The main demographic, clinical, and molecular characteristics of T-ALL cases according to NOTCH1 status are shown in Table . The median age of the total series of cases was 8 years (1-18 years), and a predominance of males was observed, with a 2.6:1 male/female ratio. Immunophenotyping analyses by flow cytometry were available for 130 cases, and in 8 cases the T-cell diagnosis were confirmed by immunohistochemistry with CD45+/CD3+. Regarding T-cell maturation, we observed a predominance of T-IV subtype (38.0%). Overall, 60/138 patients (43.5%) presented with NOTCH1 mutations; additional molecular alterations observed were FBXW7 mutations (21/110; 19.1%), SIL-TAL1 fusion (37/129; 28.7%), TLX3 ectopic expression (10/119; 8.4%), KRAS (10/105; 9.5%) and PTEN mutations (9/96; 9.4%). In 78.3% of cases, the coexistence of NOTCH1 mutations and other molecular alterations was observed, including FBXW7 (n = 13), SIL-TAL1 (n = 19), TLX3 (n = 6), PTEN (n = 5), and KRAS (n = 4) mutational. However, no statistical association was disclosed between NOTCH1 mutations status and any other variable analyzed.
Clinical, demographic and laboratorial features of T-ALL patients according to NOTCH1 status, 2001-2008
n number of cases, mut mutated, wt wild-type, WBC white blood cells count at diagnosis. a 8 cases without information about mediastinal mass, being 4 with NOTCH1 mutation and 4 wild-type; b classification according to EGIL criteria, flow cytometry was not performed in 8 cases, 2 presented NOTCH1 mutation and 6 wild-type; c there were 22 cases without CD10 status evaluated, 7 with NOTCH1 mutation and 15 wild-type; pos, positive; neg, negative
Considering that NOTCH1 and FBXW7 belong to the same signalling pathway, patients with mutations in both genes (NOTCH1 and/or FBXW7) were analyzed together as a NOTCH1-FBXW7 mutated group. In total, 62 patients (56.4%) presented with NOTCH1-FBXW7 mutations. Patients with FBXW7, KRAS or PTEN mutations shared other molecular alterations along with NOTCH1 mutations, such as FBXW7 mutation and SIL/TAL1 (n = 5), FBXW7 mutation and TLX3 (n = 1), FBXW7 and KRAS mutations (n = 2), FBXW7 and PTEN mutations (n = 1). We also observed KRAS mutation and SIL/TAL1 (n = 1), KRAS mutation and TLX3 (n = 2), PTEN mutation and SIL-TAL1 (n = 1), PTEN and KRAS (n = 1), and one case exhibited PTEN, KRAS and SIL-TAL1 alterations.
The complete descriptions of NOTCH1
mutations sequences in HD and PEST domains are shown in Additional file 1
: Table S1. Single nucleotide polymorphism (SNP) already described, recurrent mutations as well as previously unreported mutations were observed. Thirty-eight new mutations were found in the HD domains and 15 in PEST domain, representing 63.1% (53/84) of all mutations found. Among these new mutations there were no SNPs, since we have checked and excluded any SNP possibility through NCBI SNP database and 1000 genomes data. The NOTCH1
SNPs found in this work have been excluded from all subsequent mutation analyses; and they were mentioned in Additional file 1
: Table S1 only for descriptive purposes.
The main demographic and clinical features of T-ALL patients with different types of NOTCH1 mutations are summarized in Table . Mutations were not randomly distributed, with a predominance of point mutations in the HD domain, and complex mutations in the PEST domain (p = 0.014). Also, nonsense mutations were more frequent in the PEST domain and missense mutations in the HD domain (p < 0.0001). Seven cases presented with mutations in both HD and PEST domains, with a predominance of complex mutations (22.2%). No significant statistical differences were observed with respect to age groups, gender and T-ALL subtypes, in respect to any type of NOTCH1 mutation. High WBC was observed in patients harbouring complex mutations, while patients with point mutations exhibited lower WBC (p = 0.035). Point mutations were more frequent in non-white patients (p = 0.053); where a higher frequency of mutations in the HD domain was observed (89.7% versus only 10.3% in PEST domain, p = 0.028). Missense mutations were more frequent in CD10+ cases (p = 0.042). Eleven out of 14 nonsense mutations were caused by complex mutations, the other three by point mutations. Thirty out of 46 missense mutations were caused by point mutations and the remaining 16 by complex mutations (p = 0.004, data not shown).
Main demographic and clinical features of T-ALL patients according to type and classification of NOTCH1 mutations
Overall survival of the 138 cases was 50.7% (Figures ). Mean length of the follow-up was 68.4 months (95% CI 58.9-77.8 months). For 68 patients who evolved to death, the follow-up was 0-76 months (median 9), with 9 deaths occurring in the first 3 days after diagnosis and 57.4% of deaths (39 patients) occurring in the first 12 months of treatment. For 70 patients alive, the follow-up was 15-124 months (median 73.5), with 75% of patients with 94.5 months follow-up. No differences in OS were observed in patients treated with GBTLI or BFM-based protocols (52.5% vs. 45.9%, respectively; p = 0.639).
Figure 1 Kaplan-Meier overall survival curves for T-ALL patients. (A) Overall survival (OS) of T-ALL patients. In this analysis were included all 138 patients with T-ALL. (B) NOTCH1 type of mutation (point mutations vs. complex mutations) OS curve. For the construction (more ...)
The results of the OS univariate analyses of T-ALL cases considering variables such as age, WBC at onset of disease, T-ALL cellular subtypes, CD10 expression, skin colour, response to induction therapy, mutational status of NOTCH1
, presence of SIL-TAL1
fusion, and ectopic expression of TLX3
are shown in Table , Figure and Additional file 2
: Figure S1.No statistically significant impact in OS was found for the following variables: age, WBC, T-ALL subtypes, CD10 expression, skin colour, mutational status of FBXW7
, and ectopic expression of TLX3
Univariate analysis of overall (OS) and event-free survival (EFS) in T-ALL patients
Test of equality of survival distribution for the differences of NOTCH1 status (mutated vs. wild-type) showed no statistical significant results (53.3% vs. 48.7%; p = 0.479). On the other hand, carriers of NOTCH1 complex mutations (indels) exhibited a favourable OS when compared with carriers of point mutations (66.7% vs. 42.4%; p = 0.031; HR 2.33, CI95% 1.05-5.18) (Figures ), NOTCH1 wild-type patients showed an intermediate OS rate (48.7%). OS differences between complex and point NOTCH1 mutations were more marked in the patients treated with the BFM protocols backbone strategies (27.3% vs. 62.5%; p = 0.041). For NOTCH1-FBXW7 combined mutations no impact on OS was observed (58.1% vs. 47.9%), mutated in NOTCH1-FBXW7 vs. WT; p = 0.319 showed in Figure .
The presence of SIL-TAL1 fusion was predictive of a worse outcome (OS 37.8% vs. 56.5%; p = 0.012; HR 1.89, CI95% 1.13-3.17) (Figure ). Multivariate Cox analysis showed that the type of NOTCH1 mutations (point vs. complex mutations) was not an independent prognostic factor (p = 0.769), compared with SIL-TAL1 fusion (p = 0.04; HR 2.87; 95% CI 1.39-5.92) and induction response (p = 0.10; HR 2.69; 95% CI 1.27-5.71), even when adjusted by treatment protocol.
In 80 cases, the initial response to induction phase (day 30-33) was recorded, with 52.5% of cases exhibiting induction failure, independent on protocol allocation. Of the 38 cases that achieved clinical remission, 26 (72.5%) maintained the initial response and 12 (27.5%) relapsed during maintenance therapy and died. Of 42 cases with induction failure 11 (26.2%) evolved to death and the remaining 31 were submitted to re-induction therapy (14 with secondary remission).
Induction therapy response was significantly associated with a longer overall survival, both in univariate and multivariate analyses (p < 0.0001; HR 2.86; 95% CI 1.38-5.94) as shown in Table . However, no significant association was found between any of the molecular alterations and induction response (data not shown).
Ninety-five patients had sufficient clinical data for conducting EFS analysis (9 cases that evolved to death previously to treatment due to severity of disease were excluded of this analysis). All variables evaluated in the OS analysis were also included in EFS investigations. Results of EFS analysis are described in Table .
EFS exhibited no difference according to NOTCH1-FBXW7 status (40.5% Mutated, 48.5% WT, p = 0.286) (Figure ). For patients bearing complex NOTCH1 mutations the EFS was better than for those presenting point mutations (30.4%, 40% respectively, p = 0.289), albeit not statistically significant (Figure ). EFS was significantly worse in patients bearing SIL-TAL1 (18.2% vs. 43.8%; p = 0.004) (Figure ), and also in those patients with no response to induction therapy (32.4% vs. 70.3%, p < 0.0001) (Figure ). Multivariate Cox analysis identified only SIL-TAL1 (HR 3.12; 95% CI 1.48-6.57) and induction failure (HR 3.07; 95% CI 1.44-6.55) as independent negative prognostic factors.
Figure 2 Kaplan-Meier event-free survival curves (EFS) for T-ALL patients. For the EFS endpoint we performed survival analysis in 95 out of 138 T-ALL patients. (A) EFS curve for patients harbouring NOTCH1-FBXW7 mutations vs. WT cases. In the construction of this (more ...)