Next generation sequencing as a tool for pharmacogenomic profiling: Nine novel potential genetic markers for targeted therapy in childhood acute lymphoblastic leukemia

Uvod/Cilj Sekvenciranje nove generacije (SNG) omogućilo je genomsko profilisanje svakog bolesnika. Nova saznanja u oblasti farmakogenomike omogućavaju primenu podataka dobijenih ovom metodom u cilju otkrivanja novih mogućih genetičkih markera za ciljanu terapiju mnogih, posebno malignih bolesti. Cilj ovog istraživanja je bio da se primenom SNG odre- di genetski profil akutne limfoblastne leukemije (ALL) kod dece u cilju procene mogućih molekularnih meta za ciljanu terapiju. Metode Analizirali smo DNK uzorke 17 bolesnika obolelih od ALL dečjeg doba koristeći ciljano SNG. Napredne bioinformatičke metode su korišćene da identifikuju nove mutacije u analiziranim genima i da predvide njihov uticaj i farmakogenomski potencijal. Rezultati Identifikovali smo devet genskih varijanti koje do sada nisu opisane u relevantnim bazama podataka. U navedenim varijantama identifikovane su dve 'besmislene' varijante, ABL1 p.Q252* i AKT1 p.W22*, jedna varijanta koja pomera okvir čitanja, STK11 p.G257fs*28, i šest nesinonimnih varijanti. Kreirali smo trodimenzionalni model za četiri proteina koji bi bili produkt novih nesinonimnih varijanti. Analizirali smo farmakogenomski potencijal svake varijante i otkrili da su dve, STK11 c.1023G gt T/ p.L341F i ERBB2 c.2341C gt T/ p.R781W, mogući kandidati za ciljanu terapiju. Zaključak Nove varijante otkrivene u ovoj studiji pripa- daju uglavnom genima povezanim sa Ras signalnim putem, koji je često zahvaćen mutacijama u ALL kod dece. Farmakogenomsko profilisanje svake dečje ALL biće nezamenljivo za nove terapijske pristupe. Detekcija i inicijalna analiza novih genskih varijanti, koja je predstavljena u ovoj studiji, postaće standardna procedura za dizajniranje i razvoj individualizovane terapije za decu obolelu od ALL.Introduction/Objective Next generation sequencing (NGS) technology has enabled genomic profiling of each patient. Growing knowledge in pharmacogenomics makes it possible to use NGS data for discovery of novel potential genetic markers for targeted therapy of many diseases, especially cancers. The aim of this study was to use targeted NGS to make a genetic profile of childhood acute lymphoblastic leukemia (cALL) in order to evaluate potential molecular targets for targeted therapy. Methods We analyzed DNA samples from 17 cALL patients using NGS targeted sequencing. Advanced bioinformatic analysis was used to identify novel mutations in analyzed genes and to predict their effect and pharmacogenomic potential. Results We identified nine variants that have not been previously reported in relevant databases, including two stop-gain variants, ABL1 p.Q252* and AKT1 p.W22*, one frameshift, STK11 p.G257fs*28, and six missense variants. We created three-dimensional models of four proteins harboring novel missense variants. We analyzed pharmacogenomic potential of each variant and found that two of them, STK11 c.1023G gt T/ p.L341F and ERBB2 c.2341C gt T/ p.R781W, are suitable candidates for targeted therapy. Conclusion Most new variants detected in this study were found in the genes associated with Ras signaling pathway, which is frequently mutated in cALL patients. Pharmacogenomic profiling of each cALL will be indispensable for novel therapy approaches. Detection and initial analysis of novel variants, presented in this study, will become a standard procedure for the design and development of individualized therapies for children with ALL, leading to improved patient outcomes

Importance of genotyping of Thiopurine S-methyltransferase in children with acute lymphoblastic leukaemia during maintenance therapy

Uvod. Tiopurin-S-metiltransferaza (TPMT ) je enzim koji katalizuje inaktivaciju merkaptopurina, leka koji se široko primenjuje u lečenju akutne limfoblastne leukemije (ALL) kod dece. Kada se osobe s nedostatkom TPMT leče standardnim dozama merkaptopurina, kod njih se razvija teška i po život opasna mijelotoksičnost. Cilj rada. Cilj rada je bio da se utvrdi da li kod dece s ALL koji su nosioci mutacije u genu za TPMT individualizovanjem doziranja merkaptopurina može da se smanji mijelotoksičnost terapije, te da li broj tandemskih ponovaka (engl. variable number of tandem repeats - VNTR) u promotoru gena za TPMT ima uticaja na efekte terapije merkaptopurinom. Metod rada Metodima lančane reakcije umnožavanja DNK (engl. polymerase chain reaction - PCR) ispitano je 50 nasumično odabrane dece lečene ALL IC-BFM 2002 protokolom na najčešće mutacije u genu za TPMT. Za 20 dece je PCR metodima određen VNTR genotip. Ispitanicima je tokom faze održavanja beležen broj nedelja kada su terapiju dobijali u punim ili smanjenim dozama, kao i broj nedelja bez terapije. Rezultati Među 50 dece bilo je 29 dečaka (58%) i 21 (42%) devojčica, uzrasta od 1,8 do 17,3 godine (medijana 6,2 godine). Utvrđeno je četvoro (8%) heterozigotnih nosilaca mutacija, kod kojih je otkrivena TPMT*3A varijanta. Posle 12, 14, 16 i 19 nedelja lečenja smanjenim dozama merkaptopurina bolesnici su, zbog dobrog podnošenja terapije, postepeno počeli da primaju punu dozu leka. Nije bilo odlaganja terapije. Smanjenje kumulativne doze merkaptopurina za bolesnike sa TPMT mutacijama bilo je 7,8%, 7,4%, 11,2% i 16,6%. Između dece bez TPMT mutacija i heterozigota nije za- beležena statistički značajna razlika u trajanju lečenja punim (53,6 nasuprot 55,7 nedelja) i smanjenim dozama merkaptopurina (19,9 nasuprot 15,2 nedelje). Otkrivenih VNTR bilo je između četiri i sedam. Većina bolesnika imala je različit broj VNTR na homolognim hromozomima. Najčešće uočen polimorfizam bio je VNTR*5. Nije zabeležena korelacija između nasleđivanja TPMT i VNTR genotipa. Zaključak Farmakogenetskim principima u lečenju ALL dece može se postići napredak u podnošenju lečenja merkaptopurinom.INTRODUCTION Thiopurine S-methyltransferase (TPMT) is an enzyme that catalyses the inactivation of mercaptopurine (MP) which is widely used in the treatment of acute lymphoblastic leukaemia (ALL) of childhood. Potentially fatal myelotoxicity may develop after standard doses of MP in TPMT deficient patients. OBJECTIVES To establish if individually tailored doses of MP can reduce myelotoxicity in ALL patients carrying mutations in the TPMT gene. To establish if variable number of tandem repeats (VNTR) genotype influences the treatment effects of MP. METHOD Fifty randomly selected patients treated according to ALL IC-BFM 2002 protocol were tested for most frequent TPMT gene mutations using PCR based methods. VNTR genotype was determined in 20 children by PCR methods. During the maintenance phase, we recorded the number of weeks when therapy was applied in either full doses, reduced doses or when patients were without any therapy. RESULTS Fifty children were examined, 29 boys (58%) and 21 girls (42%); age ranged from 1.8-17.3 years (median 6.2 years). Four patients (8%) were heterozygous for TPMT mutations, all of them carrying the TPMT*3A variant. After 12, 14, 16 and 19 weeks of therapy with reduced doses of MP, the patients switched to full doses due to good tolerance. There was no therapy omission. Cumulative dose of MP was reduced for 7.8%, 7.4%, 11.2% and 16.6%, respectively, in patients with TPMT mutations. No significant difference was found between children with no mutations and TPMT heterozygotes regarding full dose therapy (53.6 vs. 55.7 weeks, respectively) and reduced dose therapy (19.9 vs. 15.2 weeks respectively). The number of detected VNTRs ranged from four to seven. The majority of patients had different number of VNTRs on homologous chromosomes. Most frequently detected polymorphism was VNTR*5. No correlation was found between TPMT and VNTR genotype inheritance. CONCLUSION Obeying pharmacogenetic principles in the treatment of childhood ALL may improve the tolerance of therapy with MP

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

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

Wilms tumor (wt)1 gene expression in children with acute leukemia in Serbia

Acute leukemias constitute the most common malignancy in childhood, accounting for 25-35% of all cancer in children. They are divided into acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Genetic susceptibility is known to play a major role in childhood leukemias. Wilms tumor (WT)1 is a zinc finger transcription factor involved in regulating the process of cell differentiation; it has been implicated in a wide range of human neoplasms. WT1 overexpression in the bone marrow at diagnosis is reported to be an independent negative prognostic factor in adults and children with AML. The aim of the present investigation was to determine the expression of WT1 in the bone marrow of children with AML and ALL in Serbia and its possible impact on patient survival. We determined bone marrow WT1 expression levels by reverse -transcription polymerase chain reaction (RT-PCR) at diagnosis in 20 children with AML and 20 children with ALL (16 B-ALL and 4 T-ALL), as well as 15 age- and sex-matched controls who were evaluated for immune thrombocytopenic purpura (ITP). For children with AML, follow-up samples were also analyzed one month after treatment initiation and at variable later timepoints of control punctures. The results were normalized based on WT1 expression in controls. We found that children with AML had significantly higher WT1 expression at diagnosis (median SD: 139.42 244.03) than those with ALL (1.18 54.37; Mann -Whitney U=82; p lt 0.01) and ITP (0.76 1.01; U=32; p lt 0.01). Patients with T-ALL had higher WT1 expression than those with B-ALL, though significance was not reached due to subgroup size; differences between AML subgroups according to the French-American-British (FAB) classification were also below the level of significance, though a tendency toward higher values in M3 and M4 leukemias was notable. There was also a tendency toward higher values in 14 children with AML who were still alive after a median follow-up of 1.5 years (181.42 192.52) than in 6 who succumbed to the disease (104.29 354.87). All children with AML who had WT1 expression 1 month after diagnosis below the fourth quartile (10 of 10) were still alive, while only 2 of 5 with 1 -month WT1 expression in the fourth quartile survived (Fisher's exact test: p=0.0952). Taken together, our results support a role for WT1 in the diagnostic workup in children with acute leukemia, although it needs to be considered in view of a complex and indvidualized context

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

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

Immunoglobulin genes and T-cell receptors as molecular markers in children with acute lymphoblastic leukaemia

Uvod. Akutna limfoblastna leukemija (ALL) je maligna klonalna bolest i jedna od najčešćih neoplazmi dečjeg uzrasta. Primenom savremenih protokola postižu se visok stepen remisije i dugogodišnja stopa preživljavanja. Uvođenjem metoda molekularne genetike omogućeni su submikroskopska klasifikacija i praćenje minimalne rezidualne bolesti (MRB), odgovorne za nastanak recidiva. Cilj rada. Cilj rada je bilo ispitivanje učestalosti rearanžmana genskih lokusa za teške lance imunoglobulina (IgH) i T-ćelijski receptor (TCR) i njihova korelacija s kliničkim parametrima. Metode rada. Ispitano je 41 dete obolelo od ALL kojima je na dijagnozi rađena molekularna detekcija rearanžmana genskih lokusa za IgH i TCR metodom lančanog umnožavanja DNK (PCR). Posmatranje razvoja MRB je rađeno u indukcionoj fazi lečenja, kada se očekuje morfološki odgovor na terapiju. Rezultati. Rearanžman genskog lokusa za IgH zabeležen je kod 82,9%, a za TCR kod 56,1% ispitanika. Posle 33 dana indukcione terapije rearanžmani za IgH genski lokus su zabeleženi kod 39%, a za TCR kod 36,5% dece. Zaključak. Otkrivanje genetskih aberacija na molekularnom nivou i njihova korelacija sa standardnim prognostičkim parametrima ukazala je na značaj nove stratifikacije rizičnih grupa, koje zahtevaju promenu intenziteta hemioterapije. Praćenje MRB se pokazalo preciznim prediktivnim faktorom u nastanku recidiva bolesti. PR Projekat Ministarstva nauke Republike Srbije, br. 143051.Introduction. Acute lymphoblastic leukaemia (ALL) is a malignant clonal disease, one of the most common malignancies in childhood. Contemporary protocols ensure high remission rate and long term free survival. The ability of molecular genetic methods help to establish submicroscopic classification and minimal residual disease (MRD) follow up, in major percent responsible for relapse. Objective. The aim of the study was to detect the frequency of IgH and TCR gene rearrangements and their correlation with clinical parameters. Methods. Forty-one children with ALL were enrolled in the study group, with initial diagnosis of IgH and TCR gene rearrangements by polimerase chain reaction ( PCR). MRD follow-up was performed in induction phase when morphological remission was expected, and after intensive chemiotherapy. Results. In the study group IgH rearrangement was detected in 82.9% of children at the diagnosis, while TCR rearrangement was seen in 56.1%. On induction day 33, clonal IgH rearrangements persisted in 39% and TCR rearrangements in 36.5% of children. Conclusion. Molecular analysis of genetic alterations and their correlation with standard prognostic parameters show the importance of risk stratification revision which leads to new therapy intensification approach. MRD stands out as a precise predictive factor for the relapse of disease

Variants in TPMT, ITPA, ABCC4 and ABCB1 genes as predictors of 6-mercaptopurine induced toxicity in children with acute lymphoblastic leukemia

Uvod: Akutna limfoblastna leukemija je najčešća maligna bolest decjeg doba. Optimalna upotreba antileukemijskih lekova dovela je do smanjenja toksičnosti i neželjenih događaja, kao i do povećanja stope preživljavanja. Tiopurinski lekovi, uključujući 6-merkaptopurin, predstavljaju najčešće korišćene antileukemijske lekove u lečenju dece obolele od akutne limfoblastne leukemije, koji se koriste u toku faze održavanja. Persona lizovana terapija 6-merkaptopurinom, prilagođena TPMT genotipu svakog pacijenta, već je implementirana u terapijske protokole. Cilj ove studije je bio da ispita ulogu varijanti u genima TPMT, ITPA, ABCC4 i ABCB1 kao prediktora ishoda i toksičnosti uzrokovane 6-merkaptopurinom u toku faze održavanja u lečenju pedijatrijske akutne limfoblastne leukemije. Metode: U studiju je bilo uključeno 68 dece obolele od akutne limfoblastne leukemije. Pacijenti su lečeni primenom A LL IC-BFM 2002 ili A LL IC-BFM 2009 protokola. Toksičnost i neželjeni događaji su posmatrani i beleženi upotrebom surogat markera (broj nedelja bez terapije, broj epizoda leukopenije i prosecna doza 6-merkaptopurina) a probabilisticki modeli su korišćeni za predikciju ukupne toksicnosti uzrokovane primenom 6-merkaptopurina. Rezultati: Studija je potvrdila da pacijenti oboleli od akutne limfoblastne leukemije koji imaju neaktivni TPMT alel zahtevaju redukciju doze 6-merkaptopurina. Varijante u genima ITPA i ABCC4 nisu bile povezane sa toksicnošću koja je uzrokovana primenom 6-merkaptopurina u toku faze održavanja. Nosioci varijante u genu ABCB1 su ispoljili veću hepatotoksicnost. Probabilisticki model Neural net kojim su posmatrane sve analizirane genske varijante se pokazao kao najbolji predikcioni model. Primenom ovog modela bilo je moguće razlikovati A LL pacijente sa lošom i dobrom tolerancijom 6-merkaptopurina u 71% slucajeva (AUC=0,71). Zaključak: Ova studija doprinosi dizajniranju panela farmakogenetskih markera koji bi se koristili za predikciju toksicnosti uzrokovane primenom tiopurinskih lekova kod dece obolele od akutne limfoblastne leukemije.Background: Acute lymphoblastic leukemia is the most common childhood malignancy. Optimal use of anti leukemic drugs has led to less toxicity and adverse reactions, and a higher survival rate. Thiopurine drugs, including 6-mercaptopurine, are mostly used as antileukemic medications in the maintenance phase of treatment for children with acute lymphoblastic leukemia. For those patients, TPMT genotype-tailored 6-mercaptopurine therapy is already implemented in the treatment protocols. We investigated the role of TPMT, ITPA, ABCC4 and ABCB1 genetic variants as predictors of outcome and 6-mercaptopurine induced toxicity during the maintenance phase of treatment in pediatric acute lymphoblastic leukemia. Methods: Sixty-eight children with acute lymphoblastic leukemia were enrolled in this study. Patients have been treated according to A LL IC-BFM 2002 or ALL IC-BFM 2009 protocols. Toxicity and adverse events have been monitored via surrogate markers (off-therapy weeks, episodes of leukopenia and average 6-mercaptopurine dose) and a probabilistic model was employed to predict overall 6-mercaptopurine related toxicity. Results: We confirmed that patients with acute lymphoblastic leukemia that carry inactive TPMT allele(s) require 6mercaptopurine dose reduction. ITPA and ABCC4 genetic variants failed to show an association with 6-mercapto - purine induced toxicity during the maintenance phase. Carriers of ABCB1 variant allele experienced greater hepatotoxicity. The probabilistic model Neural net which considered all the analysed genetic variants was assessed to be the best prediction model. It was able to discriminate ALL patients with good and poor 6-mercaptopurin tolerance in 71% of cases (A U C = 0 .7 l). Conclusions: This study contributes to the design of a panel of pharmacogenetic markers for predicting thiopurineinduced toxicity in pediatric ALL

Importance of pharmacogenetic markers in the methylenetetrahydrofolate reductase gene during methotrexate treatment in pediatric patients with acute lymphoblastic leukemia

Despite remarkable progress in survival of children with acute lymphoblastic leukemia (ALL) which has reached about 85%, early toxicity and relapse rate remain issues that need to to be resolved. Genetic variants are important factors influencing the metabolism of cytotoxic drugs in ALL treatment. Variants in genes coding for methotrexate (MTX)-metabolizing enzymes are under constant scientific interest due to their potential impact on drug toxicity and relapse rate. We investigated methylenetetrahydrofolate reductase (MTHFR) c.677C gt T and MTHFR c.1298A gt C variants as pharmacogenetic markers of MTX toxicity and predictors of relapse. The study enrolled 161 children with ALL, treated according to the current International Berlin-Frankfurt-Munster group (BFM) for diagnostics and treatment of leukemia and lymphoma protocols. Genotyping was performed using PCR-RFLP and allele-specific PCR assays. Our results revealed similar distributions of MTHFR c.677C gt T and MTHFR c.1298A gt C genotypes among 104 healthy individuals as compared to pediatric ALL patients. A lower incidence of early MTX toxicity was noted in the MTHFR c.677TT genotype (p=0.017), while MTHFR c.1298A gt C genotypes were not associated with MTX toxicity. Carriers of any MTHFR c.677C gt T and MTHFR c.1298A gt C genotypes did not experience decreased overall survival (OAS) or higher relapse rates. Genetic variants in the MTHFR gene are not involved in leukemogenesis in pediatric ALL. The presence of the MTHFR c.677TT genotype was recognized as a predictive factor for decreased MTX toxicity during the intensification phase of therapy. Neither MTHFR c.677C gt T nor MTHFR c.1298A gt C genotypes correlated with an increased number of toxic deaths or relapse rate. Our study emphasizes the importance of implementing pharmacogenetic markers in order to optimize pediatric ALL therapy

Application of targeted next generation sequencing for the mutational profiling of patients with acute lymphoblastic leukemia

lt b gt Uvod: lt /b gt Akutna limfoblastna leukemija (ALL) je najčešće maligno oboljenje kod dece, dok je kod odraslih njena učestalost mnogo niža. U današnjoj kliničkoj praksi kao najvažnije metode stratifikacije pacijenata u određene grupe rizika koriste se metode identifikacije citogenetičkih aberacija i malog broja molekulanih markera. Tehnologija sekvenciranja nove generacije (SNG) obezbeđuje veliku količinu podataka koji doprinose razjašnjavanju mutacionog profila dečje (dALL) i adultne ALL (aALL). lt b gt Metode: lt /b gt Uzorci DNK iz 34 dALL i aALL pacijenata analizirani su primenom SNG ciljanog sekvenciranja ("TruSeq Amplicon Cancer Panel - TSACP") kojim se sekvenciraju "hotspot" mutacije u 48 gena povezanih sa kancerom. lt b gt Rezultati: lt /b gt Identifikovano je ukupno 330 varijanti u kodirajućim regionima, od kojih je samo 95 njih za posledicu imalo potencijalnu promenu u proteinu. Posmatrano kod pojedinačnih pacijenata, detektovane mutacije su pretežno remetile Ras/RTK signalni put (STK11, KIT, MET, NRAS, KRAS, PTEN). Pored toga, identifikovano je 5 pacijenata sa istom mutacijom u HNF1A genu, koja je uzrokovala poremećaje u Wnt i Notch signalnom putu. Kod dva pa cijenta otkrivene su varijante u NOTCH1 genu. Nije detektovano istovremeno prisustvo varijanti u HNF1A i NOTCH1 genu, dok su geni uključeni u Ras/RTK signalni put pokazali tendenciju ka akumuliranju mutacija. lt b gt Zaključak: lt /b gt Naši rezultati pokazuju da ALL sadrži Mali broj mutacija, bez značajnih razlika između dALL i aALL (medijana po pacijentu 2 odnosno 3). Detektovane mutacije izazivaju poremećaje u nekoliko ključnih signalnih puteva, prvenstveno Ras/RTK kaskade. Ova studija doprinosi ukupnom znanju o mutacionom profilu ALL, što vodi ka boljem razumijevanju molekularne osnove ovog oboljenja.lt b gt Background: lt /b gt Acute lymphoblastic leukemia (ALL) is the most common cancer in children, whereas it is less common in adults. Identification of cytogenetic aberrations and a small number of molecular abnormalities are still the most important risk and therapy stratification methods in clinical practice today. Next generation sequencing (NGS) technology provides a large amount of data contributing to elucidation of mutational landscape of childhood (cALL) and adult ALL (aALL). lt b gt Methods: lt /b gt We analyzed DNA samples from 34 cALL and aALL patients, using NGS targeted sequencing TruSeq Amplicon - Cancer Panel (TSACP) which targets mutational hotspots in 48 cancer related genes. lt b gt Results: lt /b gt We identified a total of 330 variants in the coding regions, out of which only 95 were potentially protein-changing. Observed in individual patients, detected mutations predominantly disrupted Ras/RTK pathway (STK11, KIT, MET, NRAS, KRAS, PTEN). Additionally, we identified 5 patients with the same mutation in HNF1A gene, disrupting both Wnt and Notch signaling pathway. In two patients we detected variants in NOTCH1 gene. HNF1A and NOTCH1 variants were mutually exclusive, while genes involved in Ras/RTK pathway exhibit a tendency of mutation accumulation. lt b gt Conclusions: lt /b gt Our results showed that ALL contains low number of mutations, without significant differences between cALL and aALL (median per patient 2 and 3, respectively). Detected mutations affect few key signaling pathways, primarily Ras/RTK cascade. This study contributes to knowledge of ALL mutational landscape, leading to better understanding of molecular basis of this disease

Expression pattern of long non-coding RNA growth arrest-specific 5 in the remission induction therapy in childhood acute lymphoblastic leukemia

Uvod: Ekspresija duge nekodirajuće RNK G AS5 je izme - njena u mnogim kancerima zbog njene uloge u apoptozi i inhibiciji rasta ćelije. G AS5 interaguje sa glukokortikoidnim receptorom, što je čini potencijalnim farmakotranskripcionim markerom značajnim za glukokortikoidnu terapiju. Naš cilj u ovoj studiji je bio da analiziramo ekspresiju GAS5 tokom indukcione terapije kod dečje akutne limfoblastne leukemije (ALL), u kojoj se koriste glukokortikoidni lekovi, i da te rezultate povežemo sa odgovorom na terapiju. Metode: Nivo ekspresije G AS5 u mononuklearnim ćelijama periferne krvi izolovanih od 29 dece obolelih od ALL, određen je metodologijom RT-qPCR, i to u momentu dijagnoze, 15. i 33. dana indukcione terapije. Rezultati: Naši rezultati su pokazali da postoje interindivi - dualne razlike u ekspresiji GAS5 kod pacijenata, i to u svim analiziranim tačkama. Kod svakog ALL pacijenta GAS5 ekspresija je 15. dana bila visa u odnosu na ekspresiju u momentu dijagnoze (p lt 0,0005). Nivo ekspresije GAS5 je 33. dana bio niži u poređenju sa 15. danom (p lt 0,0005), ali je i dalje bio značajno visi u odnosu na momenat dijagnoze kod većine pacijenata (p = 0,001). Pacijenti čiji je broj blasta u perifernoj krvi 8. dana bio ispod 100 po mikrolitru periferne krvi, imali su viši nivo ekspresije GAS5 (p = 0,016) i niži odnos ekspresija merenih 15. dana i u momentu dijagnoze (p = 0,009). Zaključak: Nasi rezultati ukazuju da bi nivo ekspresije GAS5 mogao da bude marker terapijskog odgovora u indukcionoj terapiji kod dece obolele od ALL.Background: Long non-coding RNA growth arrest-specific 5 (GAS5) is deregulated in many cancers because of its role in cell growth arrest and apoptosis. Additionally, GAS5 interacts with glucocorticoid receptor, making it a potential pharmacotranscription marker of glucocorticoid (GC) therapy. In this study, we aimed at analysing G AS5 expression in the remission induction therapy phase of childhood acute lymphoblastic leukemia (ALL), in which GCs are mandatorily used, and to correlate it with therapy response. Methods: G AS5 expression was measured in peripheral blood mononuclear cells taken from 29 childhood ALL patients at diagnosis, on day 15 and day 33 of remission induction therapy using RT-qPCR methodology. Results: Our results have shown interindividual differences in G AS5 expression at all time points. For each ALL patient, G A S5 expression was higher on day 15 in comparison to its level at diagnosis (p lt 0.0005). On day 33, the level of G A S5 expression decreased in comparison with day 15 (p lt 0.0005), but it was still significantly higher than at diagnosis for the majority of patients (p=0.001). Patients whose number of blasts on day 8 was below 100 per mL of peripheral blood had a higher GAS5 expression at diagnosis (p=0.016), and lower ratio day 15/diagnosis (p=0.009). Conclusions: Our results suggest that the expression level of GAS5 could be a potential marker of therapy response in remission induction therapy of childhood ALL