Clinical impact of small TP53 mutated subclones in chronic lymphocytic leukemia

Abstract

TP53 mutations are strong predictors of poor survival and refractoriness in chronic lymphocytic leukemia (CLL) and have direct implications for disease management. Clinical information on TP53 mutations is limited to lesions represented in >20% leukemic cells. Here, we tested the clinical impact and prediction of chemorefractoriness of very small TP53 mutated subclones. The TP53 gene underwent ultra-deep-next generation sequencing (NGS) in 309 newly diagnosed CLL. A robust bioinformatic algorithm was established for the highly sensitive detection of few TP53 mutated cells (down to 3 out of ∼1000 wild-type cells). Minor subclones were validated by independent approaches. Ultra-deep-NGS identified small TP53 mutated subclones in 28/309 (9%) untreated CLL that, due to their very low abundance (median allele frequency: 2.1%), were missed by Sanger sequencing. Patients harboring small TP53 mutated subclones showed the same clinical phenotype and poor survival (hazard ratio = 2.01; P = .0250) as those of patients carrying clonal TP53 lesions. By longitudinal analysis, small TP53 mutated subclones identified before treatment became the predominant population at the time of CLL relapse and anticipated the development of chemorefractoriness. This study provides a proof-of-principle that very minor leukemia subclones detected at diagnosis are an important driver of the subsequent disease course.

Figure 1

Molecular profile of subclonal TP53 mutations. (A) Allele frequency of the 85 TP53 mutations identified by ultra-deep-NGS. Mutations are ordered according to their allelic abundance. Mutations that tested positive (clonal mutations: gray bars) and negative (subclonal mutations: red bars) by Sanger sequencing are indicated. (B) Prevalence of TP53 lesions according to their clonal representation in the study cohort of 309 newly diagnosed CLL (for each category, the crude number of patients is represented). (C) Comparison of the molecular profile of subclonal mutations from the CLL study cohort (n = 50) vs clonal mutations from public CLL databases (n = 257; see Zenz et al). p, P values by Fisher's exact test corrected for multiple hypothesis testing. (D) Comparison of the transition/transversion profile between subclonal TP53 substitutions from the CLL study cohort (n = 48) and clonal TP53 substitutions from public CLL databases (n = 210; see Zenz et al). p, P values by Fisher's exact test corrected for multiple hypothesis testing. (E) Schematic diagram of the TP53 protein with its conserved functional domains. Color-coded shapes indicate the position of subclonal TP53 mutations from the CLL study cohort (n = 50; red shapes) and clonal TP53 mutations from public CLL databases (n = 257; gray shapes; see Zenz et al). Hot spot codons recurrently affected by both subclonal and clonal TP53 mutations are highlighted. (F) Residual CDKN1A transactivation capacity of subclonal TP53 missense substitutions from the CLL study cohort (n = 39; red box) vs clonal TP53 missense substitutions from public CLL databases (n = 193; gray box; see Zenz et al). The band inside the box is the median value. The bottom and top of the box are the 25th and 75th quartiles. The ends of the whiskers are the second percentile and the 98th percentile. p, P value by Mann-Whitney test.

Figure 2

Experimental validation of subclonal TP53 mutations identified by ultra-deep-NGS. (A) Representation of the variant frequency of 2 exemplificative subclonal TP53 mutations (c.743G>A p.R248Q and c.673-2A>T) of very low allelic abundance (<0.5%). The first bar of the graphs shows the variant allele frequency in the discovery ultra-deep-NGS experiment. The second and third bars show the variant allele frequency in independent ultra-deep-NGS validation experiments. The number of mutated read outs of the total number of reads covering the variant position is reported. (B) Conventional agarose-gel electrophoresis of the AS-PCR products. Patient 10642, harboring the subclonal TP53 c.743G>A p.R248Q missense substitution (left), and patient 7561, harboring the subclonal TP53 c.673-2A>T splice site mutation (right), are represented. After AS-PCR for the mutant allele, a mutation-specific band is amplified from the patient sample and from the mutated plasmid DNA (positive control). No bands are amplified from the wild-type plasmid DNA and the wild-type genomic DNA from a healthy donor (negative controls), thus confirming the specificity of the assay. (C) Due to their low clonal abundance (<0.5%), the subclonal TP53 c.743G>A p.R248Q missense substitution (left) and the subclonal TP53 c.673-2A>T splice site mutation (right) are not detectable by conventional Sanger sequencing in patient 10 642 and patient 7561, respectively. Asterisks point to the positions of the subclonal variants.

Figure 3

Kaplan-Meier estimates of OS of patients harboring small TP53 mutated subclones. (A) Comparison of OS from CLL diagnosis between patients harboring solely subclonal TP53 mutations, cases harboring clonal TP53 mutations, and cases harboring an unmutated TP53 gene. (B) Comparison of OS from CLL diagnosis between patients harboring solely subclonal TP53 mutations, cases harboring solely clonal TP53 lesions (ie, mutations or deletions), cases harboring clonal TP53 lesions coexisting with subclonal TP53 mutations, and cases harboring a wild-type TP53 gene. (C) Comparison of OS from first treatment between patients harboring solely subclonal TP53 mutations, cases harboring clonal TP53 mutations, and cases harboring an unmutated TP53 gene. p, P values by log-rank test.

Figure 4

Longitudinal analysis of clonal evolution in CLL patients harboring small TP53 mutated subclones. Graphical illustration of the kinetics of the TP53 mutated populations in 4 representative CLL patients who required treatment at diagnosis and who have been longitudinally investigated by deep-NGS. The x-axis represents time and the y-axis represents allele frequency. TP53 mutations and 17p13 deletion are represented by color-coded circles. The size of the circles is proportional to the allele frequency of the lesion. Arrows indicate the time point at which tumor samples were collected. The relationship between sample collection and treatments is also indicated. BR, bendamustine, rituximab; CLB, chlorambucil; CR, complete response according to the IWCLL-NCI criteria; FCM, fludarabine, cyclophosphamide, mitoxantrone; FCR, fludarabine, cyclophosphamide, rituximab; PD, progressive disease according to the IWCLL-NCI criteria; PR, partial response according to IWCLL-NCI criteria; RDHAP, rituximab, dexamethasone, high-dose cytarabine, cisplatin; Richter, Richter syndrome.