Using both polymerase chain reaction–restriction fragment length polymorphism and ‘broad‐range’ denaturing‐gradient gel electrophoresis/DNA sequencing analysis, 19 disease‐causing mutations were identified, corresponding to mutation detection rate of 97%. The most frequent ones were L48S (21%), R408W (18%), P281L (9%), E390G (7%) and R261Q (6%), accounting for 60% of all mutant alleles. The genotype–phenotype correlation was studied in homozygous and functionally hemizygous patients. We found that the most frequent mutation, L48S, was exclusively associated with the classical (severe) PKU phenotype. The mutation E390G gave rise to mild PKU. For the mutation R261Q, patients had been recorded in two phenotype categories. Considering allele frequencies, PKU in Serbia and Montenegro is heterogeneous, reflecting numerous migrations over the Balkan Peninsula.

Polymorphisms of tumor-necrosis factor-α−308 and lymphotoxin-α + 250: Possible modulation of susceptibility to apoptosis in chronic lymphocytic leukemia and non-Hodgkin lymphoma mononuclear cells

Tumor necrosis factor alpha (TNF-α) and lymphotoxin alpha (LT-α) have been shown to play an important role in the pathogenesis of limphoproliferative disease. Both cytokines regulate cell-survival and cell-death in leukemic cells. TNF-α and LT-α are highly produced in chronic lymphotic leukemia (CLL) and non-Hodgkin lymphoma (NHL) patients. Genetic polymorphism within regulatory regions of these cytokine genes can alter their expression levels. This study investigates an influence of TNF-α−308 and LT-α + 250 polymorphisms on the activity of alkaline DNase in mononuclear cells of both patient groups as a potent biochemical marker of DNA fragmentation in the terminal phase of apoptosis. Study was performed on mononuclear cells of CLL and NHL patients. SNP were obtained by PCR-RFLP method. The activity of alkaline DNase was measured by spectrophotometric method. The study provided evidence of the influence of TNFG/A genotype and A alleles in the susceptibility to NHL, since the association of LT-αG/G genotype with CLL was observed. High-producing TNF-α−308/LT-α + 250 heterozygous haplotype is associated with high NHL incidence. The investigated SNP influence the activity of alkaline DNase in CLL and NHL patients. The observed polymorphisms may modulate susceptibility of leukemic cells to apoptosis by way of DNase activity.

Incidence of FLT3 and nucleophosmin gene mutations in childhood acute myeloid leukemia: Serbian experience and the review of the literature.

AbstractMutations in the fms-like tyrosine kinase 3 (FLT3) gene (internal tandem duplication (ITD) and point mutation in the tyrosine kinase domain, FLT3/D835) as well as the nucleophosmin (NPM1) gene are the most common abnormalities in adult acute myeloid leukemia (AML). Their significance in pediatric AML is still unclear. In this study we evaluated the frequency of FLT3 and NPM1 mutations in childhood AML. We also examined clinical features and outcome of these patients. FLT3 and NPM1 mutations were analysed in 42 and 37 childhood AML patients, respectively, using polymerase chain reaction (PCR) and direct sequencing. FLT3 mutations were detected in 4/42 patients (9.5%). The frequencies of FLT3/ITD and FLT3/D835 were the same, 2/42 (4.7%). NMP1 mutations were found in 1/37 patients (2.7%). FLT3 gene mutations were correlated with induction failure. Here we report the results of the study of FLT3 and NPM1 gene mutations in childhood AML patients in Serbia. Low frequencies of these molecular markers point out that these abnormalities are rare in this cohort of patients. Comparative study of data on NPM1 mutations in childhood AML revealed that various NPM1 gene mutation types are associated with childhood AML. Our findings as well as previously reported data, contributes to a hypothesis of different biology and etiology of adult and childhood AML. More extensive studies of NPM1 and FLT3 mutations in childhood AML are needed to determine their biological and clinical importance.

Clinical features of the most common fusion genes in childhood acute lymphoblastic leukemia

Contemporary protocols ensure high-remission rate and long-term free survival in children with acute lymphoblastic leukemia (ALL), but small percentage of patients is still incurable. Molecular genetic methods helped to establish submicroscopic classification as well as minimal residual disease follow-up, considered to be responsible for relapse. Our study enrolled 70 pediatric patients with de novo ALL, analyzed using reverse transcriptase-polymerase chain reaction for the presence of four major risk-stratifying translocations (BCR/ABL, MLL/AF4, TEL/AML1, and E2A/PBX1). Bone marrow samples were collected at diagnosis, at the end of induction phase, and after intensive chemotherapy with the aim to establish the correlation between chromosomal aberration, clinical features, and treatment response. Presenting the results of this study, we offer another evidence of variable incidence and clinical characteristics of ALL subtypes.

Expression of Bcl2L12 in chronic lymphocytic leukemia patients: association with clinical and molecular prognostic markers

Dysregulation of apoptosis is a distinctive feature of chronic lymphocytic leukemia (CLL), although a unique mechanism underlying apoptosis resistance of CLL B lymphocytes has not been identified yet. Aberrant expression as well as genetic and epigenetic alterations of numerous genes involved in different pathways of apoptosis regulation has been described in CLL. Here, we report the expression analysis of Bcl2L12 (Bcl2-like 12), a novel apoptotic gene belonging to Bcl2 family, in 58 Serbian CLL patients. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) analysis revealed a significant overexpression of Bcl2L12 mRNA in CLL samples compared to non-leukemic samples, implying its role in the pathogenesis of the disease. Receiver operating characteristic (ROC) analysis showed that Bcl2L12 expression efficiently discriminates CLL cases from healthy controls. However, relatively homogenous Bcl2L12 mRNA expression among patients did not reflect their clinical characteristics (with the exception of lactate dehydrogenase status and time from diagnosis to treatment) and failed to show association with the most informative prognostic markers, namely the mutational status of rearranged immunoglobulin heavy chain variable region genes, CD38 and lipoprotein lipase gene (LPL) expression.

Novel transcriptional regulatory element in the phenylalanine hydroxylase gene intron 8

We present the first transcriptional regulatory element found in a PAH gene intron. The element is located in the PAH gene intron 8, acts as an enhancer specifically in the hepatoma cell line, and binds GATA-1 transcription factor. Herein the presented data could unlock a new area for the analysis of PAH gene expression and could contribute to refining genotype-phenotype correlation.