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

CRLF2 expression associates with ICN1 stabilization in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy with few molecular alterations showing a consensual prognostic value. CRLF2 overexpression was recently identified in high-risk T-ALL patients. For these cases, no genomic abnormality was found to be associated with CRLF2 overexpression. IKZF1 has been recently shown to be a direct transcriptional regulator of CRLF2 expression. Moreover, it is known that NOTCH1 antagonizes IKZF1 in T-ALL. In light of these pieces of evidence, we reasoned that IKZF1 binding perturbation and CRLF2 upregulation could be associated in T-ALL. We evaluated two independent series of pediatric T-ALL cases (PHOP, n&#x2009;=&#x2009;57 and TARGET, n&#x2009;=&#x2009;264) for the presence of common T-ALL molecular abnormalities, such as NOTCH1/FBXW7 mutations. We also assessed CRLF2 and IKZF1 gene expression. CRLF2 overexpression was observed in 14% (PHOP) and 16% (TARGET) of T-ALL patients. No correlation was found between mRNA expression of CRLF2 and IKZF1 in both cohorts. Interestingly, we show that patients with mutations affecting NOTCH1-PEST domain and/or FBXW7 had higher CRLF2 expression (P&#x2009;=&#x2009;.04). In summary, we demonstrate for the first time that only mutations resulting in ICN1 (intracellular domain of NOTCH1) stabilization are associated with CRLF2 overexpression

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; &lt;19 years) were analyzed throughout multiparametric flow cytometry. Mutations in&nbsp;N/KRAS, NOTCH1, FBXW7&nbsp;as well as&nbsp;STIL-TAL1&nbsp;and&nbsp;TLX3&nbsp;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&ndash;1272), T-ALL was relatively low, with MFI 43.2 (1.9&ndash;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&ndash;866.3), whereas mCD3/TCR&gamma;&delta;pos&nbsp;cases had higher expressions, MFI 99.9 (16.4&ndash;866.3).&nbsp;NOTCH1mut&nbsp;and&nbsp;STIL-TAL1&nbsp;were associated with low CD44 expression, whereas&nbsp;N/KRASmut&nbsp;and&nbsp;FBXW7mut&nbsp;cases had intermediate expression. In relation to clinical features, CD44 expression was associated with tumor infiltrations (p&nbsp;= 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.</p

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

Elda Pereira Noronha, Lu&iacute;sa Vieira Code&ccedil;o Marques, Francianne Gomes Andrade, Ingrid Sardou-Cezar, Filipe Vicente dos Santos-Bueno, Carolina Da Paz Zampier, Eug&ecirc;nia Terra-Granado, Maria S Pombo-de-OliveiraPaediatric Haematology-Oncology Program, Research Centre, Instituto Nacional de C&acirc;ncer, Rio de Janeiro, RJ, BrazilPurpose: 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&lt;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 NOTCH1wt, (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.Keywords: T-lymphoid/myeloid mixed phenotype acute leukemia, early T-cell precursor lymphoblastic leukemia, NOTCH1 mutation