The Broad Spectrum of TP53 Mutations in CLL: Evidence of Multiclonality and Novel Mutation Hotspots

Abstract

TP53 aberrations are a major predictive factor of resistance to chemoimmunotherapy in chronic lymphocytic leukemia (CLL), and an assessment of them before each line of treatment is required for theranostic stratification. Acquisition of subclonal TP53 abnormalities underlies the evolution of CLL. To better characterize the distribution, combination, and impact of TP53 variants in CLL, 1,056 TP53 variants collected from 683 patients included in a multicenter collaborative study in France were analyzed and compared to UMD_CLL, a dataset built from published articles collectively providing 5,173 TP53 variants detected in 3,808 patients. Our analysis confirmed the presence of several CLL-specific hotspot mutations, including a two-base pair deletion in codon 209 and a missense variant at codon 234, the latter being associated with alkylating treatment. Our analysis also identified a novel CLL-specific variant in the splice acceptor signal of intron 6 leading to the use of a cryptic splice site, similarly utilized by TP53 to generate p53psi, a naturally truncated p53 isoform localized in the mitochondria. Examination of both UMD_CLL and several recently released large-scale genomic analyses of CLL patients confirmed that this splice variant is highly enriched in this disease when compared to other cancer types. Using a TP53-specific single-nucleotide polymorphism, we also confirmed that copy-neutral loss of heterozygosity is frequent in CLL. This event can lead to misinterpretation of TP53 status. Unlike other cancers, CLL displayed a high proportion of patients harboring multiple TP53 variants. Using both in silico analysis and single molecule smart sequencing, we demonstrated the coexistence of distinct subclones harboring mutations on distinct alleles. In summary, our study provides a detailed TP53 mutational architecture in CLL and gives insights into how treatments may shape the genetic landscape of CLL patients.

Figure 1

Workflow diagram of the study.

Figure 2

Validation of the FILO dataset. (a) The frequency of TP53 variants in the FILO dataset is similar to those included in UMD_CLL. The occurrence of each TP53 variant extracted from UMD_TP53 is shown on the y-axis of the graph for three datasets: UMD_CLL: all TP53 variants from CLL patients included in the UMD_TP53 database, excluding FILO data (5173 variants); FILO_NGS/FILO_Sanger: data from the subsets of FILO patients analyzed via NGS or Sanger sequencing, respectively. Single-nucleotide variants (SNV) are more frequent than frameshift mutations (Indel). (b) The majority of TP53 variants in the FILO dataset are either pathogenic (P) or likely pathogenic (LP). TP53 variants in both datasets were classified according to the pathogenicity data included in the UMD_TP53 database (B: benign; LB: likely benign; VUS: variant of uncertain significance). (c) More than 50% of TP53 found in CLL, in both UMD_CLL and the FILO dataset, are certified deleterious TP53 variants. UMD_CLL includes several benign polymorphisms (SNP) that were removed for all subsequent analyses (Supplementary table S4). (d) VAF is similar depending on the frequency of the TP53 variants. The number of reported cases for each TP53 variant is indicated in the x-axis. The y-axis corresponds to the VAF of each TP53 variant. (e) VAF is similar among the 3 classes (P, LP, and VUS) of TP53 variants. The y-axis corresponds to the VAF of each TP53 variant. The Mann–Whitney U test showed that there were no statistical differences across the three groups, P, LP, and VUS.

Figure 3

Variant NM_000546_c.626_627del (NP_000537_p.Arg209LysfsTer6) is a hotspot mutation in CLL. (a) Distribution of frameshift mutations at each codon of the TP53 protein in various types of cancer. Only insertions and deletions are analyzed. The same scale is used for all the analyses to emphasize the CLL hotspot (see Supplementary Figure S5 for more information). AML/MDS: acute myeloid Leukemia and myelodysplastic syndrome. Lung: NSCLC and SCLC. (b) Frequency of NM_000546_c.626_627del (NP_000537_p.Arg209LysfsTer6) in various cancer types included in the UMD_TP53. (c) Potential hairpin structure associated with an inverted repeat in regions 626–628. Two potential mutational events can lead to the same mutation with deletion of either AG or GA depicted by the three arrows. In both the UMD_T53 database and FILO dataset, variant NM_000546_c.626_627del was detected using different methodologies (conventional Sanger sequencing or NGS) precluding any methodological bias.

Figure 4

CLL hotspot mutations in intron 6 splice acceptor signal. (a) Frequency of TP53 mutation in various splice signals included in UMD_TP53 (left panel), UMD_CLL (middle panel) and FILO (right panel). Variants at position NM_000546_c.673-2A are shown in red. (b) Distribution of mutations in the 20 splice signals of the 10 introns of the TP53 gene. (c) Mutational events at position NM_000546_c.673-2 in various types of cancer. UMD_TP53^∗: whole TP53 database without CLL; CRC: colorectal carcinoma; MDS: myelodysplastic syndrome.

Figure 5

Alternative splicing and mutation consequences in TP53 intron 6. (a) In unstressed normal cells, full-length wild-type p53 (NP_000537.3, 393 residues) derives from a splice event occurring between exons 6 and 7 (in blue) of the major RNA transcript (NM_000546) using the major splice acceptor site (in green). Upon specific stress, an alternative splice occurs between a cryptic acceptor splice site (in red) localized in the 3′ region of intron 6 leading to the synthesis of a shorter TP53 isoform (TP53psi). (b) Mutations at position NM_000546_c.673-2 lead to the inactivation of the original acceptor site and the utilization of the cryptic splice acceptor used to generate TP53psi. This consequence has been observed with RNA sequencing analysis in multiple tumors or cell lines bearing variants at position NM_000546_c.672-2A [28, 29]. (c) Putative TP53 protein variants resulting from various events leading to a truncation of TP53. P53psi or putative variants resulting from a mutation at position NM_000546_c.673-2 bear a new carboxy terminus rising from the translation of intron 6 and finishing with the stop codon in the beginning of exon 7, which is translated in a different reading frame compared to wt p53 (highlighted in yellow). The putative protein, NP_000537_p.Arg209LysfsTer6, expressed by the hotspot variant NM_000546_c.626_627del ends in exon 6 with 5 extra amino acids (highlighted in green). NM_000546_c.637C>T, a hotspot variant found in every type of cancer, truncates TP53 at codon 213 (NP_000537_p.Arg213Ter).

Figure 6

CLL patients are polymutated. (a) TP53 cancer types classified according to OncoTree were analyzed for tumors carrying two (DM), three (MM3), or more than three (MM4+) TP53 variants. Lymphoid tumors were split into two subgroups including (CLL+) or excluding (CLL-) CLL patients. (b) Distribution of the number of mutations in tumors from the NGS subset of the FILO dataset. (c) Cumulated VAF from polymutated patients from the NGS subset of the FILO dataset. (d) TP53 variants are distributed on different chromosomes in the tumor of patient AVC-62. Manual examination of the sequence alignment of NGS data was performed for each exon. For 3 pairs of variants and 1 triplet, TP53 variants are in a trans configuration. (e) SMRT sequencing shows that TP53 mutations are on different alleles for patients SW3 and SW6. Standard NGS analysis is shown on the left. No allelic distribution can be inferred from this type of analysis. SMRT sequencing (right) provides an accurate picture of the allelic distribution of each TP53 variant, as well as the remaining wt allele. The frequencies of the different alleles are shown in brackets. Green triangle: TP53 variants identified by both types of analyses. Red triangle: TP53 variants not detected by long-range sequencing (see also Supplementary Figure S10a to S10e for more patients analyzed by SMRT sequencing).

Figure 7

CLL-specific TP53 variants are observed predominantly in low VAF polymutated patients. (a) Frequency of patients with one (SM), two (DM), or more than two (MM) TP53 mutations per tumor. (b–d) Frequency of individual TP53 variants in tumors bearing one (SM), two (DM), or more than two (MM) TP53 variants in the FILO dataset (b), UMD_CLL (c), or the dataset of Catherwood et al. (e, f) VAF distribution for classical TP53 hotspot variants (e) or CLL-specific variants (f) in different datasets.

Box 1

TP53 variants in CLL: unresolved questions and potential research studies.