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NOTCH1 mutations in CLL
mutated for NOTCH1 i.e., NOTCH1 M, if exceeding a variant allele frequency (VAF) of 1%.15,16
Redistribution lymphocytosis and nodal response
The redistribution lymphocytosis was calculated as percent variation of ALC over the baseline values. Nodal response was cal- culated as percent reduction in sum of the product of diameter (SPD) values on the major lymph node regions over the baseline measurement, as reported previously.17
Additional details on the employed procedures and methods are reported in the Online Supplementary Materials and Methods.
Results
NOTCH1 mutations and BAX/BCL-2: correlations with other biological parameters
Sixty-five patients were NOTCH1 M (65 of 180, 36.11%), with VAF levels >1 (Online Supplementary Table 2S). With regard to the distribution of VAF levels, 21 patients had VAF between 1% and 10%, seven patients between 10.5% and 20% and 37 patients above 20%. Fifty-six NOTCH1 M cases bore a single mutation, eight cases two mutations and one case three mutations. NOTCH1 M cases were classified as follows: 45 delCT, six frameshift other than delCT (FS), 8 3’-UTR and six considering both missense (one) and non- sense (five) mutations (Online Supplementary Table S2). Seventy-four patients showed a BAX/BCL-2 ratio lower than 1.5 (74 of 113, 65.5%). NOTCH1 M were significantly associated with SPD ratio<1.5: in fact, 34 of 38 NOTCH1 M patients showed BAX/BCL-2 ratio less than 1.5 (P=0.0001). Moreover, NOTCH1 M were strongly correlated with CD49d expression: 51 patients were both NOTCH1 M and CD49d ≥30% (P=0.0001). Furthermore, a significant corre-
lation was found between a lower BAX/BCL-2 ratio and CD38>30% (41 of 54; P=0.030) as well as between CD38>30% and NOTCH1 M (27 of 38 patients; P=0.0004) (Table 2; Online Supplementary Table S3).
Trisomy 12 was confirmed to be strongly correlated with NOTCH1 M (18 of 23; P=0.0002). There was only a trend towards significant association between NOTCH1 M and IGHV UM status (48 of 62; P=0.08). On the other hand, IGHV UM status was correlated with lower BAX/BCL-2 ratio (58 of 81; P=0.030). TP53 M and/or del17p were found in 66 of 178 patients (37.1%). Noteworthy, 23 of 178 patients (13%) were simultaneously NOTCH1 and TP53 mutated. The distribution of clinical and biological prog- nostic factors according to NOTCH1 M is shown in Table 2. The distribution of prognostic factors according to the BAX/BCL-2 ratio and CD38 was obtained in 113 patients from Rome and shown in Table 2 and the Online Supplementary Table S3.
Relevance of NOTCH1 mutations as biological prognostic parameter
The mean peripheral lymphocyte percentage change from baseline, calculated at 3 months on ibrutinib, was lower in NOTCH1 M patients than in NOTCH1 wild-type (WT) patients (14% vs. 54%; P=0.022, Mann-Whitney test), thus confirming a reduced redistribution lymphocytosis (Figure 1A).
Moreover, the mean percent SPD change, calculated at 6 months on ibrutinib, was lower in NOTCH1 M patients than in NOTCH1 WT patients (53% vs. 80%; P<0.0001, Mann- Whitney test), confirming a significant poor nodal response (Figure 1B). Moreover, we compared NOTCH1 M plus lower BAX/BCL-2 ratio versus NOTCH1 M plus higher BAX/BCL-2 ratio with respect to redistribution lymphocy-
A B
Figure 1. Box plots by NOTCH1 wild-type and mutated groups showed significant lower redistribution lymphocytosis after 3 months on ibrutinib treatment in NOTCH1 M patients (A) and equally lower sum of the product of diameter (SPD) values after 6 months in NOTCH1 M patients (B). pts: points; WT: wild-type; M: mutated; B LYMPHS: B lymphocytes.
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