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.

We analyzed mutations in four genes (FLT3, KRAS/NRAS and PTPN11) that might disrupt the RAS/mitogen activated protein kinase (MAPKinase) signaling pathway, to evaluate their prognostic value in children younger than 16 years old with B-cell precursor acute lymphoblastic leukemia (Bcp-ALL). The overall survival (OS) was determined with the Kaplan-Meier method. MAPKinase genes were mutated in 25.4% and 20.1% of childhood and infant Bcp-ALL, respectively. Children with hyperdiploidy were more prone to harboring a MAPKinase gene mutation (odds ratio [OR] 3.18; 95% confidence interval [CI] 1.07-9.49). The mean OS of all cases was 54.0 months. FLT3 and PTPN11 mutations had no impact on OS. K/NRAS mutations were strongly associated with MLL-AFF1 (OR 5.78; 95% CI 1.00-33.24), and conferred poorer OS (p = 0.034) in univariate analysis.

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DNA methylation is a labile modification associated with gene expression control and environmental adaptations. High throughput, scalable and quantitative assessments of specific DNA methylation modifications in complex genomic regions for use in large population studies are needed. The performance of Droplet Digital™ PCR (ddPCR™) was investigated for DNA methylation detection against next-generation bisulfite sequencing (NGS) to demonstrate the ability of ddPCR to detect and validate DNA methylation levels and complex patterns among neighboring CpGs in regions associated with prenatal tobacco exposure. While both techniques are reproducible, ddPCR demonstrates a unique advantage for high-throughput DNA methylation analysis in large-scale population studies and provides the specificity to accurately measure DNA methylation of target CpGs in complex regions.