Analiza transkriptoma pri otroških bolnikih s T-celično akutno limfoblastno levkemijo

Janžič, Urška (author), Trebušak Podkrajšek, Katarina (mentor), Debeljak, Maruša (comentor)

T-celična akutna limfoblastna levkemija predstavlja 10–15 % vseh akutnih limfoblastnih levkemij pri otrocih. Kljub napredku v načinu zdravljenja bolezni v zadnjih letih T-celični imunofenotip, ki je genetsko zelo heterogen, predstavlja višje tveganje za neuspešno zdravljenje bolezni kot B-celični imunofenotip. Bolniki, pri katerih se bolezen ponovi, imajo slabo prognozo, saj so relapsi pogosto odporni na zdravljenje. Delitev bolezni glede na genotip, ki bi imel pomembno napovedno vrednost za bolnike, kot je to mogoče pri B-celični akutni limfoblastni levkemiji, ne obstaja. Namen raziskave je opredeliti transkriptom T-celične akutne levkemije pri otrocih z analizo diferencialnega izražanja genov. Izolirali smo RNA iz kostnega mozga in periferne krvi otrok, ki so jim med leti 2009 in 2023 na Pediatrični kliniki v Ljubljani diagnosticirali T-celično akutno limfoblastno levkemijo. Pripravili smo knjižnice in jih sekvencirali z aparatom NovaSeq 6000, Illumina. S podatkovnim cevovodom Snakepipes in orodjema DESeq2 ter edgeR smo opredelili različno izražene gene pri bolnikih, ki so doživeli relaps v primerjavi z bolniki v remisiji. Opredelili smo tudi razlike v izražanju genov med bolniki z nezrelim in zrelim imunofenotipom ter primerjali diferencialno izražene gene pri bolnikih s T- in B-celično akutno limfoblastno levkemijo. Za ta del analize smo uporabili že pridobljene podatke sekvenciranja bolnikov z B-celično akutno limfoblastno levkemijo. Pri bolnikih s T-ALL in relapsom smo opredelili 11 genov, pri bolnikih v remisiji pa 41 genov, ki so se povečano izražali. V določenih primerih je povečano izražanje onkogenov ali zmanjšano izražanje tumorsupresorskih genov kazalo na povezavo med slabšo prognozo bolnikov z relapsom. V nekaterih primerih pa povezave niso bile jasne, saj izražanje genov v literaturi še ni bilo opisano. Rezultati analize izražanja genov pri različnih imunofenotipih so pokazali, da je povezava med imunofenotipom in genetskim profilom slaba, kar je opisano tudi v literaturi. Pri diferencialni analizi izražanja genov med bolniki s T- in B-celično akutno limfoblastno levkemijo smo opredelili največ, kar 8848 različno izraženih genov. V spletnem orodju Enrichr smo z obogatitveno analizo opredelili pot imunoregulatornih interakcij med limfoidno in nelimfoidno celico kot tisto, ki bi lahko imela vlogo pri slabšem poteku bolezni v primerjavi z B-celično akutno limfoblastno levkemijo. Da bi lahko določili izražanje genov, ki bi imelo pomembno napovedno vrednost za pediatrične bolnike s T-ALL, so potrebne nadaljnje raziskave.

• Language: Slovenian
• Keywords: T-celična akutna limfoblastna levkemija, transkriptom, izražanje genov, sekvenciranje RNA, analiza diferencialnega izražanja genov
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-160869
• Publication date in RUL: 05.09.2024

Secondary language

• Language: English
• Title: Transcriptome analysis of acute T-cell lymphoblastic leukemia pediatric patients

Abstract

T-cell acute lymphoblastic leukaemia accounts for 10–15% of all acute lymphoblastic leukaemias in children. Despite advances in the treatment of the disease in recent years, the T-cell immunophenotype, which is genetically very heterogeneous, poses a higher risk of treatment failure than B-cell immunophenotype. Patients with relapse have a very poor prognosis, as relapses are often resistant to treatment. Unlike B-cell acute lymphoblastic leukaemia, in which the immunophenotype-genotype association profile is well established as outcome predictors, T-cell acute lymphoblastic leukemia still needs further investigations.The aim of the study is a transcriptome analysis in T-cell acute lymphoblastic leukemia pediatric patients with differential gene expression analysis. We isolated RNA from bone marrow and peripheral blood of children diagnosed with T-cell acute lymphoblastic leukemia at the Pediatric clinic in Ljubljana between 2009 and 2023. Then RNA libraries were prepared and sequenced using the NovaSeq 6000, Illumina. Using the Snakepipes data pipeline, DESeq2 and edgeR, we identified differential gene expression in relapsed patients compared to patients in remission. We also identified differences in gene expression between immature and mature immunophenotype and compared differentially expressed genes in T- and B-cell acute lymphoblastic leukaemia patients. Sequencing data from patients with B-cell acute lymphoblastic leukaemia patients were used for this part of the analysis. Eleven highly expressed genes were identified in relapsed and 41 genes in remission patients. In some cases, increased expression of oncogenes or decreased expression of tumour suppressor genes could indicate a poorer prognosis of relapsed patients, in other cases the association was unclear as gene expression has not yet been described in the literature. Analysis of gene expression in different immunophenotypes showed poor association between immunophenotype and genetic profile, which is also described in the literature. In differential analysis of gene expression between T- and B-cell acute lymphoblastic leukemia patients, we identified 8,848 differently expressed genes. We identified a pathway of immunoregulatory interactions between lymphoid and non-lymphoid cells using Enrichr enrichment analysis as one that could play a role in the poorer outcome of the disease compared to B-cell acute lymphoblastic leukemia. Further research is needed to determine gene expression that would have a significant prognostic value for patients.

• Keywords: T-cell acute lymphoblastic leukemia, transcriptome, gene expression, RNA sequencing, differential gene expression analysis