305 research outputs found

    Using whole-exome sequencing data in an exome-wide association study approach to identify genetic risk factors influencing acute lymphoblastic leukemia response : a focus on asparaginase complications & vincristine-induced peripheral neuropathy

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    Le traitement de la leucémie lymphoblastique aiguë (LLA) de l’enfant, une affection d'origine maligne des cellules progénitrices lymphoïdes, s’est considérablement amélioré au cours des dernières décennies. En effet, le taux de succès du traitement a dépassé 90% dans des conditions favorables. Cependant, des toxicités liées au traitement peuvent être fatales et entrainer l’interruption ou la cessation du traitement. L'allergie, la pancréatite et la thrombose sont des complications fréquentes du traitement de la LLA et sont associées à l'utilisation de l'asparaginase (ASNase), tandis qu’une toxicité fréquente due à la vincristine (VCR) induit la neuropathie périphérique (VIPN). Étant donné que l’ajustement du schéma posologique afin d’augmenter l'efficacité et diminuer la toxicité est un processus sensible, ceci demeure un défi majeur dans plusieurs protocoles de traitement. La pharmacogénétique étudie comment des altérations de la composante génétique peuvent influer sur la variabilité interindividuelle observée dans la réponse au traitement. Une meilleure compréhension de la base moléculaire de cette variabilité pourrait améliorer considérablement les résultats du traitement, en permettant la personnalisation de ce dernier en fonction du profil génétique du patient. Des études récentes suggèrent l’avantage d’appliquer l’analyse de l’exome à la découverte de variants associés à des traits humains complexes ainsi qu’à des phénotypes de réactions médicamenteuses. L'objectif de notre travail était d'utiliser les données de séquençage pour réaliser des études d'association à l'échelle de l'exome, y compris des étapes de filtrage et de validation, afin d'identifier de nouveaux variants génétiques susceptibles de moduler le risque de développer des complications associées à ASNase et à VIPN. Douze SNP étaient associés à des complications due à l’ASNase dans la cohorte initiale, dont 3 étaient associés à une allergie, 3 à une pancréatite et 6 à une thrombose. Parmi ceux-ci, les variants rs3809849, rs11556218 et rs34708521 des gènes MYBBP1A, IL16 et SPEF2 respectivement ont été associés à des complications multiples et leur association à une pancréatite a été répliquée dans une cohorte de validation indépendante. En ce qui concerne la VCR, trois variantes ont été associées à la modulation du risque de VIPN: rs2781377 dans SYNE2, rs10513762 dans MRPL47 et rs3803357 dans BAHD1. Nous démontrons également le puissant effet combiné de la présence de plusieurs variants de risque pour chacune des toxicités étudiées et fournissons des modèles de prédiction du risque pour la pancréatite et le VIPN basés sur la méthode d’évaluation du risque génétique pondérée et qui ont été validés à l’interne. De plus, étant donné une association du polymorphisme du gène MYBBP1A avec de multiples issus de traitement, nous avons cherché à comprendre comment cette altération génétique se traduit par des variabilités de réponse aux traitements à l’ASNase. En utilisant la technique CRISPR-CAS9 pour induire l'inactivation de gènes dans des lignées cellulaires cancéreuses PANC1 (pancréatiques) nous avons testé la différence de viabilité entre les cellules inactivées et les cellules du type sauvage à la suite de la suppression du gène et du traitement par ASNase. Nos résultats suggèrent un rôle fonctionnel de ce gène dans la modulation de la viabilité, de la capacité de prolifération et de la morphologie des cellules knock-out, ainsi que dans leur sensibilité à l'ASNase, et plaident en outre pour que le gène influence l’issus du traitement de la LLA par ASNase. Le présent travail démontre que l’utilisation de l’approche de séquençage de l’exome entier dans le contexte d’une étude d’association à l’échelle de l’exome est une stratégie valide « sans hypothèse » pour identifier de nouveaux marqueurs génétiques modulant l’effet du traitement de la LLA de l’enfant, et souligne l’importance de l'effet synergique de la combinaison des locus à risque.Treatment of childhood acute lymphoblastic leukemia (ALL), a malignant disorder of lymphoid progenitor cells has improved significantly over the past decades and treatment success rates have surpassed 90% in favorable settings. However, treatment-related toxicities can be life-threatening and cause treatment interruption or cessation. Allergy, pancreatitis and thrombosis are common complications of ALL treatment associated with the use of asparaginase (ASNase), while vincristine-induced peripheral neuropathy (VIPN) is a frequent toxicity of vincristine (VCR). It is a sensitive process and a constant struggle to adjust the dosing regimen to ensure maximum efficacy and minimum toxicity. Pharmacogenetics studies show alterations in the genetic component between individuals can influence the observed variability in treatment response. A better understanding of the molecular basis of this variability in drug effect could significantly improve treatment outcome by allowing the personalization of ALL treatment based on the genetic profile of the patient. Emerging reports suggest the benefit of applying exome analysis to uncover variants associated with complex human traits as well as drug response phenotypes. Our objective in this work was to use available whole-exome sequencing data to perform exome-wide association studies followed by stepwise filtering and validation processes to identify novel variants with a potential to modulate the risk of developing ASNase complications and VIPN. Twelve SNPs were associated with ASNase complications in the discovery cohort including 3 associated with allergy, 3 with pancreatitis and 6 with thrombosis. Of those, rs3809849 in MYBBP1A, rs11556218 in IL16 and rs34708521 in SPEF2 genes were associated with multiple complications and their association with pancreatitis was replicated in an independent validation cohort. As for VCR, three variants were associated with modulating the risk of VIPN: rs2781377 in SYNE2, rs10513762 in MRPL47 and rs3803357 in BAHD1. We also demonstrate a strong combined effect of harbouring multiple risk variants for each of the studied toxicities, and provide internally-validated risk-prediction models based on the weighted genetic risk score method for pancreatitis and VIPN. Furthermore, given the association of the polymorphism in MYBBP1A gene with multiple treatment outcomes, we aimed at understanding how this genetic alteration translates into differences in ASNase treatment response through cell-based functional analysis. Using CRISPR-CAS9 technology we produced gene knockout of PANC1 (pancreatic) cancer cell-lines and tested the difference in viability between the knockouts and wild-type cells following gene deletion and ASNase treatment. Our results suggest a functional role of this gene in modulating the viability, proliferation capacity and the morphology of the knockout cells as well as their sensitivity to ASNase and further advocates the implication of the gene in influencing the outcome of ALL treatment with ASNase. The present work demonstrates that using whole-exome sequencing data in the context of exome-wide association study is a successful “hypothesis-free” strategy for identifying novel genetic markers modulating the effect of childhood ALL treatment and highlights the importance of the synergistic effect of combining risk loci

    Pharmacogenomic Markers of Methotrexate Response in the Consolidation Phase of Pediatric Acute Lymphoblastic Leukemia Treatment

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    Methotrexate (MTX) is one of the staples of pediatric acute lymphoblastic leukemia (ALL) treatment. MTX targets the folate metabolic pathway (FMP). Abnormal function of the enzymes in FMP, due to genetic aberrations, leads to adverse drug reactions. The aim of this study was to investigate variants in pharmacogenes involved in FMP and their association with MTX pharmacokinetics (MTX elimination profile) and toxicity in the consolidation therapy phase of pediatric ALL patients. Eleven variants in the thymidylate synthetase (TYMS), methylenetetrahydrofolate reductase (MTHFR), dihydrofolate reductase (DHFR), SLC19A1 and SLCO1B genes were analyzed in 148 patients, using PCR- and sequencing-based methodology. For the Serbian and European control groups, data on allele frequency distribution were extracted from in-house and public databases. Our results show that the A allele of SLC19A1 c.80 variant contributes to slow MTX elimination. Additionally, the AA genotype of the same variant is a predictor of MTX-related hepatotoxicity. Patients homozygous for TYMS 6bp deletion were more likely to experience gastrointestinal toxicity. No allele frequency dissimilarity was found for the analyzed variants between Serbian and European populations. Statistical modelling did not show a joint effect of analyzed variants. Our results indicate that SLC19A1 c.80 variant and TYMS 6bp deletion are the most promising pharmacogenomic markers of MTX response in pediatric ALL patients

    Pharmacogenomic markers of glucocorticoid response in the initial phase of remission induction therapy in childhood acute lymphoblastic leukemia

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    Background. Response to glucocorticoid (GC) monotherapy in the initial phase of remission induction treatment in childhood acute lymphoblastic leukemia (ALL) represents important biomarker of prognosis and outcome. We aimed to study variants in several pharmacogenes (NR3C1, GSTs and ABCB1) that could contribute to improvement of GC response through personalization of GC therapy. Methods. Retrospective study enrolling 122 ALL patients was carried out to analyze variants of NR3C1 (rs33389, rs33388 and rs6198), GSTT1 (null genotype), GSTM1 (null genotype), GSTPI (rs1695 and rs1138272) and ABCB1 (rs1128503, rs2032582 and rs1045642) genes using PCR-based methodology. The marker of GC response was blast count per microliter of peripheral blood on treatment day 8. We carried out analysis in which cut-off value for GC response was 1000 (according to Berlin-Frankfurt-Munster [BFM] protocol), as well as 100 or 0 blasts per microliter. Results. Carriers of rare NR3C1 rs6198 GG genotype were more likely to have blast count over 1000, than the noncarriers (p = 0.030). NR3C1 CAA (rs33389-rs33388-rs6198) haplotype was associated with blast number below 1000 (p = 0.030). GSTP1 GC haplotype carriers were more likely to have blast number below 1000 (p = 0.036), below 100 (p = 0.028) and to be blast negative (p = 0.054), while GSTP1 GT haplotype and rsl 138272 T allele carriers were more likely to be blasts positive (p = 0.034 and p = 0.024, respectively). ABCB1 CGT (rs1128503-rs2032582-rs1045642) haplotype carriers were more likely to be blast positive (p = 0.018). Conclusions. Our results have shown that NR3C1 rs6198 variant and GSTP1 rs1695-rs1138272 haplotype are the most promising pharmacogenomic markers of GC response in ALL patients

    ABCB1 genetic variants in leukemias: current insights into treatment outcomes

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    Despite improvements in treatment of different types of leukemia, not all patients respond optimally for a particular treatment. Some treatments will work better for some, while being harmful or ineffective for others. This is due to genetic variation in the form of singlenucleotide polymorphisms (SNPs) that affect gene expression or function and cause inherited interindividual differences in the metabolism and disposition of drugs. Drug transporters are one of the determinants governing the pharmacokinetic profile of chemotherapeutic drugs. The ABCB1 transporter gene transports a wide range of drugs, including drugs used in leukemia treatment. Polymorphisms in the ABCB1 gene do affect intrinsic resistance and pharmacokinetics of several drugs used in leukemia treatment protocols and thereby affect the efficacy of treatment and event-free survival. This review focuses on the impact of three commonly occurring SNPs (1236C>T, 2677G>T/A, and 3435C>T) of ABCB1 on treatment response of various types of leukemia. From the literature available, some of the genotypes and haplotypes of these SNPs have been found to be potential determinants of interindividual variability in drug disposition and pharmacologic response in different types of leukemia. However, due to inconsistencies in the results observed across the studies, additional studies, considering novel genomic methodologies, comprehensive definition of clinical phenotypes, adequate sample size, and uniformity in all the confounding factors, are warranted

    Somatic Mutation Detection in Leukemia-Derived Circulating DNA: Utility in Monitoring Clonal Dynamics and Disease Response in Pediatric Acute Lymphoblastic Leukemia

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    Despite the improved outcome associated with current treatment strategies ofpediatric acute lymphoblastic leukemia (ALL), relapse still represents a major challenge. Pediatric ALL demonstrates branched evolution in response to selective pressure exerted by therapy; relapse founder clones emerge from pre-leukemic clones or minor subclones present at diagnosis. It is hence crucial to develop biomarkers capable of tracking subclones throughout therapy. Current practices for monitoring disease response in leukemia rely on the analysis of BM biopsy sample at specific time points throughout therapy. Not only the invasiveness of the BM biopsy hinders the sequential sampling, but also, the currently implied techniques are associated with a lack of sensitivity to detect subclones other than the major diagnostic clone. Somatic mutation detection in circulating-tumor DNA (Ct-DNA) offers a new venue for non-invasive studying of genetic heterogeneity and tracking clonal dynamics throughout therapy. Here, we employ targeted Next-Generation Sequencing (NGS) using a specifically designed ALL custom gene panel for Ct-DNA analysis of sequential plasma samples of 14 pediatric ALL during remission induction therapy. Utilizing 1 ml of plasma, Ct-DNA successfully captured all the clinically relevant somatic single nucleotide variants (SNVs) detected by whole exome sequencing (WES) in bone marrow (BM) biopsy samples at diagnosis. Moreover, we were able to show the ability of Ct-DNA analysis to track the change in the mutant allele fraction (MAF) across multiple time points as well as, to detect mutations in Flowcytometry (FC) MRD-negative patients. Taken together, sequential analysis of Ct-DNA in plasma demonstrates a role, as a non-invasive technique, for detecting the clonal composition as well as, tracking clonal dynamics in pediatric ALL

    Response and toxicity to cytarabine therapy in leukemia and lymphoma: From dose puzzle to pharmacogenomic biomarkers

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    Cytarabine is a pyrimidine nucleoside analog, commonly used in multiagent chemotherapy regimens for the treatment of leukemia and lymphoma, as well as for neoplastic meningitis. Ara‐C‐based chemotherapy regimens can induce a suboptimal clinical outcome in a fraction of patients. Several studies suggest that the individual variability in clinical response to Leukemia & Lymphoma treatments among patients, underlying either Ara‐C mechanism resistance or toxicity, appears to be associated with the intracellular accumulation and retention of Ara‐CTP due to genetic variants related to metabolic enzymes. Herein, we reported (a) the latest Pharmacogenomics biomarkers associated with the response to cytarabine and (b) the new drug formulations with optimized pharmacokinetics. The purpose of this review is to provide readers with detailed and comprehensive information on the effects of Ara‐C‐based therapies, from biological to clinical practice, maintaining high the interest of both researcher and clinical hematologist. This review could help clinicians in predicting the response to cytarabine‐based treatments

    Uute tiopuriinmetüültransferaasi aktiivsust mõjutavate biomarkerite otsingul

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    Väitekirja elektrooniline versioon ei sisalda publikatsiooneRavimite vähene efektiivsus ja kahjustavad kõrvaltoimed on üks farmakoteraapia oluline probleem. Farmakogenoomika on saamas personaalmeditsiini oluliseks osaks, mille eesmärk on aidata arstil määrata patsiendile ravim või ravimidoos vastavalt tema individuaalsele geneetilisele profiilile. Tiopuriinmetüültransferaas (TPMT) on ensüüm, mis inaktiveerib lapseea leukeemia ja autoimmuunhaiguste ravis ning siirdamisjärgse äratõukereaktsiooni vältimiseks kasutatavaid tiopuriinravimeid. Uuringutest on selgunud, et TPMT aktiivsuse alusel võib inimesed jagada kolme rühma; ~0,3% indiviididest omab väga madalat ensüümi aktiivsust, ligikaudu 11%-l on ensüümi aktiivsus osaliselt vähenenud (nn keskmine) ja 89%-l on ensüümi aktiivsus normaalne. Seega teoreetiliselt on umbes 11% patsientidest ohustatud kõrvaltoimete tekkest tiopuriinravimite standarddooside kasutamisel. Hoolimata mitmetest teadaolevatest TPMT ensüümi aktiivsust vähendavatest TPMT geeni variantidest, on kliinilises praktikas suureks probleemiks TPMT genotüübi-fenotüübi erinevused, eriti nn keskmise ensüümi aktiivsusega indiviidide seas – vähenenud ensüümi aktiivsusega inimesed omavad normaalset TPMT genotüüpi ja vastupidi. See fakt annab alust oletada, et lisaks teadaolevatele TPMT geenivariantidele on veel faktoreid, mis ensüümi aktiivsust mõjutavad. Antud töö kirjeldab TPMT genotüübi-fenotüübi uuringut, mille käigus analüüsiti hetkel teadaolevate TPMT geeni markerite seost ensüümi aktiivsusega ning otsiti uusi, nii mitte-geneetilisi kui ka geneetilisi biomarkereid. Töö tulemusena tuvastasime uue mitte-geneetilise biomarkeri (SAM), mis mõjutab TPMT aktiivsust. Edasised kinnitavad uuringud on olulised selle juurutamiseks kliinilisse kasutusse. Samas, me ei leidnud uusi geneetilisi markereid ja ei kinnitunud teaduskirjanduses varem avaldatud geneetiliste markerite seos (peale TPMT geeni) TPMT ensüümi aktiivsusega. Hetkeseisuga ei ole võimalik kliinilises praktikas kasutada TPMT ensüümi aktiivsuse ja ravimvastuse ennustamiseks ainult TPMT genotüüpi. Hoolimata juhenditest geneetiliste markerite kasutamise ja tulemuste tõlgendamise kohta, tuleb arstidel jälgida patsiendi ravi kulgu kasutuses olevate raviskeemide alusel.The lack of efficiency and severe adverse reactions of drugs are serious problem in pharmacotherapy. Pharmacogenomics is becoming a part of personalized medicine, the practice of administering treatments based on the individual’s genomic profile and informing treatment decisions and allowing for more accurate and efficient selection of therapies that are best suited for specific patients. Thiopurine methyltransferase (TPMT) is an enzyme that inactivates thiopurine drugs, which are used to treat acute lymphoblastic leukemia, autoimmune diseases and to prevent rejection of transplanted organs. It has been shown in population studies that TPMT activity is trimodally distributed: approximately 0.3%, 11%, and 89% have deficient/low, intermediate, and normal TPMT activity, respectively, indicating that some 11% of individuals in this population may be prone to adverse drug events. Aside from identifiable inactivating variants of TPMT, there are additional geno- and phenotype variances (e.g., individuals without known genetic variants, but with intermediate TPMT activity and vice versa), especially among individuals with intermediate TPMT activity. This indicates that there are non-genetic and genetic biomarkers other than TPMT genotype influencing TPMT activity. The aim was to carry out a genotype-phenotype association study of TPMT activity to analyze the correlation between known TPMT variants and enzyme activity. In addition, new genetic and non-genetic biomarkers influencing variation in TPMT activity were investigated. As a result, we showed for the first time that SAM is an important modulator of TPMT activity. Therefore prospective studies are necessary to evaluate the clinical usefulness of determining SAM levels as a predictive factor of thiopurine therapy response. Additionally, our results indicate that TPMT genetics have a fundamental impact on TPMT activity in humans and provide little support for the proposal that other genes may significantly contribute to the inter-individual variability of TPMT activity. Although our data confirm that TPMT genotype is a robust predictor of TPMT activity in most individuals, TPMT genotype alone is insufficient to predict TPMT activity reliably. Despite guidelines of how to use genetic markers and interpret the results from genetic analysis, doctors should follow the course of the therapy of every individual according to the existing regimen

    Cell lines and animal model in the analysis of pharmacogenomics markers in childhood acute lymphoblastic leukemia

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    La leucémie aiguë lymphoblastique (LAL) est le cancer pédiatrique le plus fréquent. Elle est la cause principale de mortalité liée au cancer chez les enfants due à un groupe de patient ne répondant pas au traitement. Les patients peuvent aussi souffrir de plusieurs toxicités associées à un traitement intensif de chimiothérapie. Les études en pharmacogénétique de notre groupe ont montré une corrélation tant individuelle que combinée entre les variants génétiques particuliers d’enzymes dépendantes du folate, particulièrement la dihydrofolate réductase (DHFR) ainsi que la thymidylate synthase (TS), principales cibles du méthotrexate (MTX) et le risque élevé de rechute chez les patients atteints de la LAL. En outre, des variations dans le gène ATF5 impliqué dans la régulation de l’asparagine synthetase (ASNS) sont associées à un risque plus élevé de rechute ou à une toxicité ASNase dépendante chez les patients ayant reçu de l’asparaginase d’E.coli (ASNase). Le but principal de mon projet de thèse est de comprendre davantage d’un point de vue fonctionnel, le rôle de variations génétiques dans la réponse thérapeutique chez les patients atteints de la LAL, en se concentrant sur deux composants majeurs du traitement de la LAL soit le MTX ainsi que l’ASNase. Mon objectif spécifique était d’analyser une association trouvée dans des paramètres cliniques par le biais d’essais de prolifération cellulaire de lignées cellulaires lymphoblastoïdes (LCLs, n=93) et d’un modèle murin de xénogreffe de la LAL. Une variation génétique dans le polymorphisme TS (homozygosité de l’allèle de la répétition triple 3R) ainsi que l’haplotype *1b de DHFR (défini par une combinaison particulière d’allèle dérivé de six sites polymorphiques dans le promoteur majeur et mineur de DHFR) et de leurs effets sur la sensibilité au MTX ont été évalués par le biais d’essais de prolifération cellulaire. Des essais in vitro similaires sur la réponse à l’ASNase de E. Coli ont permis d’évaluer l’effet de la variation T1562C de la région 5’UTR de ATF5 ainsi que des haplotypes particuliers du gène ASNS (définis par deux variations génétiques et arbitrairement appelés haplotype *1). Le modèle murin de xénogreffe ont été utilisé pour évaluer l’effet du génotype 3R3R du gène TS. L’analyse de polymorphismes additionnels dans le gène ASNS a révélé une diversification de l’haplotype *1 en 5 sous-types définis par deux polymorphismes (rs10486009 et rs6971012,) et corrélé avec la sensibilité in vitro à l’ASNase et l’un d’eux (rs10486009) semble particulièrement important dans la réduction de la sensibilité in vitro à l’ASNase, pouvant expliquer une sensibilité réduite de l’haplotype *1 dans des paramètres cliniques. Aucune association entre ATF5 T1562C et des essais de prolifération cellulaire en réponse à ASNase de E.Coli n’a été détectée. Nous n’avons pas détecté une association liée au génotype lors d’analyse in vitro de sensibilité au MTX. Par contre, des résultats in vivo issus de modèle murin de xénogreffe ont montré une relation entre le génotype TS 3R/3R et la résistance de manière dose-dépendante au traitement par MTX. Les résultats obtenus ont permis de fournir une explication concernant un haut risque significatif de rechute rencontré chez les patients au génotype TS 3R/3R et suggèrent que ces patients pourraient recevoir une augmentation de leur dose de MTX. À travers ces expériences, nous avons aussi démontré que les modèles murins de xénogreffe peuvent servir comme outil préclinique afin d’explorer l’option d’un traitement individualisé. En conclusion, la connaissance acquise à travers mon projet de thèse a permis de confirmer et/ou d’identifier quelques variants dans la voix d’action du MTX et de l’ASNase qui pourraient faciliter la mise en place de stratégies d’individualisation de la dose, permettant la sélection d’un traitement optimum ou moduler la thérapie basé sur la génétique individuelle.Acute lymphoblastic leukemia (ALL) is the most frequent malignancy of childhood. It is the principal cause of cancer–related mortality in children due to a persistent group of patients who does not respond to standard anti-cancer treatment. Susceptible patients may also suffer from number of toxicities associated with intensive chemotherapy treatment. Pharmacogenetic studies of our group, showed that particular genetic variants of the folate dependent enzymes, particularly, dihydrofolate reductase (DHFR) and thymidylate synthase (TS), major targets of methotrexate (MTX), correlate both individually and combined with increased risk of relapse in patients with childhood ALL. Furthermore, variations of ATF5 gene involved in asparagine synthetase (ASNS) regulation and of ASNS gene were associated with higher risk of ALL relapse or with ASNase related toxicity in patients who received E.coli asparaginase (ASNase). The major goal of my doctoral research project was to further understand from the functional point of view the role of genetic variations underlying therapeutic responses of childhood ALL, by focusing on two major components of ALL treatment, MTX and ASNase. My specific goal was to analyze associations found in clinical setting using cellular proliferation assay in lymphoblastoid cell lines (LCLs, n=93) and xenograft mice model of ALL. Genetic variation in TS polymorphism (homozygosity for triple repeat allele, 3R) and of DFHR haplotype *1b (defined by particular allelic combination derived from six polymorphic sites in the major and minor promoter of DHFR), on MTX sensitivity was assessed using cellular proliferation assay. Similar in vitro assay in response to E.coli ASNase was used to access the T1562C variation in the ATF5 5’UTR and particular haplotypes of ASNS gene (defined by two genetic variation and arbitrarily named haplotype *1). Xenograft mouse model was used to access the effect of TS 3R3R genotype. Analysis of additional polymorphisms in ASNS gene revealed diversification of haplotype *1 of ASNS gene in 5 subtypes, two polymorphisms (rs10486009 and rs6971012,) defining particular subtypes correlated with in vitro sensitivity to ASNase and one of them (rs10486009) seems particularly important for reducing sensitivity to ASNase in vitro, possibly providing mechanistic explanation for lower sensitivity of haplotype *1 observed in clinical setting. No association between ATF5 T1562C variation and cellular proliferation assay in response to E.coli ASNase was found. We did not observe genotype-related association when in vitro sensitivity to MTX in LCLs was analyzed. In contrast, in vivo results using xenograft mouse model demonstrated the relationship between the TS 3R/3R genotype and the resistance to MTX treatment in dose-dependent manner. Obtained results provided function explanation for the significantly higher risk of relapse seen in 3R/3R ALL patients and suggest that these patients might benefit from increase dose of MTX. Through these experiments we also showed that xenogeneic mice model can serve as a preclinical tool to explore individualized treatment options. In conclusion, the knowledge acquired through my doctoral work confirmed and/or identified some functional variants in MTX and ASNase action pathway which may facilitate dose individualization strategies, allowing for optimal treatment selection or tailoring childhood ALL therapy based on individual genetics

    Influence of variants in genes important for farmacokinetics of 6- mercaptopurine on acute lymphoblastic leukemia treatment in children

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    Akutna limfoblastna leukemija (ALL) predstavlja najčešću malignu bolest dečjeg doba. Akutna limfoblastna leukemija je klonalna maligna bolest hematopoetskog tkiva, koja se odlikuje klonalnom proliferacijom limfoblasta. a na osnovu njihovih imunofenotipskih karakteristika može se podeliti na B-ćelijske i T-ćelijske. Najčešći oblik akutne limfoblastne leukemije kod dece je B ćelijska prekursorska leukemija. Najčešće se javlja u uzrastu od druge do pete godine života i nešto je češća kod dečaka. Optimalna primena antileukemijskih lekova u sklopu jasno definisanih terapijskih protokola i precizna stratifikacija bolesnika u grupe rizika, uz maksimalnu suportivnu terapiju doveli su do značajnog porasta ukupnog preživljavanja koje sada iznosi i do 90%. Lečenje leukemije kod dece se sastoji iz četiri faze, od kojih je poslednja i najduža, faza održavanja u čijem sklopu se primenjuje lek iz grupe tiopurina – 6-merkaptopurin (6-MP). Toksični efekti ovog leka, pre svega mijelosupresija, kod izvesnog broja dece mogu dovesti do životno ugrožavajućih infekcija kao i do značajnog odlaganja terapije i povećanja rizika za nastanak recidiva bolesti. Enzim tiopurin S-metiltransferaza (TPMT) je najznačajni enzim za inaktivaciju 6-MP i poznato je da deca koja imaju sniženu aktivnost enzima, imaju izraženije toksične efekte i zahtevaju primenu manjih dozu 6-MP. Individualizovana terapija na osnovu TPMT genotipa je postala standard u lečenju akutne limfoblastne leukemije u dece. Osim enzima TPMT, postoji još nekoliko metaboličkih puteva u inaktivaciji i transportu 6-MP koji mogu uticati na povećanu toksičnost tokom primene leka. Inozin trifosfat pirofosfataza je drugi enzim, kodiran od strane ITPA gena, koji inaktiviše 6-MP, i nedovoljna aktivnost ovog enzima takođe doprinosi pojavi izražene mijelotoksičnosti tokom primene 6-MP. P-glikoprotein, kodiran od strane ABCC4 gena, i Multidrug Resistance Protein 4, kodiran od strane ABCB1 gena su transmembranski proteini koji funkcionišu kao efluks pumpe i izbacuju ksenobiotike iz ćelija. Povećana odnosno smanjena aktivnost ovih proteina može dovesti do smanjenog terapijskog efekta lekova odnosno do povećane toksičnosti tokom primene 6-MP. Cilj ovog istraživanja je bio da utvrdi učestalost varijacija u genima TPMT, ITPA, ABCC4 i ABCB1 kod dece obolele od akutne limfoblastne leukemije i njihov uticaj na intenzitet toksičnih efekata 6-MP kao i na ukupno preživljavanje...Acute lymphoblastic leukemia is the most common childhood malignancy. Acute lymphoblastic leukemia is malignant clonal disease of hematopoetic tissue and it is characterized by clonal proliferation of lymphoblasts, which can be of B-cell or T-cell origin. The most prevalent type of acute lymphoblastic leukemia in children is B-cell precursor leukemia. It is most commonly seen in children between age of two and five and there is slight male predominance. Optimal use of antileukemic drugs in the settings of clearly defined therapeutic protocols and precise risk stratification with maximal supportive therapy have led to significant improvement in overall survival, which is now near 90%. Leukemia treatment consists of four phases and last and longest phase is maintenance phase. In this phase, the most important drug is 6-mercaptopurine (6-MP) which belongs to the group of thiopurine drugs. 6-MP induced toxicity, myelosupression as the most prominent one, can in one number of children lead to life-threatening infections as well as significant therapy delay which contributes to increased risk for relapse. Enzyme thiopurine S-methyltransferase (TPMT) is most important inactivation enzyme for 6-MP and it is well established that children with TPMT deficiency have more pronounced toxicity and require lower dose of 6-MP. Personalized therapy based on TPMT genotype has become standard in treatment of acute lymphoblastic leukemia in children. Besides inactivation pathway that utilizes TPMT there are several other metabolic pathways for inactivation and transport mechanisms that can influence toxicity profile of 6-MP. Inosine triphosphate pirophosphatase (ITPA), which is coded by ITPA gene, is one of those 6-MP inactivation enzymes which, when deficient, can contribute to profound myelotoxicity. P-glycoprotein, coded by ABCC4 gene, and Multidrug Resistance Protein 4, coded by ABCB1 gene, are transmembrane transporters that works as efflux-pumps and removes xenobiotics from cells. Increased or reduced activity of these efflux-pumps can lead to reduced therapeutic effect or increased toxicity of 6-MP. Goal of this study was to determine frequency of variations in genes TPMT, ITPA, ABCC4 and ABCB1 in children with acute lymphoblastic leukemia and their influence on 6-MP induced toxicity as well as on overall survival..

    From genomic variation to personalized medicine

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