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Objectives: To analyze the overall survival (OS) and disease-free survival (DFS) in pediatric patients diagnosed with Ph-negative B-cell Acute Lymphoblastic leukemia (B-ALL) who underwent treatment with Blinatumomab for relapse/refractory disease. Materials and Methods: This is a retrospective cohort study that included pediatric patients aged ≤ 18 years with Ph-negative B-ALL in first hematological remission with relapsed/refractory, treated at the Hospital da Criança de Brasília José Alencar (HCB) between 2017 and 2024. Clinical, pathological, and therapeutic data were collected. The analysis of OS and DFS was performed using the Kaplan-Meier method, considering the period until death as OS or until relapse/death as DFS, from the diagnosis or initiation of treatment with Blinatumomab. Fisher's exact test was used to evaluate the association between general data and outcomes, considering the small sample size. The study was approved by the hospital's Research Ethics Committee [4.825.280]. Results: 16 patients with B-ALL were included in the study, with 10 (62.5%) were male, with a mean diagnosis age of 6.81 years The probability of overall survival was 105.20 months (median = 118.00). The average time to death from the use of Blinatumomab was 55.80 months, with a final cumulative survival of 0.63. For patients with pre-Blinatumomab positive minimal residual disease (MRD), the average time to death was 34.54 months. For patients with negative MRD, it was not possible to calculate survival due to the absence of deaths. The final cumulative survival for pre-Blinatumomab MRD was 0.53. After treatment, the average time to death was 54.00 months for positive MRD and 32.50 months for negative MRD, with cumulative survivals of 0.87 (positive) and 0.57 (negative), respectively. The estimated probability of DFS, from diagnosis to relapse/death, was 72.86 months (median = 75.00) The estimated mean duration from the start of Blinatumomab was 22.28 months (median = 25.00), with a final cumulative survival of 0.25 for pre-Blinatumomab MRD, the mean time to death was 25.84 months, and for negative MRD it was 70 months. After treatment, the mean duration was 54.00 months for positive MRD and 32.50 months for negative MRD, with cumulative survivals of 0.87 (positive) and 0.57 (negative). The Log Rank (Mantel-Cox) test did not find a significant difference between pre (p = 0.12) and post (p = 0.51) Blinatumomab survival curves. The Fisher's Exact test was used to evaluate the association between general data and outcomes; considering the small sample size, no quantitative variable showed significant differences. At the end of the study, 7 patients (43.75%) were in remission, 6 (37.5%) were in relapse, and 3 (18.75%) died. The treatment of one patient was discontinued due to neurotoxicity. Discussion: Other studies have already demonstrated promising results for pediatric patients with relapsed/refractory B-ALL, such as long-term OS improvement and better response rates for negative MRD, supporting the findings of this study. Conclusion: The inclusion of Blinatumomab in new therapeutic regimens for Ph-negative B-ALL is a promising approach to achieving better outcomes in patients with poor prognoses. However, it is important to note that neurotoxicity may be a significant adverse event associated with the use of Blinatumomab.

Introdução: Neoplasias de células mastocíticas (CM) compõem um grupo heterogêneo de doenças, mas com apresentação clínica semelhante, sendo a histopatologia e biologia molecular (BM) essenciais para diagnóstico diferencial. Na Leucemia Mieloide Aguda (LMA) core binding fator, frequentemente CM estão aumentadas, sendo parte dessas leucemias secundária a Mastocitose Sistêmica (MS). Esses achados já foram relatados em adultos, mas é raro em crianças. A mutação D816V está relacionada à LMA e é o principal componente para diferenciá-la da Leucemia Mielomastocítica Aguda (LMMA). O objetivo desse trabalho foi relatar um caso de LMA com MS e com fusão RUNX1-RUNX1T1 mutação no éxon 8 do gene KIT em uma paciente pediátrica. Metodologia: Dados clínicos e laboratoriais foram obtidos por prontuário médico. A análise da medula óssea (MO) utilizou: citometria de fluxo para Imunofenotipagem (IFT) e Doença Residual Mínima (DRM), banda G para citogenética, RT-PCR para fusão gênica e sequenciamento Sanger para o gene KIT. Caso: Paciente com 6 anos, feminino, procedente da Guiana, admitida em dezembro de 2022 com história de febre e dor abdominal. A mãe é portadora de HIV, fazendo uso de retroviral durante toda a gestação, sem saber referir qual medicamento. Ao exame físico mostrava discretas petéqueas em tronco e abdome, exoftalmia à esquerda com hiperemia e edema de partes moles. Exames laboratoriais mostraram: funções renal e hepática preservadas, sorologia negativa para HIV, mas positivo para CMV, EBV e Parvovírus; foi visto ainda alterações hematológicas (Hb: 5,3 g/dL, HTC: 16,3%, Plaquetas: 16.000/mm3, Leucometria: 38.260/mm3) com 89% de blastos. A IFT da MO mostrou a presença de duas populações de células blásticas de linhagem mieloide: I- 39,1% e II- 28,7%. Além disso, foi observada a presença de 3,73% de CM. A citogenética mostrou 46,XX,der(8)t(8;21)(q22;q22)[12]/ 46,XX,t(8;21)(q22;q22)[2]/46,XX[6] e a BM evidenciou fusão RUNX1-RUNX1T1, e as mutações A) KIT :c.1249_1250insCTTTTT p.Ile416_Leu417insProPhe e B) KIT :c.2447A>T p.Asp816Val nos éxons 8 e 17 do gene KIT, respectivamente. A paciente foi diagnosticada com LMA com MS sendo tratada pelo protocolo GELMAI como alto risco. Após o primeiro MAG, a DRM mostrou 22,9% da população I e 0,67% da II, além de 22,6% de CM. A mutação B não estava presente, mas mantinha a A e a fusão RUNX1-RUNX1T1. Já após o ADE, a DRM mostrava ausência de blastos, mas 19,46% de CM e a BM se mantinha igual à avaliação anterior. A presença de CM, sem blastos, e com fusão RUNX1-RUNX1T1 se manteve até a última avaliação (dia 02.06.2023). A mutação A foi detectada até a avaliação pós ADE. A paciente foi encaminhada para transplante de medula óssea, e no momento está aguardando o procedimento. Discussão: O caso apresentado mostra um achado raro por se tratar de um paciente pediátrico. A mutação B foi essencial para diagnosticá-la como uma LMA com MS e não como LMMA. Mesmo após o desaparecimento dos blastos e da mutação B, as CM permanecem, assim como a fusão RUNX1-RUNX1T1. Isto leva a crer que a fusão está presente apenas nas CM, juntamente com a mutação A. Este é o primeiro caso relatado de uma LMA com MS, com mutação no éxon 8 do KIT em um paciente pediátrico. Conclusão: A análise de BM foi essencial para o diagnóstico. Maiores estudos são necessários para avaliar se a fusão RUNX1-RUNX1T1 estava presente apenas nas CM.

Introduction: The recent active use of immunotherapy based on CD19/CD3 bispecific T-cell engager (blinatumomab), brings significant problems on monitoring Minimal Residual Disease (MRD) based on Multiparameter Flow Cytometry (MFC), due to possible CD19 downregulation by tumor cells. This phenomenon breaks the well-established approach of MFC strategy analysis, based gating on CD19 positivity. Also, the selective pressure of the CD19-directed immunotherapy increases the incidence of the so-called lineage switch, which has become one of the possible mechanisms of BCP-ALL resistance to immunotherapy. At present, there is no consensus approach to MFC-MRD monitoring after blinatumomab therapy for BCP-ALL. There are several described changes in MRD methodology, including the addition of other early B-lineage antigens (e.g., CD22, CD24 and CD79a) or only the modification of the gating strategy using conventional sets of antibodies. Aim: Evaluated two different antibody panels to monitor MFC-MRD in patients Relapsed/Refractory (R/R) B-cell ALL after blinatumomab therapy. Materials and methods: All Bone Marrow (BM) samples the MRD detection was performed with panel EuroFlow BCPALL MRD protocol standardized and added two more tubes (8 colors) with early B-lineage antigens according to previously described in literature (e.g., Tube 1: CD66b/CD22/CD34/CD19/CD24/CD38/CD10/CD45; Tube 2: CD20/CD22/CD34/CD19/CD79a/CD38/CD10/CD45). All BM samples were studied following the EuroFlow SOPs with the acquisition at least ≥5×106 events per samples and in both panels. The cytometer FACSCanto 8-color and Infinicyt software was used for acquisition and analyzed data files. The study enrolled real-world children in public health care systems and was approved by the local ethics committees. Results: A total of 54 BM samples obtained from 11 children -48% M and 52% F with a median age of 10-years (0‒19) with R/R B-cell ALL were studied at different time points after treatment with blinatumomab, including: pré-transplant (n = 24) and post-transplant (n = 30). As controls without use of blinatumomab were include 04 BM from children with B-cell ALL at D15 of treatment with standard chemotherapy protocol. MRD were detected in 35% (19/54) of BM studied and of these 2/11 relapsed. We found 100% agreement between the two panels used. Only one sample was discordant at first time, but upon review it was noted that this CD19 negative population were very early CD19- normal B cell progenitors. Discussion: The wide implementation of CD19-directed therapy in the treatment in protocol for R/R BCP-ALL significantly complicated the rather routine procedure of MRD monitoring. Despite the subjective impressions by these investigators that other B-cell antigens such as CD22, CD24 and cyCD79a brings more confidence to analyze B-cell ALL MRD samples from patients treated with anti-CD19 therapy, for this studied cohort, we did not find difference on the results of MRD with the standard EuroFlow panel and the alternative panel. Conclusion: No difference was found on the MRD detection with the standard EUFOLOW panel and the alternative panel. Although introduction of CD22, and CD24 markers, with 8 colors strategy, it was not advantageous. Using 8 colors strategy, it may be recommended to investigate the comparative financial impact of the EuroFlow, and the alternative panel in the context of Blinatumomab therapy.

Objetivos: O estudo tem o objetivo de avaliar a implementação de protocolo de tratamento para leucemia linfoblástica (LLA) com estratificação de risco baseado em pesquisa de doença residual mínima (DRM). Material e métodos: Realizou-se estudo observacional e analítico com delineamento longitudinal retrospectivo. Incluíram-se pacientes com LLA, com idade entre 1 e 18 anos, que realizaram tratamento no Hospital da Criança de Brasília (HCB) entre 06 de 2018 e 06 de 2021. Nos dias 15, 33 e 78, realizou-se avaliação de DRM por citometria de fluxo de 8 cores de nova geração (CFNG) em medula óssea. Os pacientes foram estratificados para os braços de tratamento conforme descrito a seguir. Baixo Risco (BR): LLA-B; 5%; DRM D78 >0,01%; presença das translocações BCR-ABL ou MLL-AF4. Risco intermediário (RI): demais pacientes. Para casos específicos, aceitaram-se ajustes das classificações fora dos critérios descritos. O teste de Log Rank (Mantel-Cox) foi utilizado para comparação de curvas de sobrevida global (SG) e livre de doença (SLD). Resultados: Foram incluídos 82 pacientes, sendo 78 com LLA-B e 4 com LLA-T. Desses, 15 foram classificados como BR, 50 RI e 17 AR. Houve 14 óbitos (17,07%) e 4 recaídas (4,87%), sendo duas (2,43%) isoladas em SNC. Dos óbitos, 5 (35,71%) estavam classificados como RI e 9 (64,29%) como AR; não houve óbitos no grupo BR. Houve diferença estatística significativa para a SG entre os grupos de classificação de risco (p

We have developed a simple, rapid, high-throughput RBD-based ELISA to assess the humoral immunity against emerging SARS-CoV-2 virus variants. The cDNAs of the His-tagged RBD proteins of the virus variants were stably engineered into HEK cells secreting the protein into the supernatant, and RBD purification was performed by Ni-chromatography and buffer exchange by membrane filtration. The simplified assay uses single dilutions of sera from finger-pricked native blood samples, purified RBD in 96-well plates, and a chromogenic dye for development. The results of this RBD-ELISA were confirmed to correlate with those of a commercial immunoassay measuring antibodies against the Wuhan strain, as well as direct virus neutralization assays assessing the cellular effects of the Wuhan and the Omicron (BA.5) variants. Here, we document the applicability of this ELISA to assess the variant-specific humoral immunity in vaccinated and convalescent patients, as well as to follow the time course of selective vaccination response. This simple and rapid assay, easily modified to detect humoral immunity against emerging SARS-CoV-2 virus variants, may help to assess the level of antiviral protection after vaccination or infection.