Mutations in Specific NOTCH1 Domains Convey a Distinct Phenotype in Adult Acute T-Lymphoblastic Leukemia.
Authors: Thibaut Julia , Goekbuget Nicola , Martus Peter , Burmeister Thomas , Schwartz Stefan , Hoelzer Dieter , Thiel Eckhard , Hofmann Wolf K. , Baldus Claudia D. 
Published: April 2020

The NOTCH1 gene encodes a transmembrane receptor important for the T-lineage commitment. Activating NOTCH1 mutations had been found in approximately 56% of pediatric T-lymphoblastic leukemia (T-ALL) samples and were associated with a favourable outcome. Here the frequency and the association of NOTCH1 mutations to clinical features, as well as molecular markers were analyzed for the first time in a large cohort of adult T-ALL patients. Genomic DNA of pretreatment samples of 126 adult T-ALL patients enrolled on the GMALL protocols 05/93 and 06/99 were analyzed by direct sequencing for the presence of NOTCH1 mutations in the heterodimerization domain (HD-C, HD-N), the polypeptide enriched in proline, glutamate, serine and threonine (PEST; divided into PEST-1 and PEST-2) domain, and the transactivation domain (TAD). In addition, leukemic blasts were molecularly characterized for mRNA expression of HOX11, HOX11L2, ERG, and BAALC by real-time RT-PCR. Immunophenotyping was performed differentiating T-ALL into three subtypes (early, thymic, mature). NOTCH1 mutations were identified in 72 (57%) of the 126 patients including mutations in the HD (n=56), in the TAD (n=5), and in the PEST (n=20) domain. Eleven patients had more than one mutation, including 3 patients with HD and TAD, and 6 patients with coexisting HD and PEST mutations. T-ALL patients carrying mutations revealed similar clinical characteristics as compared to NOTCH1 wild type cases with respect to age, sex, CNS involvement, white blood count. Moreover, no differences were seen in the response to induction therapy or survival between NOTCH1 wild type and mutated cases (in the overall groups as well as in subgroups analyses). In the expression pattern of HOX11, HOX11L2, BAALC, or ERG there were no significant differences between the two NOTCH1 groups. Patients with mutated NOTCH1 showed a higher frequency of a thymic immunophenotype (65% vs. 39% for unmutated cases; P=0.013). Furthermore, a significant association was observed between the expression of specific surface antigens and the NOTCH1 genotype: NOTCH1 mutated cases were significantly associated with the expression of CD10 (P&lt;0.001) and CD1a (P&lt;0.001) as well as lack of CD117 (P=0.004) and CD34 expression (P=0.04). Interestingly, mutations in the HD-N domain were highly associated with the expression of CD10 (P&lt;0.001), whereas mutations in the PEST-1 region were characterized by CD1a positivity (P&lt;0.001). In contrast, no significant associations were seen for the HD-C, TAD, or the N-terminal PEST-2 domain to the surface marker expression. In conclusion, NOTCH1 mutations occur in all molecular defined subtypes (HOX11, HOX11L2, ERG, BAALC); thus these data suggest that NOTCH1 activation promotes transformation of T-cell progenitors as an early step independent of the additional, subsequent genetic alterations. Moreover, activating NOTCH1 mutations in adult T-ALL display a distinct leukemic phenotype suggesting that mutations in specific NOTCH1 domains arrest thymocytes at a distinct maturation stage. Recognition of this specific immunophenotype may direct the detection for NOTCH1 mutations and the guidance of treatment strategies as the high frequency of NOTCH1 mutations in adult T-ALL makes an ideal target for pharmacological interventions.