Apoptosis Resistance and NOTCH1 Mutations Impair Clinical Outcome in Chronic Lymphocytic Leukemia (CLL) Patients Treated with Ibrutinib

Abstract

Ibrutinib, an oral, selective inhibitor of Bruton's tyrosyne kinase (BTK), demonstrated superior overall response rate (ORR), progression-free survival (PFS) and overall survival (OS) compared with chemoimmunotherapy in CLL both in first line and in relapsed/refractory patients (Byrd, 2015). Moreover, no significant difference was observed for PFS across most genomic subtypes and PFS was significantly better for ibrutinib-treated patients in first/second line vs later lines of therapy (Brown, 2017). However, the mechanisms underlying ability of ibrutinib to kill CLL cells are not well understood. Deng et al (2017) found that BTK inhibition enhances mitochondrial bcl-2 dependence and therefore the basal level of apoptosis measured through bax/bcl-2 ratio before therapy may be important to test sensitivity to ibrutinib. Even more, NOTCH1 mutations (M) trigger apoptosis resistance (Rosati, 2009) and thus may confer poor response to ibrutinib. The primary aims of our research were: 1) to verify the correlations of bax/bxcl-2 and NOTCH1 M with other biological and clinical prognosticators in patients treated with ibrutinib; 2) to evaluate the impact of bax/bcl-2 and NOTCH1 M on ORR, PFS and OS; 3) to confirm bax/bcl-2 and NOTCH1 M as independent prognostic factors. Therefore, we evaluated the efficacy of ibrutinib, in a real-life contest, on 72 patients, median age 66 years (42-85), median number of previous regimens 2 (0-4; 13.9% previously untreated). Patients received 420 mg oral ibrutinib once daily until progression or occurrence of unacceptable side effects. Median follow up on ibrutinib was 12 months. ORR was 91% [complete response (CR): 20%, partial response (PR): 42%, PR with lymphocytosis (PR-L): 29%]. The estimate 1-year PFS and OS were 70% and 72%, respectively. Nineteen patients (26%) discontinued ibrutinib therapy due to progression (n= 8), adverse events (n=9) or due to other reasons (n=2). Five out of 6 patients with Richter's syndrome (RS) had baseline del(17p)/ TP53 mutations (M). PFS was significantly better for patients in first/second line vs later lines of therapy (p=0.045). Bax/bcl-2 was calculated by flow cytometry, dividing mean florescence intensity (MFI) of bax by MFI of bcl-2 on CLL cells. The threshold was set at the median value >1.5 (range 0.44-5.10). The presence of NOTCH1 M was investigated with ARMS PCR for c.75447545delCT and by Sanger sequencing of NOTCH1 exon 34. Eighteen patients were NOTCH1 M (25%) and 42 had bax/bcl-2<1.5 (58.3%). All 6 patients who underwent Richter's syndrome had bax/bcl-2 <1.5 (p=0.030). NOTCH1 M were strongly correlated with trisomy 12 (10/18; p=0.00002) and with CD49d >30% (16/18; p=0.024). There was no significant correlation between NOTCH1 M and bax/bcl-2 as well as between bax/bcl-2 and del(17)p/ TP53 M . With regard to clinical outcome, lack of response/progression was significantly correlated both with NOTCH1 M (p=0.009) and bax/bcl-2<1.5 (p=0.017), but not with del(17)p/ TP53 M (p=0.09). Significant shorter PFS was observed in NOTCH1 M patients (42% vs 76% at 24 months, p=0.002, Figure) and in bax/bcl-2 ratio<1.5 cases (55% vs 91% at 24 months, p=0.006, Figure). On the contrary, del(17)p/ TP5 M patients did not show significant worse PFS (p=0.26). Moreover, patients with lower bax/bl-2 showed significant shorter OS (63% vs 91% at 24 months, p=0.020). Interestingly, NOTCH1 M and bax/bcl-2 showed synergistic prognostic properties, since NOTCH1 M and bax/bcl-2<1.5 identified a subset at worse prognosis with regard to PFS (43% vs 86% at 24 months, p=0.001, Figure). In our hands, bax/bcl-2 and NOTCH1 M were powerful prognosticators for patients treated with ibrutinib, showing additive clinical effects. In multivariate analysis of PFS, bax/bcl-2 (HR: 0.1; p=0.03) together with NOTCH1 (HR:5.0; p=0.007), were confirmed as independent prognostic factors. Therefore, bcl-2 antagonists, such as venetoclax, could be combined with BTK inhibitors, in order to overcome apoptosis resistance in patients treated with ibrutinib, particularly within the CLL subset characterized by lower bax/bcl-2 ratio and/or NOTCH1 M. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.