Single Cell Analysis Revealed a Coexistence of NOTCH1 and TP53 Mutations Within the Same Cancer Cells in Chronic Lymphocytic Leukaemia Patients

The clinical course in chronic lymphocytic leukaemia (CLL) patients is diverse, reflecting the heterogeneous biological background of this disease (Guièze & Wu, 2015). Among reported defects, mutations in NOTCH1 and TP53 genes represent potent CLL progression drivers and contribute to disease chemo-refractoriness (Fabbri et al, 2011; Malcikova et al, 2015). Although parallel occurrence of NOTCH1 mutations and TP53 defects has been noted in CLL patients (Weissmann et al, 2013; Stilgenbauer et al, 2014), the clonal composition of these coexisting aberrations has not been yet studied. To clarify this phenomenon, we examined CLL patients with concurrently detected hotspot NOTCH1 and TP53 mutations using single cell analysis. To determine the simultaneous occurrence of c.7541_7542delCT NOTCH1 mutation and TP53 mutations, we studied a cohort of 282 CLL patients containing 193 untreated patients (Table SI) supplemented by 89 TP53-mutated patients (48 treated). The study design is shown in Fig S1 and molecular and statistical analyses performed are described in the Supplementary material. To disclose the observed mutations’ coexistence, we analysed single flow-sorted CD19+ cells from 10 NOTCH1-TP53-mutated patients using multiplex polymerase chain reaction followed by Sanger sequencing (workflow in Supplementary Methods). In the primary untreated cohort examined, the NOTCH1 mutation was detected in 10% (20/193) of patients, while TP53 mutations were found in 11% (22/193) of patients (Table SI). The concurrent presence of mutations in both genes was noted in 2·6% (5/193) of patients; this frequency was higher than in other CLL cohorts reported (1·2–1·5%) (Weissmann et al, 2013; Baliakas et al, 2015), reflecting the more unfavourable profile of our patients. To further explore the NOTCH1 mutation occurrence in TP53-mutated CLL, we extended the originally analysed cohort by adding 89 patients carrying TP53 mutations. Taking all TP53-mutated cases together, we found the NOTCH1 mutation in 17% (19/111) of TP53-mutated patients (Table SII). Using the data obtained from genomic DNA sequencing, 4 of these NOTCH1-TP53-mutated patients had NOTCH1 and/or TP53 mutations detected in ~100% of the allelic fraction, indicating their coexistence within the same cells. The clonal composition in the remaining NOTCH1-TP53-mutated patients manifesting a lower proportion of mutations was impossible to determine directly. Therefore, we analysed single CD19+ cells in 8 of these patients with available material and remarkably, the cells carrying the NOTCH1 and TP53 mutations simultaneously were found in all of them. By merging genomic DNA sequencing and single cell analysis data in the NOTCH1-TP53-mutated patients with proven coexistence of the mutations, we inferred various clonal composition profiles (Table 1; Fig S2). In the light of previous studies demonstrating the dynamic clonal evolution process in treated CLL (Landau et al, 2015; Ojha et al, 2015), we considered whether the proportion of concurrent NOTCH1 and TP53 mutations might change during the disease course. Therefore, we studied the available longitudinal samples of 12 NOTCH1-TP53-mutated patients; interestingly, clonal evolution of TP53 and/or NOTCH1 mutations after chemo-immunotherapy was proven in 9 of them. In the remaining 3 patients, the proportion of mutations in both NOTCH1 and TP53 genes remained stable irrespective of treatment (detailed description in the Supplementary results). To further explore the observed clonal evolution patterns, we examined retrospective samples of two treated NOTCH1-TP53-mutated patients carrying multiple TP53 mutations using single cell analysis and ultra-deep next generation sequencing of both genes (Fig 1). In one patient (Patient 384; Fig 1A), the simultaneous expansion of two independent clones carrying either the NOTCH1 mutation or TP53 mutation c.833C>G separately was initially noted in relapse after chemo-immunotherapy (time-points 1–4). Subsequently, in relapse after rituximab-dexamethasone treatment (time-point 5), the clone carrying the NOTCH1 mutation underwent further expansion and within this clone, the second TP53 mutation, c.569delC, occurred. The independent clone carrying the TP53 mutation c.833C>G remained stable between these time-points. In the second patient (Patient 1130; Fig 1B), the proportion of pre-existing clones carrying the TP53 mutation c.659A>G was reduced in relapse after an ofatumumab-dexamethasone treatment, while the second independent NOTCH1-mutated clone with simultaneously occurring TP53 mutation c.536A>G expanded (time-points 1–2). However, this NOTCH1-TP53-mutated clone diminished after subsequent alemtuzumab treatment and only the original NOTCH1-wild-type clone carrying the TP53 mutation c.659A>G dominated again in relapse (time-point 3). To study whether the concurrent NOTCH1 mutation might have a clinical impact in TP53-mutated CLL, we assessed overall survival (OS) and time to first treatment (TTFT) in corresponding patients. However, neither OS nor TTFT was significantly different among the analysed patients with respect to the NOTCH1 mutation status (P = 0·8445 and P = 0·1049, respectively). Detailed survival analysis data are shown in Supplementary results and Fig S3. As previously reported, mutations in both TP53 and NOTCH1 genes have been associated with increased risk of Richter's syndrome (RS) with their presence being detected a long time before CLL transformation (Fabbri et al, 2011; Villamor et al, 2013). In our TP53-mutated cohort (median follow-up 74 months), RS developed in 11% (12/111) of patients, 6 of whom had a simultaneous NOTCH1 mutation proven in peripheral blood cells. Interestingly, in longitudinal DNA samples available from 3 of these 6 NOTCH1-TP53-mutated patients, the NOTCH1 mutation was detectable a long time before RS manifestation (80, 72 and 59 months), while the TP53 mutations occurred later and closely preceded transformation (0, 4 and 11 months before RS diagnosis). In the untreated cohort of 171 patients without TP53 mutations (median follow-up 61 months), RS was diagnosed during the CLL course in only one of the 156 NOTCH1-TP53-wild-type patients, and no evidence of disease transformation was proven in 15 patients manifesting only the NOTCH1 mutation. These data indicate that patients carrying solely the NOTCH1 mutation have a lower risk of RS transformation than NOTCH1-TP53-mutated patients. However, we could not evaluate this suggestion due to the small number of RS patients. In summary, we detected the hotspot c.7541_7542delCT NOTCH1 mutation in 17% of TP53-mutated patients and proved that both genes' defects preferentially affect the same CLL cells. In the NOTCH1-TP53-mutated patients we observed a marked clonal heterogeneity and therapy-related clonal evolution and suggested that the simultaneous presence of these mutations might contribute to the development of Richter's syndrome. The authors would like to thank Lenka Jurackova, Jitka Kabathova, Ondrej Letocha and Filip Pardy for help with experimental analysis, Jana Kotaskova and Hana Plesingerova for clinical data processing and Matthew Smith for language editing. The study was supported by research grants IGA MH CR NT/13493/2012 and NT/13519/2012, AZV-MZ-CR 15-31834A, TACR (TE02000058/2014-2019), MSMT under the project CEITEC 2020 (LQ1601) and MH CR - RVO (FNBr, 65269705). SP and JMal were principal investigators with primary responsibility for the paper; BK, JMal, KB, MB, KP, JS, ED, HSF and VN performed experiments for the study; MD, YB and JMay collected patients’ samples; LR and NT provided statistical analyses and analysed NGS data; SP and MT coordinated the research, BK and JMal wrote the manuscript. The authors declare no conflict of interests. Data S1. Supplementary material Table SI. Characterization of 193 untreated CLL patients according to the presence of mutations in NOTCH1 and TP53 genes Table SII. Characterization of 111 TP53-mutated CLL patients according to the NOTCH1 hotspot mutation presence Fig S1. Study design Fig S2. Schematic representation of clonal CLL populations in 12 NOTCH1-TP53-mutated patients analysed Fig S3. Clinical outcome of NOTCH1-mutated patients in the untreated and TP53-mutated cohorts. Impact of the NOTCH1 hotspot mutation on OS (A, n = 181) and TTFT (B, n = 193) in untreated patients; OS (C, n = 121) and TTFT (D, n = 127) in untreated patients with unmutated IGHV genes only; OS (E, n = 106) in TP53-mutated patients; TTFT (F, n = 69) in TP53-mutated patients with TP53 mutation presence detected before treatment initialization. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.