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A detecção de doença residual mínima (DRM) é um dos fatores prognósticos independentes mais importantes na leucemia linfoblástica aguda de precursor B (LLA-pB). Entretanto, o papel do microambiente medular (em especial das células estromais mesenquimais e endoteliais) e sua relação com a DRM e a sobrevida livre de doença durante o tratamento ainda é pouco explorada na literatura. Uma menina de 2 anos foi admitida no Instituto de Puericultura e Pediatria Martagão Gesteira (IPPMG-UFRJ), Rio de Janeiro, Brasil em 05/03/2015. Na investigação clínica ela apresentava hepatoesplenomegalia, leucometria total de 28 × 109/L., hemoglobina de 9.2g/L, contagem de plaquetas de 34 × 109/L, 49% de blastos compatíveis com LLA-pB por imunofetipagem de sangue periférico e exames moleculares negativos para BCR-ABL, ETV6-RUNX1 e rearranjo do gene MLL. AIEOP- Foi tratada de acordo com o protocolo BFM ALL 2009, com boa resposta a prednisona, final de indução com DRM positiva de 0.05% e D+78 com níveis indetectáveis de DRM. Em todos os pontos de avaliação de DRM (D+15, D+33 e D+78) a paciente apresentava níveis elevados de células estromais endoteliais (52%, 38% e 40%) – dentro do compartimento de células estromais totais, comparados com valores controles (mediana de 20%, 4%–29%). Quartoze meses após o diagnóstico (10/05/2016) recai com 85% de blastos na medula óssea (MO), momento esse em que se realiza a troca para o protocolo terapêutico AIEOP-BFM-REZ 2002. Cinco meses após o início deste protocolo (14/10/2016) alcança níveis indetectáveis de DRM quando em 10/03/2017 a doença volta a progredir, observando-se a presença de 7,5% de blastos na MO. Na sequência, foi encaminhada para transplante realizado em 24/04/2017, após DRM indetectável pré-TMO realizada em 19/04/2017. Quatro meses depois (25/08/2017) ela voltava a apresentar 8,2% de blastos na MO quando foi encaminhada para o serviço de origem para reinfusão seriada de linfócitos do doador. A partir de 16/10/2018 não são observados blastos circulantes na MO até o dia 30/05/2019 quando recai isoladamente em sistema nervoso central, vindo à óbito por progressão de doença refratária em 06/06/2019. O papel do microambiente medular, particularmente das células estromais mesenquimais e endoteliais na determinação da resposta à terapia ainda é pouco investigada. Em estudo recente nosso grupo avaliou a distribuição dessas células em pacientes com LLA-pB e sua relação com o status de DRM e desfecho do paciente demonstrando que a presença de elevados percentuais de endoteliais (>32%), especialmente no D+78 está associada com menor sobrevida livre de doença, independente do status da DRM, emergindo portanto como um fator prognóstico adverso. Baseado nessas informações, neste estudo de caso, a pior evolução clínica da paciente observada ao longo do tratamento pode estar também associada ao enriquecimento de células endoteliais observadas. Esse impacto prognóstico desfavorável pode ser sugestivo do papel das células endoteliais no suporte à sobrevivência das células leucêmicas, uma vez que a ativação de células endoteliais promove sua adesão às células leucêmicas, condição que também confere vantagem proliferativa, diminuição da suscetibilidade à quimioterapia e consequentemente progressão da doença. Investigações adicionais são necessárias para entender melhor os mecanismos envolvidos na patogênese da doença.

Despite all knowledge acquired regarding the mutational profile of pediatric myelodysplastic syndrome (MDS), the somatic genomic landscape underlying that disease remains unclear. We evaluated the presence of somatic variants in 37 genes related to myeloid malignancies through targeted NGS in 20 Brazilian patients with refractory cytopenia of childhood (RCC). Only 15% (3/20) of patients showed at least one somatic driver mutation – all in genes coding to regulators of cell signaling (TP53 and CBLB) or epigenetics (ASXL1 and DNMT3A). Interestingly, those variants were identified in patients with no detected clonal chromosomal abnormalities.

After the exclusion of iron deficiency and β-thalassemia, molecular research for α-thalassemia is recommended to investigate microcytic anemia. Aiming to suggest more efficiently the molecular analysis for individuals with a greater chance of having a symptomatic form of the disease, we have developed and validated a new decision tool to predict the presence of two or more deletions of α-thalassemia, increasing considerably the pre-test probability. The model was created using the variables: the percentage of HbA2, serum ferritin and mean corpuscular volume standardized by age. The model was trained in 134 patients and validated in 160 randomly selected patients from the total sample. We used Youden’s index applied to the ROC curve methodology to establish the optimal odds ratio (OR) cut-off for the presence of two or more α-globin gene deletions. Using the OR cut-off of 0.4, the model’s negative predictive value (NPV) was 96.8%; the cut-off point accuracy was 85.4%; and the molecular analysis pre-test probability increased from 25.9% to 65.4% after the use of the proposed model. This tool aims to assist the physician in deciding when to perform molecular studies for the diagnosis of α-thalassemia. The model is useful in places with few financial health resources.

BACKGROUND AND OBJECTIVES: We present an updated birth weight-for-gestational-age portrait, based on nearly 8 million observations of an ethnic-mixed population. It comprises the first comprehensive charts with Brazilian data. This contribution intends to assist clinicians in classifying fetal growth, to provide a reference for investigations of predictors and to show the consequences of small and large patterns for gestational age delivery. Most of the reference data for assessing birth weight for gestational age deal with insufficient sample size, especially at low gestational age. Population-based studies with considerably large sample size refer to data collected more than 15 years ago. METHODS: We accomplished a population-based study on births in all the Brazilian states from 2003 to 2005. Results were based on 7,993,166 singletons. We constructed the 3rd, 5th, 10th, 25th, 50th, 90th, 95th and 97th smoothed percentiles curves and gender-specific tables from 22 to 43 completed weeks. RESULTS: The resulting tables and graphical representation provide a gender-specific reference to access the birth weights distribution according to the gestational age in the Brazilian population. CONCLUSIONS: This is the first population-based reference constructed on a developing country data. These charts could provide an important tool to improve clinical assessment of growth in newborns.BACKGROUND E OBJETIVOS: Apresentamos um retrato atualizado de peso-por-idade-gestacional, baseado em quase 8 milhões de observações em uma população etnicamente misturada. Estas constituem as primeiras tabelas com dados brasileiros. Esta contribuição pretende dar assistência aos clínicos na classificação do crescimento fetal, e prover uma referência para pesquisas de prognósticos e consequências em partos com padrões pequenos e grandes para a idade gestacional. A maior parte dos dados de referência para estimar peso-por-idade-gestacional sofre de tamanho de amostra insuficiente, especialmente em baixa idade gestacional. Os estudos populacionais com uma amostra de tamanho considerável se referem a dados coletados há mais de 15 anos. MÉTODOS: Nós realizamos um estudo populacional baseado em nascimentos em todos os estados brasileiros de 2003 a 2005. Os resultados foram baseados em 7.993.166 nascimentos de gravidez única. Nós construímos curvas suavizadas e tabelas gênero-específicas de 22 a 43 semanas completas para os percentiles 3°, 5°, 10°, 25°, 50°, 90°, 95° e 97°. RESULTADOS: As tabelas e representações gráficas resultantes proveem uma referência gênero-específica para acessar a distribuição de peso ao nascimento de acordo com a idade gestacional na população brasileira. CONCLUSÕES: Esta é a primeira referência populacional construída com dados de um país em desenvolvimento. Estas tabelas podem prover uma importante ferramenta para melhorara avaliação clínica do crescimento em recém-natos.

Pediatric cancer is a relatively rare and heterogeneous group of hematological and non-hematological malignancies which require multiple procedures for its diagnostic screening and classification. Until now, flow cytometry (FC) has not been systematically applied to the diagnostic work-up of such malignancies, particularly for solid tumors. Here we evaluated a FC panel of markers for the diagnostic screening of pediatric cancer and further classification of pediatric solid tumors. The proposed strategy aims at the differential diagnosis between tumoral vs. reactive samples, and hematological vs. non-hematological malignancies, and the subclassification of solid tumors. In total, 52 samples from 40 patients suspicious of containing tumor cells were analyzed by FC in parallel to conventional diagnostic procedures. The overall concordance rate between both approaches was of 96% (50/52 diagnostic samples), with 100% agreement for all reactive/inflammatory and non-infiltrated samples as well as for those corresponding to solid tumors (n = 35), with only two false negative cases diagnosed with Hodgkin lymphoma and anaplastic lymphoma, respectively. Moreover, clear discrimination between samples infiltrated by hematopoietic vs. non-hematopoietic tumor cells was systematically achieved. Distinct subtypes of solid tumors showed different protein expression profiles, allowing for the differential diagnosis of neuroblastoma (CD56(hi)/GD2(+)/CD81(hi)), primitive neuroectodermal tumors (CD271(hi)/CD99(+)), Wilms tumors (>1 cell population), rhabdomyosarcoma (nuMYOD1(+)/numyogenin(+)), carcinomas (CD45(-)/EpCAM(+)), germ cell tumors (CD56(+)/CD45(-)/NG2(+)/CD10(+)) and eventually also hemangiopericytomas (CD45(-)/CD34(+)). In summary, our results show that multiparameter FC provides fast and useful complementary data to routine histopathology for the diagnostic screening and classification of pediatric cancer.

In mammalian cells, heme can be degraded by heme-oxygenases (HO). Heme-oxygenase 1 (HO-1) is known to be the heme inducible isoform, whereas heme-oxygenase 2 (HO-2) is the constitutive enzyme. Here we investigated the presence of HO during erythroid differentiation in human bone marrow erythroid precursors and K562 cells. HO-1 mRNA and protein expression levels were below limits of detection in K562 cells. Moreover, heme was unable to induce HO-1, at the protein and mRNA profiles. Surprisingly, HO-2 expression was inhibited upon incubation with heme. To evaluate the physiological relevance of these findings, we analyzed HO expression during normal erythropoiesis in human bone marrow. Erythroid precursors were characterized by lack of significant expression of HO-1 and by progressive reduction of HO-2 during differentiation. FLVCR expression, a recently described heme exporter found in erythroid precursors, was also analyzed. Interestingly, the disruption in the HO detoxification system was accompanied by a transient induction of FLVCR. It will be interesting to verify if the inhibition of HO expression, that we found, is preventing a futile cycle of concomitant heme synthesis and catabolism. We believe that a significant feature of erythropoiesis could be the replacement of heme breakdown by heme exportation, as a mechanism to prevent heme toxicity.

The existence of loss and gain of chromosomes, known as aneuploidy, has been previously described within the central nervous system. During development, at least one-third of neural progenitor cells (NPCs) are aneuploid. Notably, aneuploid NPCs may survive and functionally integrate into the mature neural circuitry. Given the unanswered significance of this phenomenon, we tested the hypothesis that neural differentiation induced by all-trans retinoic acid (RA) in pluripotent stem cells is accompanied by increased levels of aneuploidy, as previously described for cortical NPCs in vivo. In this work we used embryonal carcinoma (EC) cells, embryonic stem (ES) cells and induced pluripotent stem (iPS) cells undergoing differentiation into NPCs. Ploidy analysis revealed a 2-fold increase in the rate of aneuploidy, with the prevalence of chromosome loss in RA primed stem cells when compared to naïve cells. In an attempt to understand the basis of neurogenic aneuploidy, micronuclei formation and survivin expression was assessed in pluripotent stem cells exposed to RA. RA increased micronuclei occurrence by almost 2-fold while decreased survivin expression by 50%, indicating possible mechanisms by which stem cells lose their chromosomes during neural differentiation. DNA fragmentation analysis demonstrated no increase in apoptosis on embryoid bodies treated with RA, indicating that cell death is not the mandatory fate of aneuploid NPCs derived from pluripotent cells. In order to exclude that the increase in aneuploidy was a spurious consequence of RA treatment, not related to neurogenesis, mouse embryonic fibroblasts were treated with RA under the same conditions and no alterations in chromosome gain or loss were observed. These findings indicate a correlation amongst neural differentiation, aneuploidy, micronuclei formation and survivin downregulation in pluripotent stem cells exposed to RA, providing evidence that somatically generated chromosomal variation accompanies neurogenesis in vitro.