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.

Citometria de fluxo multiparamétrica na determinação da expressão proteica e transcrição gênica de CRLF2 na leucemia linfoblástica aguda de células precursoras B / Multiparametric flow cytometry in the determination of protein expression and gene transcription of CRLF2 in B cell precursor acute lymphoblastic leukemia

Introdução: A leucemia linfoblástica aguda de células precursoras B (LLA-cpB) apresenta biomarcadores citogenético-moleculares clássicos com prognóstico bem definidos. Trinta por cento desses casos não possuem nenhuma dessas anormalidades e são referidas como B-others e 50% desse subgrupo possui perfil de expressão gênica semelhante a BCR-ABL1, sendo chamados então, de BCRABL1-like. Este subgrupo possui diferenças genéticas características como mutações nos genes JAK1/2, IL7Rα, e CRLF2, além da expressão gênica aumentada deste último. O CRLF2 forma um heterodímero com a cadeia alfa do receptor de Interleucina 7 (IL- 7Rα) ou CD127 formando o receptor da citocina TSLP. Estudos envolvendo o CRLF2 têm apresentado resultados controversos, devido a não utilização de técnicas padronizadas para sua detecção. Objetivo: Estabelecer um algoritmo de testes combinados imunofenotípicos e moleculares capazes de predizer o status do CRLF2 e sua associação com subtipos moleculares e celulares em amostras de crianças com LLA-cpB. Métodos: Análise do painel de anticorpos monoclonais CD10FITC/ CD127PE/CD45PerCP-CY5.5/ CD19PECY7 / CRLF2APC em 138 amostras de LLA-cpB ao diagnóstico (≤ 18 anos), quanto à expressão celular de CRLF2 e CD127 em blastos leucêmicos, avaliando percentual e intensidade mediana de fluorescência (IMF). Para avaliar a ploidia desses casos foi utilizado o índice de DNA por citometriade fluxo multiparamétrica (CFM) e a expressão gênica do CRLF2 foi avaliada por RTq-PCR. Já as alterações moleculares ETV6-RUNX1, E2A-PBX1, BCR-ABL1, r-KMT2A foram avaliadas por RTPCR. Para a avaliação da distribuição das variáveis categóricas foram aplicados os testes Exato de Fisher e Qui-Quadrado...

Introduction: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) presents classical cytogenetic molecular biomarkers with well-defined prognosis. Thirty percent of these cases do not have any of these abnormalities and are referred to as B-others and 50% of this subgroup has a BCRABL1similar gene expression profile, then called BCR-ABL1-like. This subgroup has characteristic genetic differences as mutations in the JAK1 / 2, IL7Rα, and CRLF2 genes, as well as increased geneexpression of the latter. CRLF2 forms a heterodimer with the alpha chain of the Interleukin 7 receptor (IL-7Rα) or CD127 forming the TSLP cytokine receptor. Studies involving the CRLF2 have presented controversial results due to the use of non-standardized techniques for their detection. Objective: To establish an algorithm of combined immunophenotypic and molecular tests capable of predicting the CRLF2 status and its association with molecular and cellular subtypes in children samples with BCPALL.Methods: Analysis of the monoclonal antibodies panel CD10FITC/CD127PE /CD45PerCPCY5.5 /CD19PE-CY7/ CRLF2APC in 138 samples of diagnostic-ALL (≤ 18 years) on cell expressionof CRLF2 and CD127 in leukemic blasts, evaluating percentage and median intensity of fluorescence (MIF). To evaluate the ploidy of these cases the DNA index was used by multiparametric flow cytometry (MFC) and the CRLF2 gene expression was evaluated by RTq-PCR. The molecular alterations ETV6-RUNX1, E2A-PBX1, BCR-ABL1, r-KMT2A were evaluated by RT-PCR. For theevaluation of the categorical variables distribution, Fisher's Exact and Qui-Square tests were applied. For the comparison of continuous variables, the non-parametric Mann-Whitney test (comparison of two groups) or Kruskal wallis (more than two groups) was used...