Introdução: A leucemia linfoide aguda (LLA) \"BCR-ABL1-like\" exibe um perfil de expressão gênica semelhante ao observado em pacientes com LLA BCR-ABL1+. Este subtipo representa até 15% de todos os casos de LLA de linhagem B na população pediátrica e é frequentemente associado à presença de deleção total ou parcial do gene IKZF1. As isoformas de domínio negativo têm sido associadas a um aumento na chance de falha de resposta ao tratamento e tem sido associada com pior prognóstico. Objetivos: Analisar a presença de deleções do gene IKZF1 e a expressão de suas isoformas em amostras de medula óssea ao diagnóstico de crianças com LLA por técnica simplificada e de baixo custo de RT-PCR (reverse transcription polymerase chain reaction) e avaliar a associação desta alteração com fatores clínicos, biológicos e sobrevida. Metodologia: Foram analisadas 137 amostras de medula óssea colhidas ao diagnóstico de crianças com LLA, sendo 100 amostras de LLA de linhagem B, 35 de linhagem T e 2 na qual não foi possível a definição do imunofenópo, todas classificadas e tratadas segundo o protocolos GBTLI-99. A presença de deleções das isoformas do gene IKZF1 foi analisada por técnica de RT-PCR e confirmadas por sequenciamento automático. Associação entre deleção do IKZF1 e as variáveis idade, número de glóbulos brancos, grupo de risco, subgrupo molecular, presença de doença residual mínima e evento (recidiva ou óbito) foi analisada pelo teste exato de Fisher. Sobrevida livre de eventos, sobrevida livre de doença e sobrevida global foram avaliadas por curvas de Kaplan-Meier e teste log-rank. Análise multivariada por modelo de regressão de Cox foi utilizada para testar a independência dos fatores prognósticos. Resultados: Deleção total ou parcial no gene IKZF1 foi observada em 27/100 amostras de LLA B-derivada, sendo 15 evidenciando hiperexpressão das isoformas 6 ou 10, em 9 a expressão das isoformas foi de fraca intensidade e em 3 houve deleção total do gene. Nas amostras de LLA T-derivada foram observadas 3 alterações sendo 2 hiperexpressões da isoforma Ik6 e uma deleção total do gene. Presença de expressão forte das isoformas Ik6/Ik10 de IKZF1 foi associada na LLA B-derivada com pior sobrevida livre de eventos (SG), sobrevida livre de doença (SLD) e sobrevida global (SG)(P<0,001)). A presença de qualquer deleção do gene teve impacto apenas na SG (P=0,003). A sobrevida livre de eventos em 5 anos foi de 78,1 ± 4,6% versus 32 ± 12,4 % (P< 0,001), para os grupos sem e com expressão forte das isoformas Ik6/Ik10 forte de IKZF1 respectivamente, com risco relativo de evento desfavorável de 6.034 (95% IC: 2,105 - 17,295) para a presença da deleção. Análise multivariada por modelo de regressão de Cox nas LLA de linhagem B mostrou que expressão forte das isoformas Ik6/Ik10 foi o principal fator prognóstico independente (P<0.001) quando analisada em associação com idade, imunofenótipo (ausência de CD10), status da medula óssea no D28 da terapia de indução (M2/M3) e presença de DRM no D28 da indução tanto para SLE, quanto para SLD e SG. Nossos dados sugerem que o uso de técnica simplificada e de baixo custo para análise de deleções do gene IKZF1 é capaz de detectar pacientes com maior risco de recidiva, podendo ser útil na estratificação de pacientes com LLA de linhagem B em futuros protocolos de tratamento. Estudos multicêntricos com maior número de casos são necessários para confirmação destes resultados. / Introduction: Acute lymphoblastic leukemia (ALL) \"BCR-ABL1-like\" exhibits a gene expression profile similar to that observed in patients with BCR-ABL1 + ALL. This subtype accounts for up to 15% of all B lineage ALL cases in the pediatric population and is often associated with the presence of total or partial deletion of the IKZF1 gene. Negative domain isoforms have been associated with an increased chance of treatment failure and have been associated with poorer prognosis. Objectives: To analyze the presence of deletions of the IKZF1 gene and the expression of its isoforms in bone marrow samples to the diagnosis of children with ALL by simplified technique and lowcost RT-PCR (reverse transcription polymerase chain reaction) and to evaluate the association of this with clinical, biological and survival factors. Methodology: It has been analyzed 137 bone marrow samples collected at the diagnosis of children with ALL, 100 samples of ALL B lineage , 35 of T lineage and 2 in which it was not possible to define the immunophenotype, all classified and treated according to GBTLI protocols -99. The presence of deletions of the IKZF1 gene isoforms was analyzed by RT-PCR technique and confirmed by automatic sequencing. The association between IKZF1 deletion and the variables age, white blood cell count, risk group, molecular subgroup, presence of minimal residual disease and event (recurrence or death) were analyzed by the Fisher exact test. Event-free survival, disease-free survival and overall survival were assessed by Kaplan-Meier curves and log-rank test. Multivariate analysis by Cox regression model was used to test the independence of prognostic factors. Results: Total or partial deletion in the IKZF1 gene was observed in 27/100 samples of B-derived ALL, 15 of which showed hyperexpression of isoforms 6 or 10, in 9 the expression of the isoforms was of low intensity and in 3 there was total deletion of the gene . In the T- derived ALL samples, 3 alterations were observed, 2 hyperexpressions of the Ik6 isoform and a total deletion of the gene. The presence of strong Ik6/Ik10 isoform expression was associated in B-derived ALL with worse event-free survival (SLE), disease-free survival (SLD), and overall survival (OS) (P <0.001). The presence of any deletion of the gene had an impact only on the SLE (P = 0.003). The 5-year event-free survival was 78.1 ± 4.6% versus 32 ± 12.4% (P <0.001), for the groups with and without a strong deletion of IKZF1 respectively, with relative risk of unfavorable event of 6,034 (95% CI: 2,105 - 17,295) for the presence of the deletion. Multivariate analysis by Cox regression model in B lineage ALL showed that the strong expression of Ik6 / Ik10 isoforms was the main independent prognostic factor (P <0.001) when analyzed in association with age, immunophenotype (absence of CD10), marrow status bone in D28 of induction therapy (M2 / M3) and presence of DRM in induction D28 for both SLE, SLD and SG. Our data suggest that the use of simplified and low-cost IKZF1 gene deletion analysis is capable of detecting patients at higher risk of relapse and may be useful in the stratification of patients with B lineage ALL in future treatment protocols. Multicentric studies with a greater number of cases are necessary to confirm these results.

Introdução: A leucemia linfoide aguda (LLA) \"BCR-ABL1-like\" exibe um perfil de expressão gênica semelhante ao observado em pacientes com LLA BCR-ABL1+. Este subtipo representa até 15% de todos os casos de LLA de linhagem B na população pediátrica e é frequentemente associado à presença de deleção total ou parcial do gene IKZF1. As isoformas de domínio negativo têm sido associadas a um aumento na chance de falha de resposta ao tratamento e tem sido associada com pior prognóstico. Objetivos: Analisar a presença de deleções do gene IKZF1 e a expressão de suas isoformas em amostras de medula óssea ao diagnóstico de crianças com LLA por técnica simplificada e de baixo custo de RT-PCR (reverse transcription polymerase chain reaction) e avaliar a associação desta alteração com fatores clínicos, biológicos e sobrevida. Metodologia: Foram analisadas 137 amostras de medula óssea colhidas ao diagnóstico de crianças com LLA, sendo 100 amostras de LLA de linhagem B, 35 de linhagem T e 2 na qual não foi possível a definição do imunofenópo, todas classificadas e tratadas segundo o protocolos GBTLI-99. A presença de deleções das isoformas do gene IKZF1 foi analisada por técnica de RT-PCR e confirmadas por sequenciamento automático. Associação entre deleção do IKZF1 e as variáveis idade, número de glóbulos brancos, grupo de risco, subgrupo molecular, presença de doença residual mínima e evento (recidiva ou óbito) foi analisada pelo teste exato de Fisher. Sobrevida livre de eventos, sobrevida livre de doença e sobrevida global foram avaliadas por curvas de Kaplan-Meier e teste log-rank. Análise multivariada por modelo de regressão de Cox foi utilizada para testar a independência dos fatores prognósticos. Resultados: Deleção total ou parcial no gene IKZF1 foi observada em 27/100 amostras de LLA B-derivada, sendo 15 evidenciando hiperexpressão das isoformas 6 ou 10, em 9 a expressão das isoformas foi de fraca intensidade e em 3 houve deleção total do gene. Nas amostras de LLA T-derivada foram observadas 3 alterações sendo 2 hiperexpressões da isoforma Ik6 e uma deleção total do gene. Presença de expressão forte das isoformas Ik6/Ik10 de IKZF1 foi associada na LLA B-derivada com pior sobrevida livre de eventos (SG), sobrevida livre de doença (SLD) e sobrevida global (SG)(P<0,001)). A presença de qualquer deleção do gene teve impacto apenas na SG (P=0,003). A sobrevida livre de eventos em 5 anos foi de 78,1 ± 4,6% versus 32 ± 12,4 % (P< 0,001), para os grupos sem e com expressão forte das isoformas Ik6/Ik10 forte de IKZF1 respectivamente, com risco relativo de evento desfavorável de 6.034 (95% IC: 2,105 - 17,295) para a presença da deleção. Análise multivariada por modelo de regressão de Cox nas LLA de linhagem B mostrou que expressão forte das isoformas Ik6/Ik10 foi o principal fator prognóstico independente (P<0.001) quando analisada em associação com idade, imunofenótipo (ausência de CD10), status da medula óssea no D28 da terapia de indução (M2/M3) e presença de DRM no D28 da indução tanto para SLE, quanto para SLD e SG. Nossos dados sugerem que o uso de técnica simplificada e de baixo custo para análise de deleções do gene IKZF1 é capaz de detectar pacientes com maior risco de recidiva, podendo ser útil na estratificação de pacientes com LLA de linhagem B em futuros protocolos de tratamento. Estudos multicêntricos com maior número de casos são necessários para confirmação destes resultados. / Introduction: Acute lymphoblastic leukemia (ALL) \"BCR-ABL1-like\" exhibits a gene expression profile similar to that observed in patients with BCR-ABL1 + ALL. This subtype accounts for up to 15% of all B lineage ALL cases in the pediatric population and is often associated with the presence of total or partial deletion of the IKZF1 gene. Negative domain isoforms have been associated with an increased chance of treatment failure and have been associated with poorer prognosis. Objectives: To analyze the presence of deletions of the IKZF1 gene and the expression of its isoforms in bone marrow samples to the diagnosis of children with ALL by simplified technique and lowcost RT-PCR (reverse transcription polymerase chain reaction) and to evaluate the association of this with clinical, biological and survival factors. Methodology: It has been analyzed 137 bone marrow samples collected at the diagnosis of children with ALL, 100 samples of ALL B lineage , 35 of T lineage and 2 in which it was not possible to define the immunophenotype, all classified and treated according to GBTLI protocols -99. The presence of deletions of the IKZF1 gene isoforms was analyzed by RT-PCR technique and confirmed by automatic sequencing. The association between IKZF1 deletion and the variables age, white blood cell count, risk group, molecular subgroup, presence of minimal residual disease and event (recurrence or death) were analyzed by the Fisher exact test. Event-free survival, disease-free survival and overall survival were assessed by Kaplan-Meier curves and log-rank test. Multivariate analysis by Cox regression model was used to test the independence of prognostic factors. Results: Total or partial deletion in the IKZF1 gene was observed in 27/100 samples of B-derived ALL, 15 of which showed hyperexpression of isoforms 6 or 10, in 9 the expression of the isoforms was of low intensity and in 3 there was total deletion of the gene . In the T- derived ALL samples, 3 alterations were observed, 2 hyperexpressions of the Ik6 isoform and a total deletion of the gene. The presence of strong Ik6/Ik10 isoform expression was associated in B-derived ALL with worse event-free survival (SLE), disease-free survival (SLD), and overall survival (OS) (P <0.001). The presence of any deletion of the gene had an impact only on the SLE (P = 0.003). The 5-year event-free survival was 78.1 ± 4.6% versus 32 ± 12.4% (P <0.001), for the groups with and without a strong deletion of IKZF1 respectively, with relative risk of unfavorable event of 6,034 (95% CI: 2,105 - 17,295) for the presence of the deletion. Multivariate analysis by Cox regression model in B lineage ALL showed that the strong expression of Ik6 / Ik10 isoforms was the main independent prognostic factor (P <0.001) when analyzed in association with age, immunophenotype (absence of CD10), marrow status bone in D28 of induction therapy (M2 / M3) and presence of DRM in induction D28 for both SLE, SLD and SG. Our data suggest that the use of simplified and low-cost IKZF1 gene deletion analysis is capable of detecting patients at higher risk of relapse and may be useful in the stratification of patients with B lineage ALL in future treatment protocols. Multicentric studies with a greater number of cases are necessary to confirm these results.

Abstract Childhood acute lymphoblastic leukemia (ALL) is the most common cancer in children. The overall survival rate has reached to 90%. However, ALL still presents a significant disease burden and is a major cause for deaths in children. Recently, both inherited germline variants related to ALL susceptibility and somatic genetic variants forming novel subgroups of ALL have been discovered. In this thesis two families with familial ALL were studied. Constitutional heterozygous microdeletion at chromosome 7p12.1p13, including IKZF1, was discovered in the first family with intellectual impairment, overgrowth, and susceptibility to childhood ALL. In the second family, constitutional chromosome translocation was revealed in two individuals with childhood ALL and, subsequently, in seven unaffected family members. The balanced reciprocal translocation t(12;14)(p13.2;q23.1) resulted in breakpoints on two genes; ETV6 on chromosome 12 and RTN1 on chromosome 14. Only a few familial and sporadic ALL cases with germline variants in either IKZF1 or ETV6 have been published, thus supporting the significant role of these constitutional variants in childhood ALL predisposition. Inherited bone marrow failure syndromes (IBMFS) may predispose to childhood leukemia, including ALL. Two unrelated patients were diagnosed with bone marrow failure without the symptoms of classical IBMFS. Neither patient had any signs of developmental delay or congenital anomalies. Exome sequencing revealed identical c.1457del(p.(Ile486fs)) mutation on the ERCC6L2 gene in both patients. A few patients with IBMFS and ERCC6L2 variants have been described in previous studies. Some of them also had congenital craniofacial anomalies and developmental delay that were not detected in the patients in this thesis. The ALL cohort study on genetic variation of mitochondrial DNA (mtDNA) included 36 children. Metabolic change where malignant cells uncouple energy production from oxidative phosphorylation (OXPHOS) is one of the established hallmarks of cancer. In the cohort in this study, 22% of patients harbored nonsynonymous variants on mtDNA in the protein-coding genes of OXPHOS enzyme complexes. The somatic non-neutral variants were found in patients with a poor prognosis cytogenetic marker. The results support the hypothesis that cancer cells harbor mtDNA variants that may affect the cell metabolism. / Tiivistelmä Akuutti lymfoblastileukemia (ALL) on lasten yleisin syöpä. Vaikka nykyisin noin 90 prosenttia paranee, ALL aiheuttaa huomattavan paljon sairastavuutta ja on merkittävä lasten kuolinsyy. Vastikään on löydetty perinnöllisiä geneettisiä muutoksia, jotka altistavat lapsuusiän ALL:lle. Tutkimuksen kohteena oli kaksi perhettä, joissa vähintään kaksi lasta on sairastunut ALL:aan. Ensimmäisessä perheessä havaittiin lapsuusiän ALL:aan sairastuneilla kehityshäiriöisillä sisaruksilla äidiltä periytyvä heterotsygoottinen deleetio kromosomissa 7p12.1p13, jossa sijaitsee IKZF1-geeni. Toisessa perheessä perinnöllinen kahden kromosomin translokaatio todettiin kahdella lapsuusiän ALL:aan sairastuneella sekä seitsemällä perheenjäsenellä. Balansoitu translokaatio t(12;14)(p13.2;q23.1) aiheuttaa katkaisukohdan ETV6-geeniin kromosomissa 12 ja RTN1-geeniin kromosomissa 14. Tähän mennessä on julkaistu vain muutamia tutkimuksia potilaista, joilla on ollut perinnöllinen muutos joko IKZF1- tai ETV6-geenissä. Näillä geeneillä oletetaan olevan tärkeä merkitys perinnöllisessä alttiudessa sairastua lapsuusiän ALL:aan. Perinnölliset luuytimen toimintahäiriöt voivat altistaa leukemialle, kuten ALL:lle. Kahdella lapsella todettiin luuytimen toimintahäiriö, mutta ei muita oireita, jotka voisivat liittyä tyypillisiin perinnöllisiin luuytimen toimintahäiriöihin. Eksomisekvensoinnissa todettiin identtinen, homotsygoottinen mutaatio c.1457del(p.(Ile486fs)) ERCC6L2-geenissä. Kirjallisuuslähteiden mukaan vain muutamalla potilaalla on todettu ERCC6L2-geenin muutoksesta johtuva luuytimen toimintahäiriö. Osalla heistä on ollut synnynnäisiä kallon ja kasvojen anomalioita sekä kehityshäiriö, jollaisia tähän tutkimukseen osallistuneilla potilailla ei todettu. Potilaskohorttitutkimuksessa tutkittiin mitokondriaalisen DNA:n (mtDNA) muutoksia ALL:aan sairastuneilla lapsilla. Syöpäsolut eivät hyödynnä mitokondrion elektroninsiirtoketjua energian tuotantoon, ja tämä aineenvaihdunnan muutos on tunnustettu syövän ominaisuus. Tutkimuksessa havaittiin, että 22 prosentilla potilaista ilmeni diagnoosivaiheessa poikkeavia mtDNA:n muutoksia, jotka olivat elektroninsiirtoketjun entsyymien alayksiköitä koodaavissa geeneissä. Muutoksia todettiin useimmiten potilailla, joilla oli leukemiasoluissa huonon ennusteen geneettinen tekijä. Havaitut muutokset voivat mahdollisesti vaikuttaa leukemiasolun energia-aineenvaihduntaan.

Great progress has been achieved in the diagnostics and therapy of childhood acute lymphoblastic leukemia (ALL) during the last few decades and the permanent cure rate for children and adolescents has risen to nearly 90%. The basic principle of ALL treatment is to split patients into several groups receiving treatment of different intensity according to exactly defined prognostic features. This is aimed at reducing both the risk of relapse and toxic complications of treatment. The development of new diagnostic methods, especially in the field of molecular genetics and flow cytometry, allowed further improvements in the risk stratification - the minimal residual disease (MRD) has become a crucial prognostic factor in modern treatment protocols for pediatric ALL as a sensitive marker of both response to therapy and subclinical leukemic involvement of various tissues of the organism. Nevertheless, there is still an intensive search for new markers that would enable even more precise characterization of the leukemic clone, and treatment strategies reflecting the biology of leukemic cells are being optimized. The first part of our study describes the monitoring and prognostic impact of MRD in peripheral blood of children with ALL with emphasis on very early time points of treatment. MRD was examined by the...