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Letters to the Editor
Treatment with ibrutinib does not induce a TP53 clonal evolution in chronic lymphocytic leukemia
Ibrutinib is active both in treatment-naïve (TN) and relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL) patients, including those with unmutated immunoglobulin heavy chain variable region (IGHV) genes and TP53 disruption.1-3 The acquisition of BTK or PLCg2 gene mutations conferring resistance, supports the existence of clonal evolution also under ibrutinib treat- ment.4-8 Whilst it is well described that subclonal TP53 mutations undergo a positive clonal selection following chemoimmunotherapy (CIT),9-11 being the main driver of treatment failure, recent studies have suggested that this might not be the case under ibrutinib.7,12-14
In order to investigate the dynamics of major and minor TP53 mutations under ibrutinib treatment, we per- formed longitudinal TP53 monitoring by deep-sequenc- ing in CLL-treated patients. Two cohorts were included: 44 TN and 14 R/R patients. A total of 216 peripheral blood (PB) samples in TN and 52 in R/R were collected at baseline and at subsequent time points during therapy. Among TN patients, 28 were males and 16 females, with a median age of 72 years (range, 54-87); they received ibrutinib plus rituximab (GIMEMA trial LLC1114), with a median ibrutinib exposure of 2.7 years (range, 1.2-3.7) and were evaluated at 6-month intervals for a median number of 5 time points (range, 3-8). R/R patients, nine males and five females, with a median age of 71 years (range, 55-80), received ibrutinib single agent after a median of 1.5 (range, 1-4) CIT lines; they were evaluated at disease progression (PD) before each line of CIT and after ibrutinib treatment (median: 4 time points; range, 2- 6), with 2.5 years ibrutinib exposure (range, 2.1-3.3). Amplicon libraries, covering the entire coding region and splice sites of TP53 gene (exon 2 to 11), were prepared according to the TruSeq Custom Amplicon Low Input Library Prep kit protocol dual strand (Illumina, San Diego, CA, USA) and paired-end sequenced on a Miseq Sequencer (Illumina). A mean coverage of 8,956x was obtained; across the target region a coverage >5,000x was obtained in >80% of the sequence in 70% of the samples. Bioinformatic analysis was performed by MiSeq Reporter (Illumina) and an in-house bioinformatics pipeline. A total concordance was observed for the vari- ants with variant allele frequency (VAF) ≥3%; variants with 1%≤VAF<3%, only identified from in-house pipeline, were manually checked on alignment files resulting from MiSeq Reporter analysis of two DNA strands, using Integrative Genomics Viewer. Variants were manually curated according to the International Agency for Research on Cancer TP53 database (http://p53.iarc.fr/). Validate polymorphism, synonymous
variants and variants mapping >2 bp outside coding exons were filtered out.
Mutations were defined major if VAF was >10% and minor if ≤10%; the latter were confirmed in an independ- ent deep-sequencing run. VAF was corrected to cancer cell fraction (CCF) by the proportion of CD19+/CD5+ cells in each sample, assessed by flow cytometry. The limit of detection (LOD) was 1% (Online Supplementary Figure S1). A mutation was considered stable when the log2 fold-change (Log2FC) of CCF values before and after ibru- tinib treatment was included between -0.5 and 0.5, decreasing or increasing if the Log2FC was <-0.5 or >0.5, respectively.
Among the 44 TN patients at baseline (T0), 27 cases (61%) resulted TP53 wild-type (WT) and 17 (39%) mutated by deep-sequencing analysis. Nine of 43 (21%) carried the del(17p) and 30 (68%) showed unmutated IGHV.
Twenty-three TP53 mutations (1.4 mutation/patient; range, 1-5) were identified: 17 (74%) were major (mean VAF 58.8%; range, 18-94.8) and six (26%) minor (mean VAF 5.3%; range, 2.1-9.2). Thirteen patients carried a sole major TP53 mutation; two cases (cases #10025, #10875) showed co-existing major and minor mutations; two cases (#9915, #10671) showed one minor mutation each and 27 none (Online Supplementary Table S1).
According to the CCF, after 2.7 years from ibrutinib treatment, nine of 23 (39%) major mutations decreased, eight of 23 (35%) major mutations persisted stable, four of 23 (17%) (1 major and 3 minor) were undetectable and two of 23 (9%) minor mutations increased (Figure 1A and B; Table 1; Online Supplementary Table S1). Novel TP53 mutations emerged during treatment neither in TP53 mutated nor WT patients.
Thus, TP53 mutated TN patients followed two main patterns: i) major TP53 mutations persisting major from T0 with a stable CCF (6 patients); ii) major TP53 muta- tions persisting major from T0 with a decreasing CCF (7 patients). In addition, in one patient (#10671) a minor TP53 mutation at T0 showed an increasing CCF, becom- ing major and in another patient a minor TP53 mutation resulted undetectable; in the two cases with a complex mutational architecture, TP53 mutations dynamics is shown in Figure 1C. No TP53 mutation was detected in 27 patients over time. Most patients are still on therapy, including those who showed an increase of TP53 muta- tion CCF (#10671, #10875) at month +20 (T20) who are still on therapy at T36 and T30, respectively. Two discon- tinued due to PD (#9795, #10239).
Among TN patients, seven TP53 mutated and 11 WT with a measurable PB residual disease (CD19+/CD5+ >20%) at T20 (n=3), T26 (n=2), T32 (n=7), T38 (n=4) and T44 (n=2), were analyzed by next-generation sequencing for BTK and PLCg2 mutations and resulted WT for both
Table 1. Dynamics of TP53 mutations in treatment-naïve under ibrutinib and relapsed/refractory patients under ibrutinib and chemoim- munotherapy.
TN patients
R/R patients, ibrutinib phase R/R patients, CIT phase*
Undetectable Decreasing
4/23 9/23 (17.5%) (39%) 11/31 8/31
(35%) (26%) 0/29 3/29
(10%)
Stable Increasing
8/23 2/23 (35%) (9%) 8/31 4/31 (26%) (13%) 6/29 7/29 (21%) (24%)
Novel
0
2
13/29 (45%)
*13 evaluable cases.TN: treatment-naïve; R/R: relapsed/refractory; CIT: chemoimmunotherapy.
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