Outcome of adolescents and young adults with acute lymphoblastic leukemia in a single center in Brazil

ABSTRACT Introduction: Acute lymphoblastic leukemia (ALL) presents a poor prognosis in adults. The adoption of pediatric protocols has been changing this scenario, especially for adolescents and young adults (AYA). Objective and method: We aimed to evaluate a consecutive series of patients treated at the State Institute of Hematology of Rio de Janeiro between 2012 and 2020, focusing on the AYA subgroup. Result: The B-ALL was the most frequent subtype (81.6%) and AYA, the predominant age group (57.7%). The median overall survival (OS) was 9.4 months. High early mortality was observed and sepsis was the main cause of death. Better OS results were noted in AYA, in comparison to over 39y (13.3 × 6.2 months, respectively), the Berlin-Frankfurt-Münster (BFM) being the protocol of choice in this group. Conclusion: The use of the pediatric protocol seems to improve the OS of AYA, however, high rates of deaths from infection were observed, demonstrating the need for advances in the Brazilian public system clinical support.

Outcome of adolescents and young adults with acute lymphoblastic leukemia in a single center in Brazil.

INTRODUCTION: Acute lymphoblastic leukemia (ALL) presents a poor prognosis in adults. The adoption of pediatric protocols has been changing this scenario, especially for adolescents and young adults (AYA). OBJECTIVE AND METHOD: We aimed to evaluate a consecutive series of patients treated at the State Institute of Hematology of Rio de Janeiro between 2012 and 2020, focusing on the AYA subgroup. RESULT: The B-ALL was the most frequent subtype (81.6%) and AYA, the predominant age group (57.7%). The median overall survival (OS) was 9.4 months. High early mortality was observed and sepsis was the main cause of death. Better OS results were noted in AYA, in comparison to over 39y (13.3 × 6.2 months, respectively), the Berlin-Frankfurt-Münster (BFM) being the protocol of choice in this group. CONCLUSION: The use of the pediatric protocol seems to improve the OS of AYA, however, high rates of deaths from infection were observed, demonstrating the need for advances in the Brazilian public system clinical support.

Multiparametric flow cytometry directing the evaluation of CRLF2 rearrangements and JAK2 status in pediatric B cell precursor acute lymphoblastic leukemia.

INTRODUCTION: This study aimed to determine whether cytokine receptor-like factor 2 (CRLF2) antigen expression evaluated using multiparametric flow cytometry (MFC) could predict the genotype of CRLF2 and Janus kinase 2 (JAK2) status for application in the diagnosis of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). METHODS: A total of 321 BCP-ALL bone marrow samples were collected, 291 at diagnosis and 13 at first relapse, while 17 samples were excluded due to low cellular viability. The CRLF2 antigen expression was evaluated using flow cytometry (percentage of positivity and median fluorescence intensity [MFI]). The CRLF2 transcript levels were assessed via quantitative reverse transcription polymerase chain reaction using SYBR Green. The CRLF2 rearrangements (CRLF2-r) were identified using the CRLF2 break-apart probe via fluorescence in situ hybridization. Sanger sequencing was performed to identify the JAK2 exon 16 mutations. RESULTS: We observed that 60 of the 291 cases (20.6%) presented CRLF2 antigen positivity, whereas the CRLF2 transcript overexpression was found in 19 of 113 cases (16.8%). The JAK2 mutation was found in four out of 116 cases (3.4%), all of which had CRLF2 ≥10% of positive cells and intermediate or high MFI (p < 0.0001). In addition, in the 13 cases with the CRLF2-r, a positive correlation was found with the CRLF2 antigen intermediate (61.5%) MFI (p = 0.017). Finally, the CRLF2-positive antigen was identified in the BCP-ALL subclones. CONCLUSION: The identification of the CRLF2 antigen using the MFC, based on the percentage of positivity and MFI values, is a useful tool for predicting JAK2 mutations and CRLF2-r.

Multiparametric flow cytometry directing the evaluation of CRLF2 rearrangements and JAK2 status in pediatric B cell precursor acute lymphoblastic leukemia

Asbtract Introduction This study aimed to determine whether cytokine receptor-like factor 2 (CRLF2) antigen expression evaluated using multiparametric flow cytometry (MFC) could predict the genotype of CRLF2 and Janus kinase 2 (JAK2) status for application in the diagnosis of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Methods A total of 321 BCP-ALL bone marrow samples were collected, 291 at diagnosis and 13 at first relapse, while 17 samples were excluded due to low cellular viability. The CRLF2 antigen expression was evaluated using flow cytometry (percentage of positivity and median fluorescence intensity [MFI]). The CRLF2 transcript levels were assessed via quantitative reverse transcription polymerase chain reaction using SYBR Green. The CRLF2 rearrangements (CRLF2-r) were identified using the CRLF2 break-apart probe via fluorescence in situ hybridization. Sanger sequencing was performed to identify the JAK2 exon 16 mutations. Results We observed that 60 of the 291 cases (20.6%) presented CRLF2 antigen positivity, whereas the CRLF2 transcript overexpression was found in 19 of 113 cases (16.8%). The JAK2 mutation was found in four out of 116 cases (3.4%), all of which had CRLF2 ≥10% of positive cells and intermediate or high MFI (p < 0.0001). In addition, in the 13 cases with the CRLF2-r, a positive correlation was found with the CRLF2 antigen intermediate (61.5%) MFI (p= 0.017). Finally, the CRLF2-positive antigen was identified in the BCP-ALL subclones. Conclusion The identification of the CRLF2 antigen using the MFC, based on the percentage of positivity and MFI values, is a useful tool for predicting JAK2 mutations and CRLF2-r.

IKZF1 deletions associate with CRLF2 overexpression leading to a poor prognosis in B-cell precursor acute lymphoblastic leukaemia.

Cytokine Receptor-Like Factor 2 (CRLF2) overexpression occurs in 5-15% of B-cell precursor acute lymphoblastic leukaemia (B-ALL). In ∼50% of these cases, the mechanisms underlying this dysregulation are unknown. IKAROS Family Zinc Finger 1 (IKZF1) is a possible candidate to play a role in this dysregulation since it binds to the CRLF2 promoter region and suppresses its expression. We hypothesised that IKZF1 loss of function, caused by deletions or its short isoforms expression, could be associated with CRLF2 overexpression in B-ALL. A total of 131 paediatric and adult patients and 7 B-ALL cell lines were analysed to investigate the presence of IKZF1 deletions and its splicing isoforms expression levels, the presence of CRLF2 rearrangements or mutations, CRLF2 expression and JAK2 mutations. Overall survival analyses were performed according to the CRLF2 and IKZF1 subgroups. Our analyses showed that 25.2% of patients exhibited CRLF2 overexpression (CRLF2-high). CRLF2-high was associated with the presence of IKZF1 deletions (IKZF1del, p = 0.001), particularly with those resulting in dominant-negative isoforms (p = 0.006). Moreover, CRLF2 expression was higher in paediatric samples with high loads of the short isoform IK4 (p = 0.011). It was also associated with the occurrence of the IKZF1 plus subgroup (p = 0.004). Furthermore, patients with CRLF2-high/IKZF1del had a poorer prognosis in the RELLA05 protocol (p = 0.067, 36.1 months, 95%CI 0.0-85.9) and adult cohort (p = 0.094, 29.7 months, 95%CI 11.8-47.5). In this study, we show that IKZF1 status is associated with CRLF2-high and dismal outcomes in B-ALL patients regardless of age.

A novel PAX5 rearrangement in TCF3-PBX1 acute lymphoblastic leukemia: a case report.

BACKGROUND: Chromosome translocations are a hallmark of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Additional genomic aberrations are also crucial in both BCP-ALL leukemogenesis and treatment management. Herein, we report the phenotypic and molecular cytogenetic characterization of an extremely rare case of BCP-ALL harboring two concomitant leukemia-associated chromosome translocations: t(1;19)(q23;q13.3) and t(9;17)(p13;q11.2). Of note, we described a new rearrangement between exon 6 of PAX5 and a 17q11.2 region, where intron 3 of SPECC1 is located. This rearrangement seems to disrupt PAX5 similarly to a PAX5 deletion. Furthermore, a distinct karyotype between diagnosis and relapse samples was observed, disclosing a complex clonal evolution during leukemia progression. CASE PRESENTATION: A 16-year-old boy was admitted febrile with abdominal and joint pain. At clinical investigation, he presented with anemia, splenomegaly, low white blood cell count and 92% lymphoblast. He was diagnosed with pre-B ALL and treated according to high risk GBTLI-ALL2009. Twelve months after complete remission, he developed a relapse in consequence of a high central nervous system and bone marrow infiltration, and unfortunately died. CONCLUSIONS: To our knowledge, this is the first report of a rearrangement between PAX5 and SPECC1. The presence of TCF3-PBX1 and PAX5-rearrangement at diagnosis and relapse indicates that both might have participated in the malignant transformation disease maintenance and dismal outcome.

Osteopontin and their roles in hematological malignancies: Splice variants on the new avenues.

Osteopontin (OPN) is a protein expressed in several tissues, including bone marrow, in which it performs distinct roles, such as modulating hematopoietic stem cell niche and bone remodeling. Most data in hematological malignancies (HMs) refers to total OPN (tOPN), comprehending the sum of distinct OPN splicing isoforms (OPN-SI), while reports describing the expression and roles of each OPN-SI are scarce. This review aims to summarize tOPN roles in HMs and provide evidence that OPN-SIs can also modulate specific functions in HMs biology. We summarize that upregulated tOPN can modulate HMs (leukemia, lymphoma and myeloma) progression, inducing cell adhesion, invasion, angiogenesis, cell differentiation and extramedullary and/or central nervous system infiltration. Based on this expression pattern, tOPN has been pointed out as a biomarker in those HMs, thus providing potential targets for therapeutic approaches. Our group found that OPN-SIs are expressed in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cell lines (unpublished data), providing early evidence that OPN-SIs are also expressed in BCP-ALL. Further studies should investigate whether these OPN-SIs can differently modulate HMs biology and their putative application as auxiliary biomarkers for HMs.

ARID5B polymorphism confers an increased risk to acquire specific MLL rearrangements in early childhood leukemia.

BACKGROUND: Acute leukemia in early age (EAL) is characterized by acquired genetic alterations such as MLL rearrangements (MLL-r). The aim of this case-controlled study was to investigate whether single nucleotide polymorphisms (SNPs) of IKZF1, ARID5B, and CEBPE could be related to the onset of EAL cases (<24 months-old at diagnosis). METHODS: The SNPs (IKZF1 rs11978267, ARID5B rs10821936 and rs10994982, CEBPE rs2239633) were genotyped in 265 cases [169 acute lymphoblastic leukemia (ALL) and 96 acute myeloid leukaemia (AML)] and 505 controls by Taqman allelic discrimination assay. Logistic regression was used to evaluate the association between SNPs of cases and controls, adjusted on skin color and/or age. The risk was determined by calculating odds ratios (ORs) with 95% confidence interval (CI). RESULTS: Children with the IKZF1 SNP had an increased risk of developing MLL-germline ALL in white children. The heterozygous/mutant genotype in ARID5B rs10994982 significantly increased the risk for MLL-germline leukemia in white and non-white children (OR 2.60, 95% CI: 1.09-6.18 and OR 3.55, 95% CI: 1.57-8.68, respectively). The heterozygous genotype in ARID5B rs10821936 increased the risk for MLL-r leukemia in both white and non-white (OR 2.06, 95% CI: 1.12-3.79 and OR 2.36, 95% CI: 1.09-5.10, respectively). Furthermore, ARID5B rs10821936 conferred increased risk for MLL-MLLT3 positive cases (OR 7.10, 95% CI:1.54-32.68). Our data do not show evidence that CEBPE rs2239633 confers increased genetic susceptibility to EAL. CONCLUSIONS: IKZF1 and CEBPE variants seem to play a minor role in genetic susceptibility to EAL, while ARID5B rs10821936 increased the risk of MLL-MLLT3. This result shows that genetic susceptibility could be associated with the differences regarding MLL breakpoints and partner genes.

Predição da fusão gênica TV6 - RUNX1 através da expressão celular de CD9 em leucemia linfoblástica aguda de células precursoras B

Introdução: A translocação cromossômica t(12;21)(p13;q22) gera a fusão gênica ETV6-RUNX1. Esta alteração é especifica de leucemias linfoblásticas agudas de células precursora B (LLA-CPB), especialmente aquelas com CD10+ e também um biomarcador de bom prognóstico. A expressão celular de CD9 tem sido sugerida como uma possível ferramenta de rastreio para predizer a existência de ETV6-RUNX1. Este estudo teve como objetivo estabelecer um algoritmo de testes utilizando inicialmente a citometria de fluxo, para direcionar e otimizar o diagnóstico dos subtipos de LLA-CPB através da correlação entre os níveis de intensidade da expressão celular de CD9 e a fusão gênica ETV6-RUNX1. Metodologia: Aspirados de medula óssea de pacientes foram analisados ao diagnóstico por citometria com painel de anticorpos estabelecidos como padrão de imunofenotipagem. Um tubo adicional consistindo de CD9/CD10/CD19 foi incorporado ao painel. Análises do percentual de células marcadas e intensidade média de fluorescência (IMF) da molécula CD9 foram utilizadas para comparação com os testes moleculares. RT-PCR e FISH foram usadas para identificar a fusão gênicaETV6 -RUNX1. Os resultados obtidos foram plotados em planilhas de dados e em seguida analisados por regressão linear para determinar a probabilidade da expressão dessa molécula predizer a fusão gênica. As análises estatísticas foram realizadas no programa GraphPad Prism 5. Curvas de ROC por “Receiver Operating Characteristic” (ROC) com pacote ROCR usando a versão 2.10., foram geradas para determinar a probabilidade da expressão dessas moléculas predizer o genótipo. Foram realizados os testes de acurácia, sensibilidade, especificidade, valor preditivo positivo (VPP) e valor preditivo negativo (VPN). Resultados: Foram incluídos 186 casos de LLA-CPB, em pacientes com idade entre 0-12 anos,no período de 2011 a 2013; 13 casos eram LLA pro-B (CD10-); 150 casos de LLA-comum (CD10+) e 23 casos como LLA pré-B (CD10+/μ+)...

Introduction: The chromosomal translocation t(12;21) (p13;q22) generates the fusion gene ETV6-RUNX1. This alteration is specific of B-cell precursor lymphoblastic leukemia (BCP-ALL), mainly those with CD10+, and is also a good prognosis biomarker. The CD9 cellular expression has been suggested as a possible screening tool to predict the existence of ETV6-RUNX1. This study aimed to establish an algorithm of tests, initially using flow cytometry, in order to optimize the diagnosis of BCP-ALL subtypes through the correlation between the cellular expression of CD9 and ETV6-RUNX1gene fusion. Methodology: Bone marrow aspirates of patients at diagnosis were analyzed by flow cytometry with antibodies established as standard immunophenotyping panel. An additional tube consisting of CD9/CD10/CD19 has beenincorporated to the panel. Percentage analysis of labeled cells and mean fluorescence intensity (MFI) of CD9 molecule were used for comparison with the molecular tests. RT-PCR and FISH were used to identify the ETV6-RUNX1gene fusion. The results were plotted on spreadsheets and then analyzed by linear regression to determine the probability of prediction of this molecule expression. Statistical analyzes were performed with GraphPad Prism 5 program. ROC curves for "Receiver Operating Characteristic" (ROC) with ROCR package using version 2.10. were performed to determine the likelihood of expression of these molecules to predict genotype. Accuracy, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated. Results: We included 186 cases of BCP-ALL diagnosed in patients with ages between 0-12 years, in the period 2011-2013. Of these, 13 cases were subclassified as pro-B-ALL (CD10-), 150 cases as common-ALL (CD10+) and 23 cases as pre-B ALL (CD10+/μ+). The ETV6-RUNX1gene fusion was found in 44/186 (23.6%) of cases of BCP-ALL, being 36 out of these classified as CD10+ subtype, type II. Seven samples with...