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G. Del Poeta et al.
the response to chemo-immunotherapy in CLL. In the CLL8 study Stilgenbauer et al.5 demonstrated that patients carrying NOTCH1 M did not benefit of the addition of rit- uximab to standard fludarabine and cyclophosphamide chemotherapy. Moreover, it has emerged that NOTCH1 M are associated with decreased duration of response in a large series of relapsed/refractory (R/R) patients treated with venetoclax.6
In a recent study, Tissino et al.7 have demonstrated that patients with CLL whose cells were characterized by high CD49d expression, underwent reduced lymphocytosis and inferior nodal response after treatment with ibrutinib. Several reports confirmed that in CLL the balance between the pro- and anti-apoptotic members of the BCL-2 family determines chemotherapy sensitivity and cell survival.8,9 Noteworthy, we demonstrated that a low BAX/BCL-2 ratio had an additive negative prognostic impact in both TP53 M and NOTCH1 M patients with CLL treated with chemo- immunotherapy.10 The recent introduction of novel B-cell receptor inhibitors such as ibrutinib and idelalisib and of novel potent oral BH3 peptidomimetics such as venetoclax in clinical practice, prompted us to evaluate the clinical impact of both NOTCH1 M and BAX/BCL-2 ratio in patients treated with targeted oral therapies and in particu- lar in those treated with ibrutinib.
The aims of this study were: i) to analyse the correlations between NOTCH1 M and other biological parameters including CD49d expression and the BAX/BCL-2 ratio; ii) to address the impact of NOTCH1 M both on redistribu- tion lymphocytosis and on nodal responses after treatment with ibrutinib; iii) to evaluate the impact of NOTCH1 M and BAX/BCL-2 ratio on the overall response rate (ORR) to ibrutinib, progression free survival (PFS) and overall sur- vival (OS); iiii) to assess whether NOTCH1 M could be considered an independent prognostic factor.
Methods
Study design and patients
In this study we retrospectively analysed 180 patients with CLL exposed to treatment with ibrutinib. Patients were recruited from three independent cohorts from Italy (Rome Tor Vergata University, Rome Cattolica Sacro Cuore University and Catania Ferrarotto Hospital), between 2014 and 2019. Informed consent was obtained in accordance with the Declaration of Helsinki. The study was performed under the Institutional Review Board of the Centro di Riferimento Oncologico (IRCSS) of Aviano (approval numbers: IRB-05-2010 and IRB-05-2015).
Patients were 122 males and 58 females with a median age of 69 years (range, 36-90). According the modified Rai staging sys- tem,11 134 patients had an intermediate risk and 46 a high risk stage. All these parameters were considered at the time ibrutinib was initiated.
All patients received 420 mg oral ibrutinib (Imbruvica; Janssen, Beerse, Belgium) once daily until progression or occur- rence of unacceptable side effects. Median number of previous chemotherapy regimens were two (range, 0-4). Patients receiv- ing first-line ibrutinib were 26 (14.4%), of whom 24 of 26 (92%) were TP53 mutated. Median follow-up was 25 months (range, 10-61). Seventy-three patients (40.6%) discontinued ibrutinib either for progression (n=42) or for adverse events (n=31) (Table 1): 32 patients were subsequently treated with venetoclax (11 for toxicity [grade 3 or 4 World Health Organization] and 21 for progression of disease), five patients
were treated with idelalisib, and the remaining 36 patients received other lines of chemotherapy (n= 12) or no therapy (n=24). The clinical characteristics of patients are reported in Table 1. The clinical assessment of patients with CLL to estab- lish diagnosis and response to therapy were based both on the International Workshop on Chronic Lymphocytic Leukemia (iwCLL) criteria.12 The clinical impact of NOTCH1 M and BAX/BCL-2 ratio on ibrutinib treatment was evaluated by measuring the kinetics of absolute lymphocyte counts (ALC), the reduction of lymphadenopathy, and the clinical outcome, as defined by ORR, PFS and OS.
Chronic lymphocytic leukemia characterization
Flow cytometry was employed for immunophenotypical CLL characterization and was performed with FACSCalibur or FACSCanto I flow cytometer. BCL-2 and BAX oncoproteins were analysed by flow cytometry in samples taken before starting ibru- tinib. BAX/BCL-2 ratio was calculated dividing mean fluorescence intensity (MFI) of BAX by MFI of BCL-2 on CLL cells, as previous- ly described.10 The threshold of positivity was set at ≥1.5. Immunoglobulin heavy-chain variable region gene (IGHV) muta- tional status was performed by NGS, as previously described.13,14 TP53 exons 2 to 11 mutational status and NOTCH1 exon 34 and 3’ untranslated (UTR) region mutational status were analysed by NGS, as previously described.4,7 CLL samples were considered
Table 1. Patient characteristics (n = 180)
No. of patients/Total cases (%)
Observation time Median age, y (range) Males
Modified Rai stage
Intermediate High
Number of previous regimens 0
1 2 3 4
NOTCH1 mutation BAX/BCL-2 ratio <1.50 Trisomy 12
11q deletion
TP53 mutations/17p deletion UM IGHV
CD38 ≥ 30%
CD49d ≥ 30%
Median follow up (months) Response to ibrutinib therapy
Complete response
Partial response
Partial response with lymphocytosis Stable disease/No response
Discontinuation Progression
Toxicity
Richter Syndrome Progression-free Survival at 2 years Overall Survival at 2 years
Overall Survival at 4 years
2014-2019 69 (36-90) 122/180 (68)
134 (74) 46 (26)
26/180 (14.5) 56/180 (31.1) 74/180 (41.1) 22/180 (12.2) 2/180 (1.1) 65/180 (36.1)
74/113 (65.5)
23/179 (13)
35/179 (20)
66/178 (37.1)
123/175 (70.3) 54/113 (47.8)
108/179 (60.3) 25 (10-61)
33/180 (18.3) 51/180 (28.3) 81/180 (45.1) 15/180 (8.3)
73/180 (40.6) 42/180 (35) 31/180 (65)
13/180 (7.2) 80% 84%
71%
y: years; IGHV: immunoglobulin heavy-chain variable region gene. No.: number.
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