Low Variant Allele Frequency (VAF) TP53 Mutation (mut) Is Not a Poor Prognostic Marker in CLL Patients Treated with Targeted Therapy

Introduction: TP53 aberration, defined by TP53 mut and/or deletion (17p) (del(17p)), has long been considered the strongest prognostic marker in CLL. Improved sensitivity and reproducibility of next-generation sequencing (NGS) has enabled reliable detection of low-burden TP53 mut below the conventional VAF cutoff of 5-10%. With technological advances, updated recommendations from the European Research Initiative for CLL eliminated a specific VAF cutoff for reporting TP53 variants (Malcikova et al. 2024). However, the clinical impact of low VAF TP53 mut remains debatable in CLL, particularly in the context of targeted therapy, with conflicting results in previous studies (Briegel et al. 2019; Pavlova et al. 2023).

Methods: We conducted a retrospective analysis of the Dana-Farber CLL database to identify CLL patients with available NGS and FISH data. When these tests were performed at multiple timepoints, we selected the first test to determine the TP53 status. The Kaplan-Meier method was used to estimate time from CLL diagnosis to first-line therapy (TT1T), time from diagnosis to second-line therapy (TT2T), and overall survival (OS). The distribution of TT1T, TT2T and OS were compared using log-rank test. To control guarantee-time bias that interferes with conventional survival analyses, we used left-censoring to adjust for time to the first NGS and/or the FISH test. The Cox proportional hazards model was used to estimate hazard ratios and confidence intervals.

Results: A total of 964 patients with NGS and FISH data were included (52% untreated). Median time from CLL diagnosis to first FISH and NGS testing was 13 and 23 months, respectively. 191 (20%) patients had TP53 aberration including 77 with concurrent TP53 mut and del(17p) (TP53/del(17p)), 35 with del(17p) alone, and 79 with TP53 mut alone. Known hotspots of the DNA-binding domain of TP53 were frequently mutated, including R209fs*6, Y220C, R248W, and R273H. The proportion of patients with multiple TP53 mut were similar in the TP53/del(17p) group (26%) and those with isolated TP53 mut (24%). Del(17p) was found in similar frequencies in groups with multiple (51%) and single TP53 mut (49%). Median VAF of TP53 mut was 18% (1%-81%) for patients with isolated TP53 mut and 29% (1%-91%) for those with TP53/del(17p). Low-VAF (<10%), high-VAF (≥10%), and wild-type (WT) TP53 were found in 28%, 53%, and 18% of patients with TP53 aberration, respectively. Concurrent del(17p) and unmutated IGHV were more frequently observed with high-VAF (56%, 68%) than with low-VAF (39%, 50%).

TP53 aberrant CLL was associated with a significantly higher proportion of patients with unmutated IGHV (62% vs 47%) and complex karyotype (≥3 abnormalities, 45% vs 10%), and shorter TT1T (median 1.2 vs 4.1 years) and 10-year OS (41% vs 77%) than those without the aberration (all p<0.05). There was no difference in TT1T or OS among subgroups with concurrent TP53/del(17p), TP53 mut alone, and del(17p) alone. Median TT1T was significantly shorter for the high-VAF group (0.3 years, p<0.001), but not the low-VAF group (1.2 years), than the WT group (4.1 years). 5-year OS was inferior in the high-VAF group (80%, p=0.01), but not the low-VAF group (93%), compared with the WT group (91%).

We hypothesized that the lack of differences in outcome between the low-VAF and the TP53 WT group was attributable to improved outcomes on targeted therapy. We conducted sensitivity analyses in 207 patients who received initial treatment with Bruton tyrosine kinase inhibitor (BTKi, n=120), B-cell lymphoma 2 inhibitor (n=35), or targeted combinations (n=52), excluding those who received chemoimmunotherapy added to targeted therapy. OS and TT2T did not differ by TP53 status. There were no OS differences among the WT, the low-VAF, and the high-VAF groups treated with first-line targeted therapy. The high-VAF group had shorter TT2T (p<0.05).

Conclusions: TP53 aberration and high-VAF TP53 mut, but not low-VAF TP53 mut, were associated with inferior OS and TT1T. Within the subgroup of patients who received first-line therapy with targeted agents (58% with a BTKi), -VAF TP53 mut continued to predict shorter TT2T. There were no OS differences among subgroups divided by TP53 aberration status or the 10% VAF cutoff when these patients received targeted therapy as initial treatment of CLL.