Transposon-mediated generation of CAR-T cells shows efficient anti B-cell leukemia response after ex vivo expansion.

CAR-T-cell therapy has shown considerable advance in recent years, being approved by regulatory agencies in US, Europe, and Japan for the treatment of refractory patients with CD19+ B-cell leukemia or diffuse large B-cell lymphoma. Current methods for CAR-T-cell production use viral vectors for T-cell genetic modification and can take up to 15 days to generate the infusion product. The development of simple and less costly manufacturing protocols is needed in order to meet the increasing demand for this therapy. In this present work, we generated 19BBz CAR-T cells in 8 days using a protocol based on the non-viral transposon-based vector Sleeping Beauty. The expanded cells display mostly a central memory phenotype, expressing higher levels of inhibitory receptors when compared with mock cells. In addition, CAR-T cells were cytotoxic against CD19+ leukemia cells in vitro and improved overall survival rates of mice xenografted with human RS4;11 or Nalm-6 B-cell leukemias. Infused CAR-T cells persisted for up to 28 days, showing that they are capable of long-term persistence and antitumor response. Altogether, these results demonstrate the effectiveness of our protocol and pave the way for a broader application of CAR-T-cell therapy.

Corrigendum: The Profile of Immunophenotype and Genotype Aberrations in Subsets of Pediatric T-Cell Acute Lymphoblastic Leukemia.

[This corrects the article .].

The Profile of Immunophenotype and Genotype Aberrations in Subsets of Pediatric T-Cell Acute Lymphoblastic Leukemia.

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/Bdel (71.4%), NOTCH1mut (47.6%) and FBXW7mut (17%). ETP-ALL had frequent FLT3mut (22.2%) and SUZ12del (16.7%) (p < 0.001), while CDKN2A/Bdel were rarely found in this subtype (p < 0.001). The early cortical T-ALL subtype had high frequencies of NOTCH1mut and IL7Rmut (71%, 28.6%, respectively), whereas, mature T-ALL with double positive CD4/CD8 had the highest frequencies of STIL-TAL1 (36.7%), LEF1del (27.3%) and CASP8AP2del (22.7%). The co-existence of two groups of T-ALL with NOTCH1mut/IL7Rmut, and with TLX3/SUZ12del/NF1del/IL7Rmut, were characterized with statistical significance (p < 0.05) but only STIL-TAL1 (pOS 47.5%) and NOTCH1WT/FBXW7WT (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.

T-lymphoid/myeloid mixed phenotype acute leukemia and early T-cell precursor lymphoblastic leukemia similarities with NOTCH1 mutation as a good prognostic factor.

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 NOTCH1mut 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.

CD44 Expression Profile Varies According to Maturational Subtypes and Molecular Profiles of Pediatric T-Cell Lymphoblastic Leukemia.

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). NOTCH1 mut and STIL-TAL1 were associated with low CD44 expression, whereas N/KRAS mut and FBXW7 mut 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.