Clinical Impact of Clonal and Subclonal TP53 Mutations in Chronic Lymphocytic Leukemia

Introduction. The clinical course of patients with chronic lymphocytic leukemia (CLL) is highly heterogeneous. The deletions/mutations of 17p/TP53 are predictors of chemorefractoriness, and for this reason, in the management algorithm of CLL patients, when present, indicate treatment with with chemo-free regimens also in the context of first-line therapy. Recent studies based on ultra-deep-next generation sequencing (NGS) have shown that TP53 mutations can be present at very low clonal abundance in tumor cell populations, although whether these mutations may have a detrimental clinical impact on disease course is still to be established.

Aim. To investigate the presence of clonal and subclonal mutations of TP53 in a large cohort of CLL cases using an ultra-deep NGS strategy, and determined their clinical relevance for patients outcome.

Methods. The study includes 590 CLL patients characterized for the deletion at chromosome 17p13 (FISH analysis) and TP53 mutations in samples before treatment. In all cases, analyses were carried out on DNA extracted from nearly pure (>90%) tumor cells. TP53 mutational status was investigated by NGS with an amplicon based strategy. Sequencing reads analysis was made by the Burrows-Wheeler Aligner-MEM algorithm and by SAMtools. Variant calling was performed using the entire pipeline established on the MiSeq Reporter software. Results were expressed as percentage of mutated DNA. The minimal allelic fraction for mutation calling was set at 1%. Synonymous variants and polymorphisms described in the Single Nucleotide Polymorphism Database (dbSNP138) were removed. Outcome variable was overall survival (OS). Clinical correlations were made using Kaplan-Meier plots and log-rank test.

Results. FISH and mutational analyses were performed in samples within 2 years from diagnosis in 92% of the cases (Figure 1A). A total of 125 TP53 mutations (Figure 1B) were found in 96 patients (11.7%). Subclonal mutations have similar molecular characteristics as their respective high frequency allele mutations supporting a comparable pathogenic effect (Figure 1B). According to a 15% cutoff of variant allele frequency (VAF), 78 cases were considered clonal and 18 subclonal (Figure 1C) for TP53 mutations (1% < VAF < 15%). In this context, cases with subclonal and clonal TP53 mutations experienced significant shorter OS than TP53 wild-type (wt) cases, without differences between clonal and subclonal cases (Figure 1E). Accordingly, ROC analysis on the same cohort identified a cutoff of >0% for the clinical impact of TP53 mutations (Figure 1E inset). Deletion of chromosome 17p was found in 180 out of 574 patients (31.3%), and using a 10% cutoff, 61 patients presented a percentage of deleted nuclei above the cutoff (Figure 1D). Using only 17p deletion data and considering the above mentioned cutoff, patients with 17p13 deletion ≥10% experienced shorter OS than wt cases, while patients with 17p13 deletion <10% experienced OS superimposable to wt cases (Figure 1F). These data were confirmed by ROC analysis that selected >9% of deleted nuclei as optimal cutoff for OS discrimination (Figure 1F inset). Given the frequent co-occurrence of TP53 mutations with 17p deletions, we also evaluated the impact of isolated TP53 mutations and 17p deletions. By using the ROC cutoffs for the definition of mutated/deleted cases, 466 cases (81.1%) presented no TP53 disruption (TP53 mutations and deletion), 47 cases (8.2%) were TP53 mutated only, 15 cases (2.6%) were 17p deleted only and 46 cases (8.1%) presented a concomitant TP53 mutation and 17p deletion. Kaplan-Meier curves demonstrated comparable significant shorter OS intervals for TP53 mutated and/or deleted CLL cases respect to wt cases, while no differences were observed between these three groups (Figure 1G).

Conclusion. By using a highly sensitive NGS approach, we have detected small subclones of TP53 in a relative high proportion of patients. TP53 mutations conferred a significant shorter OS irrespectively of VAF percent, while deletion of chromosome 17p impacted on OS only when detectable in more than 10% of nuclei. These cutoffs, once validated by prospective studies, may be employed in daily practice for the clinical management of CLL patients.

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