T-cell acute lymphoblastic leukemia (T-ALL) is a biologically heterogeneous malignancy, which reflects distinctive stages of T-cell differentiation arrest. We have revisited a cohort of pediatric T-ALL, in order to test if immunophenotypes associated with molecular alterations would predict the patient's outcome. Genetic mutations, translocations and copy number alterations were identified through Sanger sequencing, RT-PCR, FISH and multiplex ligation-dependent probe amplification (MLPA). We defined 8 immunophenotypic T-ALL subtypes through multiparametric flow cytometry: early T-cell precursor (ETP, n = 27), immature ( n = 38), early cortical ( n = 15), cortical ( n = 50), late cortical ( n = 53), CD4/CD8 double negative mature ( n = 31), double positive mature ( n = 35) and simple positive mature ( n = 31) T-ALL. Deletions (del) or amplifications (amp) in at least one gene were observed in 87% of cases. The most frequent gene alterations were CDKN2A/B del (71.4%), NOTCH1 mut (47.6%) and FBXW7 mut (17%). ETP-ALL had frequent FLT3 mut (22.2%) and SUZ12 del (16.7%) ( p < 0.001), while CDKN2A/B del were rarely found in this subtype ( p < 0.001). The early cortical T-ALL subtype had high frequencies of NOTCH1 mut and IL7R mut (71%, 28.6%, respectively), whereas, mature T-ALL with double positive CD4/CD8 had the highest frequencies of STIL-TAL1 (36.7%), LEF1 del (27.3%) and CASP8AP2 del (22.7%). The co-existence of two groups of T-ALL with NOTCH1 mut /IL7R mut , and with TLX3/SUZ12 del /NF1 del / IL7R mut , were characterized with statistical significance ( p < 0.05) but only STIL-TAL1 (pOS 47.5%) and NOTCH1 WT / FBXW7 WT (pOS 55.3%) are predictors of poor T-ALL outcomes. In conclusion, we have observed that 8 T-ALL subgroups are characterized by distinct molecular profiles. The mutations in NOTCH1/FBXW7 and STIL-TAL1 rearrangement had a prognostic impact, independent of immunophenotype.

Purpose: T-lymphoid/Myeloid Mixed phenotype acute leukemia (T/M-MPAL) is ambiguous leukemia which overlaps with early T-cell precursor lymphoblastic leukemia (ETP-ALL). We have revisited the immunophenotyping profile of T/M-MPAL and ETP-ALL to identify differences and/or similarities, as these entities represent a therapeutic challenge in clinical practice. Patients and methods: A total of 26 ETP-ALL and 10 T/M-MPAL cases were identified among 857 cases of childhood leukemia (T-ALL, n=266 and AML, n=591) before any treatment decisions. The variables analyzed were age strata, sex, clinical features, immunophenotyping, and molecular aberrations. Immunophenotyping was performed in all samples using a panel of cytoplasm and membrane antibodies to identify the lineage and blast differentiation. The mutational status of STIL-TAL1, TLX3, RUNX1, NOTCH1, FBXW7, FLT3, IL7R, RAS, KTM2A , and CDKN2A/B was tested using RT-PCR, FISH, and PCR sequencing methods. The outcomes were assessed in terms of overall survival (OS). Results: The immunophenotypes were similar in ETP-ALL and T/M-MPAL, regarding the cellular expression of CD34, CD117, CD13/CD33, and CD11b, although CD2 and HLA-DR were more frequent in T/M-MPAL ( p <0.01). aMPO positivity and myelomonocyte differentiation were definitive in separating both entities. NOTCH1, FLT3-ITD , and N/KRAS mutations as well as TLX3 and KMT2A rearrangements were found in both ETP-ALL and T/M-MPAL. Thirty-one patients received ALL protocol whereas five had AML therapy. The overall 5-year survival rate (pOS) was 56.4% for patients treated using ALL protocols. No differences were observed between T/M-MPAL (pOS of 57%) and ETP-ALL (pOS of 56%) patients. The prognostic value of NOTCH1 mut was associated with significantly better OS (pOS 90%) than NOTCH1 wt (pOS 37%) ( p =0.017). Conclusion: This research can potentially contribute to NOTCH1 as targeted therapy and prognostic assessment of T-cell mixed phenotype leukemia.

CONTEXT AND OBJECTIVES: The incidence of acute leukemia (AL) subtypes varies according to geographical distribution. The aim here was to determine the incidence of morphological and immunophenotypic AL subtypes in the state of Maranhão, Brazil, and to correlate the expression of aberrant phenotypes in children with acute lymphoblastic leukemia (ALL) with prognostic factors. DESIGN AND SETTING: Single prospective cohort study at a public oncology reference center in Maranhão. METHODS: Seventy AL cases were diagnosed between September 2008 and January 2010. For the diagnosis, complete blood cell counts, myelograms (at diagnosis and at the end of the induction phase), cytochemical analysis and immunophenotyping were performed. RESULTS: Among adult patients (n = 22), the incidence of AL types was: ALL (22.7%) and acute myeloid leukemia (AML) (77.3%). The subtype AML M0 occurred most frequently (29.4%). In children (n = 48), the types were: AML (18.7%), most frequently subtype AML M4 (33.4%); biphenotypic acute leukemia (BAL) (4.2%); and ALL (77.1%), including the subtypes B-ALL (72.9%) and T-ALL (27.1%). Among the children with ALL, there were no statistically significant differences between patients with and without aberrant phenotypes, in relation to hematological parameters and treatment response. CONCLUSION: This work demonstrates that the frequencies of AML M0 cases among adults and T-ALL cases among children in Maranhão were high. This suggests that there may be differences in AML subtype incidence, as seen with ALL subtypes, in different regions of Brazil. No association was found between the expression of aberrant phenotypes and prognostic factors, in children with ALL. -T (27,1%). Na LLA, em crianças, não se encontrou diferença estatisticamente significante entre pacientes com e sem fenótipos aberrantes, em relação aos parâmetros hematológicos e resposta ao tratamento. CONCLUSÃO: Esta pesquisa demonstra elevada frequência de casos de LMA M0 em adultos, bem como das LLA-T em crianças no Maranhão, sugerindo que podem haver diferenças na incidência dos subtipos das LMA, assim como dos subtipos de LLA, em diferentes regiões do Brasil. Não foi encontrada associação entre a expressão de fenótipos aberrantes e fatores prognósticos em crianças com LLA.

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CD44 is a glycoprotein expressed in leucocytes and a marker of leukemia-initiating cells, being shown to be important in the pathogenesis of T cell acute lymphoblastic leukemia (T-ALL). In this study, we have (i) identified the aberrant antigenic pattern of CD44 and its isoform CD44v6 in T-ALL; (ii) tested the association with different T-cell subtypes and genomic alterations; (iii) identified the impact of CD44 status in T-ALL outcome. Samples from 184 patients (123 T-ALL and 61 AML; <19 years) were analyzed throughout multiparametric flow cytometry. Mutations in N/KRAS, NOTCH1, FBXW7 as well as STIL-TAL1 and TLX3 rearrangements were detected using standard molecular techniques. CD44 expression was characterized in all T-ALL and AML cases. Compared with AML samples in which the median fluorescence intensity (MFI) was 79.1 (1–1272), T-ALL was relatively low, with MFI 43.2 (1.9–1239); CD44v6 expression was rarely found, MFI 1 (0.3-3.7). T-ALL immature subtypes (mCD3/CD1aneg) had a lower CD44 expression, MFI 57.5 (2.7–866.3), whereas mCD3/TCRγδpos cases had higher expressions, MFI 99.9 (16.4–866.3). NOTCH1mut and STIL-TAL1 were associated with low CD44 expression, whereas N/KRASmut and FBXW7mut cases had intermediate expression. In relation to clinical features, CD44 expression was associated with tumor infiltrations (p = 0.065). However, no association was found with initial treatment responses and overall survival prediction. Our results indicate that CD44 is aberrantly expressed in T-ALL being influenced by different genomic alterations. Unraveling this intricate mechanism is required to place CD44 as a therapeutic target in T-ALL.

Abstract:The gastroprotective action of the aqueous extract (AE) and the hydroalcoholic extract obtained from the leaves of Struthanthus marginatus (Desr.) Blume, Loranthaceae, were performed with in vivo models in rodents using: ethanol, indomethacin or stress-induced ulcers, determination of gastric secretion and the mucus production. The scavenger activity of AE in vitro was tested by the DPPH method. The treatment with the extracts (125-1000 mg/kg) significantly inhibited ulcerative lesions in comparison with the negative control groups in all the models evaluated and demonstrated greater effectiveness of the aqueous extract. Regarding the model of gastric secretion, a reduction in volume of gastric juice and total acidity was observed, as well as an increase in the gastric pH. The treatment of rats raised the gastric mucus production. Significant DPPH scavenging activity was evident in the AE. No sign of toxicity was observed. These results show that S. marginatus possesses gastroprotective activity. There are indications that the mechanisms involved in anti-ulcer activity are related to a decrease in acid secretion and an increase in gastric mucus content. Also, there is evidence for the involvement of antioxidant activity in the gastroprotective mechanism.

Introduction The ETV6-RUNX1 is a fusion gene associated with a good outcome in B-cell precursor lymphoblastic leukemia. Objective This study aimed to re-evaluate the CD9 cellular expression by flow cytometry (FC) as a possible tool to predict the presence of ETV6-RUNX1 . Method Childhood B-cell precursor lymphoblastic leukemia cases were included ( n = 186). The percentage of CD9-labeled cells and the median fluorescence intensity ratio were used for correlation with the molecular tests. Receiver Operating Characteristic curves were performed to determine the likelihood of the CD9 expression predicting ETV6-RUNX1. Results The ETV6-RUNX1 was found in 44/186 (23.6%) cases. Data analysis revealed that the best cutoff for CD9 percentage was 64%, with an accuracy of 0.84, whereas the best cutoff for CD9 median fluorescence intensity ratio was 12.52, with an accuracy of 0.80. A strong association was observed between the level of CD9 expression and the presence of ETV6-RUNX1 . Conclusion These data confirm that the CD9 expression could be used for risk stratification in clinical practice.

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