Chronic Lymphocytic Leukemia

NOTCH1 mutations identify a genetic subgroup of chronic lymphocytic leukemia patients with high risk of transformation and poor outcome

Abstract

NOTCH1 has been found recurrently mutated in a subset of patients with chronic lymphocytic leukemia (CLL). To analyze biological features and clinical impact of NOTCH1 mutations in CLL, we sequenced this gene in 565 patients. NOTCH1 mutations, found in 63 patients (11%), were associated with unmutated IGHV, high expression of CD38 and ZAP-70, trisomy 12, advanced stage and elevated lactate dehydrogenase. Sequential analysis in 200 patients demonstrated acquisition of mutation in one case (0.5%) and disappearance after treatment in two. Binet A and B patients with NOTCH1-mutated had a shorter time to treatment. NOTCH1-mutated patients were more frequently refractory to therapy and showed shorter progression-free and overall survival after complete remission. Overall survival was shorter in NOTCH1-mutated patients, although not independently from IGHV. NOTCH1 mutation increased the risk of transformation to diffuse large B-cell lymphoma independently from IGHV, with this being validated in resampling tests of replicability. In summary, NOTCH1 mutational status, that was rarely acquired during the course of the disease, identify a genetic subgroup with high risk of transformation and poor outcome. This recently identified genetic subgroup of CLL patients deserves prospective studies to define their best management.

Introduction

Chronic lymphocytic leukemia (CLL) is characterized by the proliferation and progressive accumulation of mature clonal B lymphocytes in bone marrow, blood and lymphoid tissues. The clinical course of the disease is highly heterogeneous, with patients requiring early treatment for disease progression and others who have an indolent course that does not affect their life expectancy.1, 2 Several characteristics of the disease including the IGHV mutational status, cytogenetics, the expression of several proteins in the leukemic lymphocytes and the response to treatment have been related to the outcome of patients.3

Whole-genome and exome sequencing have started to reveal the complex landscape of somatic mutations in CLL with the identification up to now of around 80 recurrently mutated genes with predicted functional impact.4, 5, 6 The distribution of these mutations in different clinical and biological subgroups of patients suggests that they may be relevant in determining the heterogeneous behavior of the disease. However, the clinical impact of these mutations and their stability during the course of the disease is not well known. Activating mutations of NOTCH1 have emerged as one of the most frequent somatic aberrations in CLL affecting up to 10–15% of patients.4, 6, 7, 8, 9 Virtually all these mutations generate a truncated protein lacking the C-terminal domain, that is more stable and activates the NOTCH1 signaling pathway.4 The presence of NOTCH1 mutations in CLL cells seems to be associated with adverse prognosis features and to confer an adverse prognosis.4, 6, 8, 9

In this study, we have investigated the presence of NOTCH1 mutations in a large series of CLL cases to better define their stability along the evolution of the disease, as well as their relationship with other clinical and biological features of the disease and their clinical impact, particularly their influence in the requirement of and response to therapy and the transformation to diffuse large B-cell lymphoma (DLBCL).

Methods

Patients

A total of 565 patients diagnosed with CLL according to the World Health Organization criteria10 with available DNA from samples before treatment, containing more than 30% of tumor cells, were included in the study. Clinical and biological data at diagnosis, treatment and follow-up were recorded and analyzed. Patients were predominantly males (59%) with a median age at diagnosis of 61 years and most of them in Binet stage A (81%). The distribution of prognostic factors with adverse impact on the evolution of patients was: unmutated IGHV in 46% of the patients analyzed, adverse cytogenetics (del(11)(q22.3) and del(17)(p13.1)) in 14%, high expression of CD38 or ZAP-70 in leukemic lymphocytes in 33% and 34% of patients, respectively. After a median follow-up of 6.2 years (range 0.2–27) for surviving patients, 259 patients remained untreated. The remainder received different therapies along the years, including chlorambucil (n=105), monotherapy with purine analogs (n=36), fludarabine-based polychemotherapy without rituximab (n=51), fludarabine-based polychemotherapy with rituximab (n=68), CHOP-like regimens (n=26) and other therapies (n=20). Actuarial median time to treatment (TTT) was 5.3 years. Response to treatment was evaluated according to the International Workshop on Chronic Lymphocytic Leukemia criteria.11 In patients achieving a complete response (CR), minimal residual disease (MRD) was evaluated by sensitive multiparametric flow cytometry (0.01%),12, 13 according to the International Workshop on Chronic Lymphocytic Leukemia recommendations.11 CR patients in whom no study of MRD was performed were considered as CR MRD-positive in all the analyses. Transformation to DLBCL was diagnosed by cytology in 2 cases and histology in 34 cases. In all, 204 patients died during the follow-up, with a median overall survival (OS) of 12 years.

Informed consent to participate in the study was obtained according to the guidelines of the local Ethic Committees.

Gene amplification and sequencing

DNA and RNA were extracted from mononuclear cells containing more than 30% of tumor cells. The median percent of tumor cells in the samples was 90% (range: 30–100%), with only 24 samples (4%) having less than 50% of CLL cells. PCR for IGHV was carried out according to ERIC guidelines.14, 15 IGHV sequences were aligned using Immunoglobulin database (http://www.imgt.org).

Exon 34 of NOTCH1 was amplified with forward: 5′-IndexTermATGGCTACCTGTCAGACGTG-3′/ reverse: 5′-IndexTermTCTCCTGGGGCAGAATAGTG-3′ and forward: 5′-IndexTermGAGCTTCCTGAGTGGAGAGC-3′/ reverse: 5′-IndexTermCCTGGCTCTCAGAACTTGCT-3′ primers. These amplifications cover the whole PEST domain and most of the TADD domain and include 97% of NOTCH1 mutations previously described in CLL. The sensitivity of the Sanger technique employed for assessment of the allelic representation of NOTCH1 mutational status was 10% (equivalent to 20% tumor cells carrying the mutations in heterozygosis in all tumor cells), as assessed by DNA titration experiments (n=2) by diluting genomic DNA from a heterozygous mutated sample with 99% of tumor cells into normal DNA simulating 50–10% of tumor cells. In these two cases, the allelic representation of NOTCH1 mutations was around 50% as assessed by next-generation sequencing technologies. A clonospecific PCR with a sensitivity of 3% of allelic representation was performed in cases that showed changes in NOTCH1 mutational status (see Supplementary Material and Supplementary Table 1). Exon 5 of MYD88 and exons 14, 15, 16 and 18 of SF3B1 were sequenced as previously described.4, 5 Exons 4 – 9 of TP53 were amplified as previously described by International Agency for Research on Cancer (IARC) Consortium (http://www-p53.iarc.fr). PCR products were purified and sequenced as previously described.5

Statistical analysis

Fisher’s test or non-parametric tests were employed to correlate clinical and biological variables according to NOTCH1 mutational status. The main endpoints were OS, relative survival (adjusted for expected survival in the general population), TTT, progression-free survival (PFS) from CR achievement (considering need of more treatment, transformation to DLBCL or death, whichever occurred first, as events), and incidence of transformation to DLBCL. Survival curves were plotted by the Kaplan and Meier method and compared by the log-rank test. The independent value of NOTCH1 mutations to predict the various time-to-event outcomes was assessed by multivariate Cox regression analysis. Relative survival was calculated by the cohort method described by Dickman et al.16 Estimates of expected survival were calculated by the Ederer II method17 from Spanish life tables stratified by age, sex and calendar year that were obtained from the Human Mortality Database (http://www.mortality.org).

The impact of NOTCH1 on the risk of transformation to DLBCL was evaluated by two different methods. First, a nested case–control study in which 2–4 randomly selected control patients were matched to each case patient by the IGHV mutational status and the duration of follow-up, to guarantee that controls have had the same opportunity as cases with transformation to DLBCL. The association between the NOTCH1 status and the risk of transformation to DLBCL was then assessed by logistic regression, conditional on the set of matched cases and controls. Second, we analyzed the influence of NOTCH1 on the cumulative incidence of transformation to DLBCL by taking death as a competing risk. The multivariate adjustment for other factors predicting the transformation to DLBCL was performed within the framework of competing risks by the method of Fine and Gray.18 The replicability of the nested case–control study and the competing-risks model were assessed by bootstrap resampling. For this purpose, control case selection for nested case–control analysis of DLBCL transformation was done by constraining the matching criteria of dates of diagnosis and last follow-up of the controls for a given case that anteceded the diagnosis of CLL and postdated DLBCL transformation, respectively, of that given case. The replicability of the nested case–control study and the competing-risks model analyzing the effect of NOTCH1 on the risk of DLBCL transformation were assessed by bootstrap resampling. A total of 1000 samples, the same size as the original series, were built through random extraction with reposition, so that in each sample a given patient may either not be represented at all or represented once, twice or more times. The parameters assessed by resampling were the P-values of either the subhazard ratios of the Fine and Gray’s regression or the odds ratios of the conditional logistic regression. Bootstrap resampling allows verifying that the predictive value of NOTCH1 status was not critically dependent on the particular composition of the present series.

All statistical tests were two-sided and the level of statistical significance was 0.05. All the analyses were conducted with the use of the Stata 11 software (http://www.stata.com) and the SPSS 19 software (http://www.ibm.com). For relative survival analysis, the Stata routines developed by Paul Dickman (Karolinska Institutet, Stockholm, Sweden; available at http://www.pauldickman.com) were used.

Results

Frequency of NOTCH1 mutations and sequential analysis

Of the 565 patients, 63 (11%) carried somatic mutations of NOTCH1. Of them, 54 (86%) had the dinucleotide deletion p.P2514Rfs*4, 3 patients had a p.L2482Ffs*2, 2 patients had a p.Q2394* and 1 patient each p.Q2444*, p.Q2404*, p.Q2503* and p.P2437fs*36.

To determine the stability of the NOTCH1 mutational status during the clinical evolution of the CLL, we assessed NOTCH1 mutations in two sequential samples of 200 patients with a median interval of 3.5 years (0.2–21.6 years). The disease status at the time of both samples, interval between samples and changes in NOTCH1 are described in Table 1. A change in NOTCH1 was observed in 3 of 200 patients (1.5%). Two patients with p.P2514fs*4 at diagnosis had a wild-type NOTCH1 4 and 7 years later after having received two lines of treatment (chlorambucil and fludarabine-containing therapy in one, CHOP-like chemotherapy and an autologous stem cell transplantation in the other). Another patient with unmutated NOTCH1 at diagnosis acquired a NOTCH1 mutation (p.Q2501fs*6) after 9.5 years of stable disease. In summary, only 0.5% of patients acquired a mutation in NOTCH1 during the follow-up, suggesting that in most patients the status of NOTCH1 is stable throughout the course of the disease. To better assess the changes in NOTCH1 mutational status, a more sensitive clonospecific PCR was used. Cells carrying NOTCH1 mutation were detected in samples from these three patients that were negative by Sanger.

Table 1 Sequential analysis of NOTCH1 mutations in patients with CLL

NOTCH1 mutations are associated with adverse biological and clinical features

The main biological features of the patients according to the NOTCH1 mutational status are listed in Table 2. NOTCH1-mutated CLL patients showed more frequently unmutated IGHV, as well as elevated expression of CD38 and ZAP-70. In addition, they had more frequently trisomy 12, and less frequently del(13q)(q14.3) than cases with NOTCH1-unmutated. No case carried simultaneous mutation of NOTCH1 and MYD88.

Table 2 Main biological features of 565 patients with CLL according to the NOTCH1 mutational status

NOTCH1-mutated patients had more frequently advanced Binet and Rai stages, elevated serum lactate dehydrogenase and elevated beta-2-microglobulin than unmutated patients (Table 3).

Table 3 Main clinical characteristics and outcome of the 565 patients with CLL according to the NOTCH1 mutation

TTT, response and outcome according to NOTCH1 mutational status

A total of 259 patients have not required therapy during the follow-up. This proportion was lower in patients with NOTCH1-mutated than in NOTCH1-unmutated cases (Table 3). Patients in Binet stage A and B with NOTCH1-mutated showed shorter TTT than NOTCH1-unmutated patients (median TTT 1.8 vs 6.0 years; P<0.001) (Figure 1). Of note, 17 of 63 (27%) NOTCH1-mutated patients have never received treatment after a median follow-up of 2.5 years (range: 0.6–16.5 years). The multivariate analysis of TTT, including Binet stage (A vs B), NOTCH1 and IGHV mutational status, showed that only Binet stage and IGHV mutational status were independent in predicting need of treatment.

Figure 1
figure1

TTT in Binet stage A and B CLL patients according to NOTCH1-mutated (solid line) and NOTCH1-unmutated (dashed line) (P<0·001). The 95% confidence interval for each group of patients is depicted.

The status of NOTCH1 was similar among groups of patients receiving different types of treatment. The response to therapy is listed in Table 3. Refractoriness to treatment was significantly more frequent among NOTCH1-mutated than in unmutated patients. Moreover, MRD-negative CR rates were lower in NOTCH1-mutated patients.

In all, 54 of the 115 patients achieving a CR eventually required further treatment, and 6 died without further therapy. PFS from CR achievement was shorter in NOTCH1-mutated than in unmutated patients (Table 3, Figure 2a). Unmutated IGHV and MRD-positive CR also predicted shorter PFS from CR. In a multivariate analysis, the three variables, namely NOTCH1-mutated (P=0.02; hazard ratio (HR)= 2.4), IGHV unmutated (P=0.001, HR=4.0) and MRD-positive CR (P=0.003, HR= 2.6) maintained independent value to predict failure, in the Cox model with 105 patients. In addition, the survival from CR achievement was significantly shorter in NOTCH1-mutated patients (median survival from CR: 4.9 vs 8.7 years, respectively; P=0.003; Figure 2b). The prognostic value for OS from CR was not independent from IGHV and MRD status. Similar results were observed when the analysis was restricted to patients treated with fludarabine-containing regimens (data not shown).

Figure 2
figure2

Outcome from CR achievement. (a) PFS from CR achievement according to NOTCH1-mutated (solid line) and NOTCH1-unmutated (dashed line) (P<0·001). (b) Survival from CR of NOTCH1-mutated CLL patients (solid line) and NOTCH1-unmutated CLL patients (dashed line) (P=0·003).

After a median follow-up of 6.2 years, 204 patients have died. The main variables associated with poor OS were advanced clinical stage, unmutated IGHV, high expression of CD38 and ZAP-70, adverse cytogenetics, elevated serum lactate dehydrogenase, high beta-2-microglobulin and short lymphocyte-doubling time (P<0.001 in all comparisons). Patients with NOTCH1-mutated CLL showed shorter OS when compared with unmutated patients (10-year OS: 35% vs 64%; P<0.001) (Table 3, Figure 3). NOTCH1 p2514fs*4 mutation and the other NOTCH1 mutations had similar impact on OS (Supplementary Figure 1). Multivariate analysis identified, in a model with 404 cases, the following unfavorable variables to predict OS: age (HR=1.04; P<0.001), advanced Binet stage (HR=1.7; P=0.002), high beta-2-microglobulin (HR=2.2; P<0.001) and unmutated IGHV (HR=4.2; P<0.001). NOTCH1 mutations did not reach independent prognostic value for OS.

Figure 3
figure3

OS in CLL patients according to NOTCH1-mutated (solid line) and NOTCH1-unmutated (dashed line) (P<0·001). The 95% confidence interval for each group of patients is depicted.

To analyze the impact of NOTCH1 mutations on the life expectancy of CLL patients, relative OS adjusted by general population mortality rates was calculated. As shown in Figure 4, the life expectancy of patients with NOTCH1-mutated CLL was significantly lower than that of the general population being around 50% after 10 years.

Figure 4
figure4

Relative survival of CLL patients with NOTCH1-mutated (solid line) and NOTCH1-unmutated (dashed line) CLL. The 95% interval of confidence for each cohort is plotted.

NOTCH1 mutations increase the risk of transformation to DLBCL

A total of 36 patients developed transformation to DLBCL. At 10 years from diagnosis, when 143 patients were still at risk, the cumulative incidences of transformation or death without transformation were 8% and 33%, respectively. The influence of NOTCH1 mutations on the incidence of transformation to DLBCL was investigated after adjustment for other variables associated with transformation, including high expression of CD38, trisomy 12, absence of del(13q), previous exposure to purine nucleoside analogs or anthracyclines and unmutated IGHV. Only NOTCH1-mutated (HR=5.2; P<0.001) and IGHV-unmutated (HR=3.6; P=0.006) were independently associated with a higher risk of DLBCL (n=469). At the resampling test of replicability, NOTCH1-mutated was selected as an independent predictor of transformation to DLBCL in 63% of the 1000 bootstrap samples, whereas unmutated IGHV was selected in 13%. Figure 5 shows the cumulative incidence of transformation to DLBCL according to NOTCH1 status. At 10 years from diagnosis, the cumulative incidence of transformation was 6% and 31% for NOTCH1-unmutated and NOTCH1-mutated patients, respectively.

Figure 5
figure5

Cumulative incidence of transformation to DLBCL in NOTCH1-unmutated CLL patients (a) and NOTCH1-mutated CLL patients (b) (P<0·001).

The case–control study included the 36 case patients who evolved into DLBCL and 168 who did not and were matched to the cases by the IGHV mutational status and length of follow-up. Median follow-up to diagnosis of DLBCL in case patients or to death or last follow-up in control patients was 4.5 years (range, 0.02–23) and 9.8 years (range, 0.4-39), respectively. At the conditional logistic regression, harboring NOTCH1 mutations was strongly associated with progression to DLBCL (HR: 8.0, % CI: 3.2–20, P<0.001). At the resampling test of replicability, the association between mutated NOTCH1 and progression to DLBCL was statistically significant in 79% of the 1000 bootstrap samples.

In 15 patients, NOTCH1 and TP53 were analyzed at transformation: 6 cases had mutation in NOTCH1 (40%), 2 in TP53, 5 in both genes and 2 had no mutations. In 8 cases, the status of NOTCH1 was available in samples before transformation and it was identical than at transformation (5 unmutated and 3 mutated). Regarding TP53, two patients of eight analyzed acquired the mutation at transformation (both being NOTCH1-mutated). In addition, at time of transformation simultaneous samples of DLBCL and non-transformed peripheral blood CLL were available in six patients. In all cases, NOTCH1 configuration was identical in both samples (four NOTCH1-mutated and two NOTCH1-unmutated).

Discussion

The use of whole-genome and exome sequencing has revealed the presence of recurrent somatic mutations in CLL with specific gene mutations clustering in one of the two major subgroups of CLL according to the IGHV mutational status.4, 5, 19, 20 Among the most frequently mutated genes, we and others have found NOTCH1 and SF3B1 mutations in up to 10–15% of CLL samples. Preliminary data suggested an unfavorable prognostic impact of NOTCH1 mutation.4, 6, 8, 9, 20 Thus, to gain insight into the impact of NOTCH1 mutations in CLL, we have extended our initial series and we have analyzed in depth the role of NOTCH1 mutation in the outcome of CLL patients with particular emphasis in the evaluation of the response to treatment and transformation to DLBCL.

Whether NOTCH1 mutational status remains stable or, on the contrary, it changes over time in the evolution of the disease is an important issue not well established at present. There is evidence that patients at progression and relapse have more frequently NOTCH1 mutations,6, 8, 20 as we also observed in our series (Table 1). This finding is not unexpected since NOTCH1-mutated patients have a higher risk of progression. Fabbri et al.6 observed that in 5 of 16 patients with Richter syndrome harboring NOTCH1 mutation, this alteration was not present in the CLL at diagnosis. However, the mutational status of NOTCH1 over time in the evolution of CLL before transformation has not been investigated. Herein we report that, in a large series of 200 patients analyzed by Sanger changes in NOTCH1 status were observed only in 3 patients, and more importantly, only 1 patient acquired the mutation with no evidence of CLL progression. The disappearance of NOTCH1 mutation after treatment has been previously reported in one patient.21 The use of the more sensitive clonospecific PCR demonstrated low levels of cells with NOTCH1 mutation in the three cases. The identification of these small subclones carrying the mutation may reflect the complex fluctuation of different tumor subclones in the evolution of the disease. A recent study using next-generation sequencing in three CLL patients22 has highlighted the heterogeneous patterns of subclonal evolution of the disease with many subclones present at very low frequencies evolving over the years. The clinical impact of these subclones carrying mutation at low levels will require further specific studies. Moreover, no differences in NOTCH1 status were found when comparing samples at CLL and at transformation with DLBCL. These results suggest that acquisition of NOTCH1 mutation during the evolution of the disease, although possible, is an uncommon phenomenon. Further studies should clarify the relevance of the modulation of clones carrying NOTCH1 mutations and whether clones acquiring such mutation may emerge during the follow-up.

In our non-selected CLL series, we have confirmed that NOTCH1 mutations are present in 11% of cases. This proportion is similar to that found in a recent study of a similar large series of patients.6, 8, 20 NOTCH1 mutations were associated with unmutated IGHV, and high expression of CD38 and ZAP-70, as well as more frequently trisomy 12.8, 19, 23, 24 In our study, we have closely analyzed the impact of NOTCH1 mutations on the requirement and response to treatment. Interestingly, patients with NOTCH1 mutations required therapy more frequently and earlier than patients with unmutated NOTCH1. Moreover, patients with NOTCH1 mutation showed poorer response to treatment, and shorter PFS and OS from CR achievement. Of note, NOTCH1-mutated patients who achieved CR after front-line therapy had poor outcome with 50% of them requiring further therapy within 2 years. These patients, particularly if young and fit, would be candidates to intensive or investigational treatments. However, more information is warranted from prospective clinical trials to define the real impact of NOTCH1 mutations in CLL patients.

Overall, patients with NOTCH1-mutated CLL had poor outcome in terms of OS, which is in agreement with previous reports.4, 8 Rossi et al.8 have recently observed that the impact of NOTCH1 mutations in OS is independent from IGHV. However, we could not confirm this finding in our study, in accordance with a previous smaller series.20 The small group of patients with NOTCH1-mutated IGHV-mutated in our series behaved as low-risk CLL, in sharp contrast with the poor prognosis observed by Rossi et al.8

Transformation to DLBCL is an evolving event of CLL that occurs in 0.5–1% of the patients per year. The development of DLBCL, that confers an ominous prognosis to patients, has been associated with unmutated and stereotyped immunoglobulin genes, trisomy 12, del(11)(q22.3), mutations of TP53 and CDKN2A, among others.25, 26, 27, 28 Expanding our initial previous observation,4 the results of the current study demonstrate that NOTCH1 mutation is one of the most important predictors of DLBCL development, with more than 30% of those patients having developed DLBCL at 10 years of diagnosis. Rossi et al.8, 29 have reported a similar risk of transformation in NOTCH1-mutated patients. In addition, we have observed that the higher risk of DLBCL conferred by NOTCH1 mutation is independent from the IGHV status. Acquisition of NOTCH1 mutation has been observed in some patients at transformation to DLBCL.6 None of our patients with sequential or simultaneous sample from non-transformed and transformed tissue had differences in NOTCH1. The presence of subclones with NOTCH1 mutation6 together with technical reasons could account for the discrepancies.

The molecular mechanisms by which NOTCH1 mutation confers higher risk of transformation and bad response to treatment are unknown. In our previous study, we showed that the truncated NOTCH1 protein encoded by mutated NOTCH1 is more stable, accumulates in the cell and activates the downstream NOTCH1 signaling pathway.4 NOTCH1 activation induces several cellular functions, including the activation of PI3K/Akt, MYC and NFkB signaling pathways, that promote cell proliferation, survival and angiogenesis, which may be important for the aggressive behavior and frequent transformation of CLL cells.30, 31, 32, 33, 34, 35 The relevance of NOTCH1 mutation in the CLL biology opens the possibility of designing specific new therapeutic strategies for these patients.36, 37, 38

In summary, we have shown that NOTCH1 mutation is a genetic marker that defines a high-risk group of CLL patients characterized by high risk of transformation and poor outcome. Although this newly identified subgroup of patients only represents a 10% of the whole CLL population, their increase risk in developing DLCBL and dismal prognosis deserves specific investigation in prospective clinical trials.

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Acknowledgements

We are grateful to Sara Guijarro, Silvia Martín, Cristina Capdevila, Montse Sánchez and Laura Plà for excellent technical assistance and Nathalie Villahoz and Carmen Muro for excellent work in the coordination of the CLL Spanish Consortium. We are indebted to the HCB-IDIBAPS Biobank-Tumor Bank and Hematopathology Collection for the sample procurement. We are also very grateful to all patients with CLL who have participated in this study. This study was supported by research funding from Spanish Ministry of Science and Innovation (MICINN) through the Instituto de Salud Carlos III (ISCIII) and Red Temática de Investigación del Cáncer (RTICC) (RD06-0020-0039 to E Campo and RD06-0020-0051 to AL-G), Fondo de investigaciones sanitarias (PI07/0301, MA) and Botín Foundation (CL-O).

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Correspondence to N Villamor.

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The authors declare no conflicts of interest.

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Author Contributions

LC, MC, AN, MP, VQ, XSP and CL-O performed sequencing analysis. MR, NV, MG-D, JMH, BN, E Colado and E Campo reviewed the pathological data and confirmed the diagnosis. DC, CL, SB carried out genetic and biological studies. AM-T, TB, JD, EG, PA, CR, ARP, MJT, FB and AL-G reviewed clinical data. MA prepared and supervised the bioethics requirements. AP contributed to and critically reviewed statistical analysis. E Campo and AL-G directed the research. NV, E Campo and AL-G wrote the manuscript, which all the authors approved.

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Villamor, N., Conde, L., Martínez-Trillos, A. et al. NOTCH1 mutations identify a genetic subgroup of chronic lymphocytic leukemia patients with high risk of transformation and poor outcome. Leukemia 27, 1100–1106 (2013). https://doi.org/10.1038/leu.2012.357

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Keywords

  • NOTCH1
  • chronic lymphocytic leukemia
  • DLBCL transformation

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