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.

Identification of the MYST3-CREBBP fusion gene in infants with acute myeloid leukemia and hemophagocytosis

ABSTRACT Background: Acute myeloid leukemia presenting the MYST3-CREBBP fusion gene is a rare subgroup associated with hemophagocytosis in early infancy and monocytic differentiation. The aim of this study was to define the relevant molecular cytogenetic characteristics of a unique series of early infancy acute myeloid leukemia cases (≤24 months old), based on the presence of hemophagocytosis by blast cells at diagnosis. Methods: A series of 266 infant cases of acute myeloid leukemia was the reference cohort for the present analysis. Acute myeloid leukemia cases with hemophagocytosis by blast cells were reviewed to investigate the presence of the MYST3-CREBBP fusion gene by fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction. Results: Eleven cases with hemophagocytosis were identified with hemophagocytic lymphohistiocytosis being ruled out. Six cases were classified as myelomonocytic leukemia, three as AML-M7 and two as AML-M2. In five cases, the presence of the MYST3-CREBBP fusion gene identified by molecular cytogenetics was confirmed by fluorescence in situ hybridization. All patients received treatment according to the Berlin-Frankfürt-Münster acute myeloid leukemia protocols and only one out of the five patients with the MYST3-CREBBP fusion gene is still alive. Conclusions: Our findings demonstrate that the presence of hemophagocytosis in acute myeloid leukemia was not exclusively associated to the MYST3-CREBBP fusion gene. Improvements in molecular cytogenetics may help to elucidate more complex chromosomal rearrangements in infants with acute myeloid leukemia and hemophagocytosis.

Biomarcadores somáticos nas leucemias mielóides agudas pediátricas / Somatic markers in leukemias acute myeloid pediatric

Introdução. A leucemia mielóide aguda (LMA) pediátrica apresenta uma heterogeneidade reflexo da diversidade de alterações genético-moleculares observadas nos seus diferentessubtipos. Estas anormalidades são importantes para estratificação de risco. Alguns estudos em LMA de adultos relatam que a alteração t(8;16)(p11;p13)/MYST3-CREBBP ocorre em<2% dos casos, com aspectos biológicos e clinicamente distintos, com sobrevida média em torno de 5 meses. LMA pediátrica com esta translocação está associada à eritrogocitose e altamortalidade precoce. O foco principal deste projeto foi investigar as alterações genéticas tipo I e tipo II nas LMAs e explorar associações entre si e subtipos morfológicos. Metodologia. Foram analisados 282 casos de LMA de novo (≤18 anos), encaminhados para o laboratório no período de 2008-2013, para o rastreamento das fusões RUNX1-RUNX1T1, CBF-MYH11, PML-RAR , dos rearranjos do gene MLL (MLL-r) e de mutações em KRAS, FLT3 e c-KIT.Dentre as crianças ≤24 meses, foram selecionados 15 casos com a presença da eritrofagocitose para pesquisa da fusão MYST3-CREBBP. A pesquisa das translocações cromossômicas foi realizada através de RT-PCR e/ou FISH. Mutações nos genes KRAS, FLT3e c-KIT foram rastreadas através das técnicas de RFLP e/ou sequenciamento direto. A distribuição entre as diferentes alterações de acordo com a idade, leucometria e subtipos foi avaliada através de métodos estatísticos. Resultados. LMAs com maturação mielomonocítica foram os subtipos mais frequentes observados em crianças com idade ≤5 anos e o subtipo promielocítico entre crianças e adolescentes entre 5-18 anos-idade...

Background. Pediatric acute myeloid leukemia (AML) has long been recognized for its biological heterogeneity. The broad range of abnormalities across different subtypes is important to stratify AML cases to different prognostic groups and choosing of an appropriate therapy of the disease. Studies involving adult AML have shown that the balancedchromosomal rearrangement t(8;16)(p11;p13)/MYST3-CREBBP is found in <2% of cases, with distinct clinical biological and median overall survival of 5 months. This pediatric AML subgroup is associated with erythrophagocytosis and early death. Our aim was to evaluate type I and II genetic alterations in pediatric AML and explore associations with morphological subtypes. Methods. A series of 282 samples was ascertained from 2008-2013 prior to any treatment and from different Brazilian centers. We have evaluated the prevalence of the genetic alterations RUNX1-RUNX1T1, CBF-MYH11, PML-RAR and MLLrearrangements (MLL-r), and mutations in KRAS, c-KIT and FLT3 in Brazilian childhood AML cases. Among infant AML (aged ≤24 months), 15 cases with erythrophagocytosis were selected to test the presence of MYST3-CREBBP. Mutations were detected by directsequencing; the fusion were analyzed by FISH and/or RT-PCR. Results. AML with myeloidmonocytic maturations were the most frequent subtypes observed among children aged ≤5years-old and acute promyelocytic leukemia among children and adolescents (5-18 years-old). Sixteen out of 56 cases (19%) were reclassified according to chromosomal abnormalitiesfound according to WHO classification...

Biomarcadores somáticos nas leucemias mielóides agudas pediátricas / [Somatic biomarkers in pediatric acute myeloid leukemias]

Introdução. A leucemia mielóide aguda (LMA) pediátrica apresenta uma heterogeneidade reflexo da diversidade de alterações genético-moleculares observadas nos seus diferentessubtipos. Estas anormalidades são importantes para estratificação de risco. Alguns estudos em LMA de adultos relatam que a alteração t(8;16)(p11;p13)/MYST3-CREBBP ocorre em<2% dos casos, com aspectos biológicos e clinicamente distintos, com sobrevida média em torno de 5 meses. LMA pediátrica com esta translocação está associada à eritrogocitose e altamortalidade precoce. O foco principal deste projeto foi investigar as alterações genéticas tipo I e tipo II nas LMAs e explorar associações entre si e subtipos morfológicos. Metodologia.Foram analisados 282 casos de LMA de novo (≤18 anos), encaminhados para o laboratório no período de 2008-2013, para o rastreamento das fusões RUNX1-RUNX1T1, CBF-MYH11, PML-RAR , dos rearranjos do gene MLL (MLL-r) e de mutações em KRAS, FLT3 e c-KIT.Dentre as crianças ≤24 meses, foram selecionados 15 casos com a presença da eritrofagocitose para pesquisa da fusão MYST3-CREBBP. A pesquisa das translocações cromossômicas foi realizada através de RT-PCR e/ou FISH. Mutações nos genes KRAS, FLT3e c-KIT foram rastreadas através das técnicas de RFLP e/ou sequenciamento direto. A distribuição entre as diferentes alterações de acordo com a idade, leucometria e subtipos foiavaliada através de métodos estatísticos...

Background. Pediatric acute myeloid leukemia (AML) has long been ecognized for its biological heterogeneity. The broad range of abnormalities across different subtypes is important to stratify AML cases to different prognostic groups and choosing of an appropriate therapy of the disease. Studies involving adult AML have shown that the balanced chromosomal rearrangement t(8;16)(p11;p13)/MYST3-CREBBP is found in <2% of cases, with distinct clinical biological and median overall survival of 5 months. This pediatric AML subgroup is associated with erythrophagocytosis and early death. Our aim was to evaluate type I and II genetic alterations in pediatric AML and explore associations with morphological subtypes. Methods. A series of 282 samples was ascertained from 2008-2013 prior to any treatment and from different Brazilian centers. We have evaluated the prevalence of the genetic alterations RUNX1-RUNX1T1, CBF-MYH11, PML-RAR and MLL rearrangements (MLL-r), and mutations in KRAS, c-KIT and FLT3 in Brazilian childhood AML cases. Among infant AML (aged ≤24 months), 15 cases with erythrophagocytosis were selected to test the presence of MYST3-CREBBP. Mutations were detected by directsequencing; the fusion were analyzed by FISH and/or RT-PCR. Results. AML with myeloidmonocytic maturations were the most frequent subtypes observed among children aged ≤5years-old and acute promyelocytic leukemia...