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lation between VAF by ddPCR and deep targeted next- generation sequencing (tNGS) (r2=0.999) (Online Supplementary Table S6). Consequently, one TP53mut (0.2% VAF) only identified by DMA was excluded based on SEM (Online Supplementary Figure S7C) but was in fact validated by ddPCR. However, the TP53 status remained unchanged as this patient harbored additional high and low burden mutations.
Among the 290 newly diagnosed patients, 41 patients (14%) harbored 18 high burden (VAF >10%) and 31 low burden (VAF ≤10%) mutations, including 20 minor TP53muts (VAF <1%). This resulted in 6 patients with both del(17p) and TP53mut, one with del(17p) only, 10 with high burden TP53mut only, and 25 patients with low burden TP53mut only (Figure 1A and Online Supplementary Table S4). Patients harboring only minor TP53mut were mainly older patients (>65 years) but were otherwise char- acterized as low risk according to the CLL-IPI (Online Supplementary Table S7A), whereas the distribution of high and low burden TP53muts was similar among patients stratified based on IGHV mutational status (Online Supplementary Table S7B). All mutations were located within exons 4-8 and 80% were missense mutations. Multiple high burden TP53muts were seen in 2 patients, while multiple low burden TP53muts were seen in 5 patients.
Among 61 patients at time of treatment, we identified 57 mutations in 17 patients (28%): 7 patients with high burden and 10 with low burden TP53muts, including 4 with minor TP53muts (Figure 1B and Online Supplementary Table S4). Forty-three mutations (75%) were observed in 6 out of 11 previously treated patients with a median VAF of 1.01% (IQR: 0.46-2.93). Five of the 6 patients with del(17p) also harbored TP53muts on the remaining allele at time of treatment (Online Supplementary Table S8A). All mutations were located within exons 4-9 and 72% were missense mutations (Figure 1B). Seven patients harbored
multiple TP53muts. Patients’ characteristics are summa- rized in Online Supplementary Table S8.
Prognostic impact on newly diagnosed patients
Stratifying TP53 aberrated patients into high and low burden (TP53wt vs. VAF ≤10% vs. VAF >10%), only high burden TP53ab [including del(17p)] patients showed a trend for worse OS and significantly worse TFS compared to TP53wt (Figure 2A and B) (P=0.06 and P=0.01, respec- tively). Further stratifying low burden TP53mut patients (VAF <1% vs. VAF 1-10%), still no impact on OS or TFS was seen for either group (Figure 2C and D), whereas combining the group of patients with a VAF above 1% could demonstrate a significant impact on OS and TFS compared to TP53wt (Online Supplementary Figure S8A and B). However, this was fully dependent on del(17p) patients (P=0.004 and P<0.001, respectively) (Figure 2E and F), as TP53 mutated patients without del(17p) demonstrated similar OS and TFS compared to TP53wt patients (P>0.25) (Figure 2E and F and Online Supplementary Figure S8C and D). Multiple TP53muts were observed in 7 newly diag- nosed patients without impact on OS or TFS (1 vs. >1 TP53mut; P>0.2) (data not shown), while multiple TP53ab including del(17p) resulted in a significant impact on OS and a trend for TFS (1 vs. >1 TP53ab; P=0.036 and P=0.051, respectively) (data not shown),
Prediction of treatment outcome
At time of treatment, TP53ab was significantly associat- ed with a poor OS compared to patients with TP53wt (P=0.005) (Figure 3A and data not shown). Stratifying TP53- aberrated patients into high and low burden, only high burden TP53ab patients demonstrated poor OS compared to TP53wt (P<0.001) (Figure 3A). Further stratifying low burden TP53 mutated patients (VAF <1% vs. VAF 1-10%), OS was significantly worse for patients with TP53mut with VAF 1-10% compared to TP53wt (P=0.002) (Figure
AB
Figure 1. Characterization of TP53 mutations. Number of mutations indicated in bar plots [regardless of del(17p)] and the number of patients according to TP53 status indicated in pie charts]. (A) Located within exons 4-8, 49 mutations were detected in 41 of 290 newly diagnosed patients; 6 of 7 del(17p) patients also har- bored TP53 mutations. Eighteen (37%) and 31 (63%) mutations classified as high and low burden, respectively. (B) Fifty-seven TP53 mutations within exons 4-9 were detected in 17 of 61 patients at time of treatment; 5 of 6 del(17p) patients also harbored TP53 mutations. Nine (16%) and 48 (84%) mutations classified as high and low burden, respectively. Primarily missense mutations were detected. All percentages indicate variant allele frequencies (VAF). TP53 mutation without del(17p) (TP53mut).
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