Early Thymic Progenitor Leukemia (ETP-ALL) in Adults: A Distinct Molecular Entity,

Early thymic progenitor acute lymphoblastic leukemia (ETP-ALL) has been characterized as new T-ALL subgroup characterized by a specific gene expression profile reflecting the signature of normal early thymic progenitors (ETP). ETP-ALL is also defined by a certain immunophenotype (CD1a⁻, CD8⁻, CD5^weak with expression of stem cell and/or myeloid markers). Recently, we described a high rate of FLT3 mutations in T-ALL, which were almost exclusively found in the subgroup of ETP-ALL. To further unravel additional molecular alterations we now investigated in a large cohort of adult ETP-ALL patients the NOTCH1 /FBXW7 mutation status, as NOTCH1 activation, frequently detected in T-ALL patients (50–70%), represents an attractive target for γ-secretase inhibitors. In addition, we analyzed the T-cell receptor (TCR) rearrangement status in these ETP-ALL patients. An absence of TCR rearrangement was linked to an immature T-ALL subgroup and associated with a poor prognosis in paediatric patients.

We explored a cohort of 68 ETP-ALL adult patients (55 male, 13 female, median age 38 years). These samples were selected based on flow cytometry data from a total of 1241 T-ALL samples that had been sent to the diagnostic GMALL reference laboratory between 1997 and 2007. DNA was extracted from these diagnostic specimens and the NOTCH1 mutation status was determined by direct sequencing of the N-terminal and the C-terminal regions of the HD domain, the TAD region, the N-terminal and the C-terminal region of the PEST domain. The FBXW7 mutation status was analyzed by direct sequencing of the amplified PCR products of exons 8 and 9. TCR rearrangement status was assessed by the IdentiCloneTM TCRG Gene Clonality Assay (InVivoScribe Technologies). FLT3 mutations were assessed using the FLT3 mutation assay (InVivoScribe Technologies).

We found NOTCH1 mutations in 11 of the 68 ETP-ALL patients. This rate of only 16% is significantly lower compared to a cohort of 128 non-ETP T-ALL adult patients with a NOTCH1 mutation frequency of 61%. Of the identified NOTCH1 mutations, 6 were in the HD domain, 2 in the TAD region and 3 in the PEST domain. No FBXW7 mutations were found in the 68 ETP-ALL patients compared to 12% in the cohort of non-ETP T-ALL. The analysis of the TCR rearrangement revealed that 38 ETP-ALL patients (56%) lacked clonal TCR rearrangement, while 30 patients (44%) had a monoclonal TCR rearrangement. No correlation was found between TCR status and NOTCH1 mutation status. As previously reported, ETP-ALL patients showed a high frequency of FLT3 mutations (n=23/68, 34%) and these FLT3 mutated ETP-ALL cases had a distinct immunophenotype characterized by the positivity for CD2, CD117 and CD13. Interestingly, ETP-ALL patients with FLT3 mutations predominantly lacked clonal TCR rearrangements (46%), whereas patients without FLT3 mutations showed more frequently TCR rearrangements (79%; p=0.007). FLT3 and NOTCH1 mutations were mutually exclusive. Interestingly, NOTCH1 mutations were indicative for an ETP-ALL phenotype distinct to the FLT3 mutated cases: NOTCH1 mutated patients were characterized by a lower rate of positivity for CD2 (18% vs 56%, p=0.02), CD13 (28% vs 65%, p=0.02), and CD117 (18% vs 56%, p=0.02) compared to NOTCH1 wild type patients. Likewise, patients lacking of clonal TCR rearrangement also displayed a specific immunophenotype with a higher rate of positivity for CD2 (68% vs. 27%, P<0.001) and CD13 (70% vs. 43%, P=0.023) compared to patients with a clonal TCR status.

ETP-ALL patients represent a distinct molecular subgroup of adult T-ALL patients with a yet unreported low frequency of NOTCH1 mutations and a high rate of FLT3 mutations. ETP-ALL is further characterized by a lack of clonal TCR rearrangements indicating a prothymocyte arrest of this very immature T-ALL subgroup. Moreover, within the ETP-ALL subgroup, different genetic alterations drive distinct phenotypes likely representing separate molecular entities; consequently these high risk ETP-ALL patients should be differentially treated with respect to targeted therapies. '-secretase inhibitors might likely be not effective due to the lack of activating NOTCH1 mutations. In contrast, ETP-ALL patients with a FLT3 mutation could benefit from a specific treatment with FLT3 inhibitors.

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