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NOTCH1 PEST domain mutation is an adverse prognostic factor in B‐CLL

2010; Wiley; Volume: 151; Issue: 4 Linguagem: Inglês

10.1111/j.1365-2141.2010.08368.x

1365-2141

Paolo Sportoletti, Stefano Baldoni, Laura Cavalli, Beatrice Del Papa, Ezio Bonifacio, Raffaella Ciurnelli, Alain Sylvin Bell, Ambra Di Tommaso, Emanuela Rosati, Barbara Crescenzi, Cristina Mecucci, Isabella Screpanti, P Marconi, Maria Paola Martelli, Mauro Di Ianni, Franca Falzetti,

Lymphoma Diagnosis and Treatment

B-cell chronic lymphocytic leukaemia (B-CLL) is a heterogeneous disease with a highly variable clinical course (Chiorazzi et al, 2005) and several factors at diagnosis predict prognosis and help guide treatment decisions (Seiler et al, 2006). Unmutated immunoglobulin heavy variable (IGHV) genes and high expression of zeta-chain-associated protein kinase 70 (ZAP-70) are well-known factors for poor prognosis (Rassenti et al, 2004). When they are discordant other cytogenetic and molecular biological factors such as 17p-, IGHV3-21 or microRNA signature appear helpful (Gribben, 2008). NOTCH signalling activation was recently implicated in B-CLL cell survival and apoptosis resistance (Rosati et al, 2009) and in an attempt to identify the underlying mechanism we found a NOTCH1 PEST domain mutation in a minority of B-CLL patients (Di Ianni et al, 2009). As these findings led us to hypothesize that this NOTCH1 mutation might be a novel prognostic marker in B-CLL, we extended a DNA-based sequencing screening strategy to a consecutive series of 133 unselected B-CLL patients and, when possible, correlated the results with IGHV gene status, ZAP-70 expression and clinical outcome. Given the marked structural similarities between NOTCH1 and NOTCH2 and the recent demonstration of NOTCH2 gain-of-function mutations in lymphomas (Lee et al, 2009) we also screened for the presence of NOTCH2 mutations in a sub-group of these B-CLL patients. NOTCH1 mutational status was correlated with IGHV gene status, ZAP-70 expression and clinical outcome in 111 (69 males, 42 females) of the 133 patients who were analysed. Median age at diagnosis was 60 years (range 40–84 years). According to the Binet staging system, 85/111 (76·5%) patients were in stage A, 19 (17·1%) patients were in stage B, and 7 (6·3%) patients in stage C. IGHV was unmutated in 45/111 (40·5%) patients and ZAP70 expression was >20% in 66/111(59·4%). Peripheral blood samples were collected at diagnosis, before disease progression or before any treatment. Mutations of NOTCH1 exons 26, 27 and 34 were investigated by DNA-based polymerase chain reaction as previously described (Di Ianni et al, 2009). The mutation was confirmed by independent amplicon amplification. To evaluate the clinical significance of NOTCH1 mutation we selected time from diagnosis to initial treatment (TTT) as the primary end-point. Our first step in cohort validation was to analyse TTT in relation to IGHV status and ZAP70 expression. Median TTT was significantly shorter in patients with unmutated IGHV genes (36 vs. 77 months; P = 0·0028) and in patients with ZAP70 expression >20% (45 vs. 77 months; P = 0·0135). A NOTCH1 PEST domain mutation was found in 7/133 (5·3%) B-CLL patients, and extended our previous findings to a much larger cohort. Neither NOTCH2 heterodimerization nor PEST domains were mutated in the sub-group of 73 patients, thus excluding a NOTCH2 gene mutation from involvement in altered NOTCH signalling in B-CLL. As we found a NOTCH1 mutation only in a small minority of patients, it seems other mechanisms come into play since both NOTCH1 and NOTCH2 are constitutively activated in all B-CLL patients (Rosati et al, 2009). Consequently a NOTCH1 PEST mutation in this subgroup of B-CLL patients is therefore only one of the possible mechanisms implicated in the NOTCH signalling activation. Six of the seven patients bearing NOTCH1 activating mutation had unmutated IGHV genes and ZAP70 positivity. The other had mutated IGHV and ZAP70 positivity. Strikingly, median TTT was significantly shorter in all seven patients with NOTCH1 mutation than in the 104 with wild-type NOTCH1 (24·5 vs. 63 months, P = 0·0053) (Fig 1A) and than in the 39 patients with unmutated IGHV genes and wild-type NOTCH1 (24·5 vs. 48 months, P = 0·0481) (Fig 1B). Interestingly, as patients carrying the NOTCH1 mutation showed poorer prognosis than patients with unmutated IGHV, the NOTCH1 mutation emerged as a potentially factor for poor prognosis in B-CLL. Kaplan–Meier curves of time from diagnosis to initial treatment (TTT) according to NOTCH1 mutational status in the cohort of 111 patients with B-CCL (A) and/or in patients with unmutated IGHV (B). The analysis of CD38 expression in the seven NOTCH1 mutated patients did not reveal any correlation between these two markers as only three patients harbouring the NOTCH1 mutation expressed CD38 in more than 30% of CD19-positive cells. Prognostically relevant anomalies of chromosomal regions were assessed by fluorescent in situ hybridization (FISH) on five of the seven NOTCH1 mutated patients. Two patients presented with trisomy 12, one patient with deletion ATM/11q23 and one exhibited a normal FISH pattern. Moreover, one patient presented with trisomy 12 together with TP53/17p13 and 5′IGH/14q32 deletions. These data indicated no evident correlation between specific cytogenetic aberrations and the NOTCH1 mutation, although the benign del(13q) lesion was always absent. To investigate the balance between wild-type and mutated NOTCH1 alleles in the seven patients with the NOTCH1 mutation we developed a semi-quantitative assay based on Genescan analysis. Genomic DNA was analysed with a sample from one patient without the mutation as negative control and DNA from the MOLT-4 cell line as positive control. The allelic ratio (AR) of mutant to wild-type NOTCH1 at diagnosis ranged from 9% to 45%, median 24% (Fig 2). As no correlation emerged between AR and TTT (P = 0·35) or OS (P = 0·9) the individual AR did not seem to impact upon outcome. Surprisingly, repeated analysis in two patients over a year revealed the AR remained unchanged in one and increased from 19% to 40% in the other, suggesting a growth advantage, at least in this patient, for the mutation bearing clone. Range of mutant to wild-type (WT) allelic ratio (AR) in 7 B-CLL patients with NOTCH1 mutation. Representative Genescan electropherograms show panel (A) lowest AR found in patients; panel (B) highest AR found in patients; panel (C) AR in a patient with WT NOTCH1 (negative control); panel (D) AR in MOLT-4 cell line DNA (positive control). This study showed that the NOTCH1 mutation is a marker of poor prognosis in a minority of B-CLL patients, predicting time to progression and need for therapy. However, since NOTCH1 is constitutively activated in all B-CLL patients (Rosati et al, 2009) the NOTCH1 mutation appears to be only one of the possible mechanisms implicated in NOTCH signalling activation. Despite this limitation, this study opens up new perspectives in the treatment of B-CLL. Should agents that are under investigation as suppressors of inappropriate NOTCH activation (Real & Ferrando, 2009) be approved for clinical purposes, then screening for the NOTCH1 mutation could be recommended at diagnosis of B-CLL. We would like to thank Dr Geraldine Anne Boyd for her help. This work was supported by "Associazione Umbra Leucemie e Linfomi", Perugia Italy and by "Associazione Italiana Leucemie, Linfomi e Mieloma", L'Aquila Section, L'Aquila, Italy.