Background:In B-cell precursor acute lymphoblastic leukaemia (B-ALL), the identification of additional genetic alterations associated with poor prognosis is still of importance. We determined the frequency and prognostic impact of somatic mutations in children and adult cases with B-ALL treated with Spanish PETHEMA and SEHOP protocols.Methods:Mutational status of hotspot regions of TP53, JAK2, PAX5, LEF1, CRLF2 and IL7R genes was determined by next-generation deep sequencing in 340 B-ALL patients (211 children and 129 adults). The associations between mutation status and clinicopathological features at the time of diagnosis, treatment outcome and survival were assessed. Univariate and multivariate survival analyses were performed to identify independent prognostic factors associated with overall survival (OS), event-free survival (EFS) and relapse rate (RR).Results:A mutation rate of 12.4% was identified. The frequency of adult mutations was higher (20.2% vs 7.6%, P=0.001). TP53 was the most frequently mutated gene (4.1%), followed by JAK2 (3.8%), CRLF2 (2.9%), PAX5 (2.4%), LEF1 (0.6%) and IL7R (0.3%). All mutations were observed in B-ALL without ETV6-RUNX1 (P=0.047) or BCR-ABL1 fusions (P<0.0001). In children, TP53mut was associated with lower OS (5-year OS: 50% vs 86%, P=0.002) and EFS rates (5-year EFS: 50% vs 78.3%, P=0.009) and higher RR (5-year RR: 33.3% vs 18.6% P=0.037), and was independently associated with higher RR (hazard ratio (HR)=4.5; P=0.04). In adults, TP53mut was associated with a lower OS (5-year OS: 0% vs 43.3%, P=0.019) and a higher RR (5-year RR: 100% vs 61.4%, P=0.029), whereas JAK2mut was associated with a lower EFS (5-year EFS: 0% vs 30.6%, P=0.035) and a higher RR (5-year RR: 100% vs 60.4%, P=0.002). TP53mut was an independent risk factor for shorter OS (HR=2.3; P=0.035) and, together with JAK2mut, also were independent markers of poor prognosis for RR (TP53mut: HR=5.9; P=0.027 and JAK2mut: HR=5.6; P=0.036).Conclusions:TP53mut and JAK2mut are potential biomarkers associated with poor prognosis in B-ALL patients.

BackgroundChronic lymphocytic leukemia (CLL) is a highly genetically heterogeneous disease. Although CLL has been traditionally considered as a mature B cell leukemia, few independent studies have shown that the genetic alterations may appear in CD34+ hematopoietic progenitors. However, the presence of both chromosomal aberrations and gene mutations in CD34+ cells from the same patients has not been explored.MethodsAmplicon-based deep next-generation sequencing (NGS) studies were carried out in magnetically activated-cell-sorting separated CD19+ mature B lymphocytes and CD34+ hematopoietic progenitors (n = 56) to study the mutational status of TP53, NOTCH1, SF3B1, FBXW7, MYD88, and XPO1 genes. In addition, ultra-deep NGS was performed in a subset of seven patients to determine the presence of mutations in flow-sorted CD34+CD19− early hematopoietic progenitors. Fluorescence in situ hybridization (FISH) studies were performed in the CD34+ cells from nine patients of the cohort to examine the presence of cytogenetic abnormalities.ResultsNGS studies revealed a total of 28 mutations in 24 CLL patients. Interestingly, 15 of them also showed the same mutations in their corresponding whole population of CD34+ progenitors. The majority of NOTCH1 (7/9) and XPO1 (4/4) mutations presented a similar mutational burden in both cell fractions; by contrast, mutations of TP53 (2/2), FBXW7 (2/2), and SF3B1 (3/4) showed lower mutational allele frequencies, or even none, in the CD34+ cells compared with the CD19+ population. Ultra-deep NGS confirmed the presence of FBXW7, MYD88, NOTCH1, and XPO1 mutations in the subpopulation of CD34+CD19− early hematopoietic progenitors (6/7). Furthermore, FISH studies showed the presence of 11q and 13q deletions (2/2 and 3/5, respectively) in CD34+ progenitors but the absence of IGH cytogenetic alterations (0/2) in the CD34+ cells. Combining all the results from NGS and FISH, a model of the appearance and expansion of genetic alterations in CLL was derived, suggesting that most of the genetic events appear on the hematopoietic progenitors, although these mutations could induce the beginning of tumoral cell expansion at different stage of B cell differentiation.ConclusionsOur study showed the presence of both gene mutations and chromosomal abnormalities in early hematopoietic progenitor cells from CLL patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-017-0450-y) contains supplementary material, which is available to authorized users.

The deletion of 11q (del(11q)) invariably comprises ATM gene in chronic lymphocytic leukemia (CLL). Concomitant mutations in this gene in the remaining allele have been identified in 1/3 of CLL cases harboring del(11q), being the biallelic loss of ATM associated with adverse prognosis. Although the introduction of targeted BCR inhibition has significantly favored the outcomes of del(11q) patients, responses of patients harboring ATM functional loss through biallelic inactivation are unexplored, and the development of resistances to targeted therapies have been increasingly reported, urging the need to explore novel therapeutic approaches. Here, we generated isogenic CLL cell lines harboring del(11q) and ATM mutations through CRISPR/Cas9-based gene-editing. With these models, we uncovered a novel therapeutic vulnerability of del(11q)/ ATM-mutated cells to dual BCR and PARP inhibition. Ex vivo studies in the presence of stromal stimulation on 38 CLL primary samples confirmed a synergistic action of the combination of olaparib and ibrutinib in del(11q)/ATM-mutated CLL patients. In addition, we showed that ibrutinib produced a homologous recombination repair impairment through RAD51 dysregulation, finding a synergistic link of both drugs in the DNA damage repair pathway. Our data provide a preclinical rationale for the use of this combination in CLL patients with this high-risk cytogenetic abnormality.

Chromosome 14q32 rearrangements/translocations involving the immunoglobulin heavy chain (IGH) are rarely detected in chronic lymphocytic leukemia (CLL). The prognostic significance of the IGH translocation is controversial and its mutational profile remains unknown. Here, we present for the first time a comprehensive nextgeneration sequencing (NGS) analysis of 46 CLL patients with IGH rearrangement (IGHR-CLLs) and we demonstrate that IGHR-CLLs have a distinct mutational profile with recurrent mutations in NOTCH1, IGLL5, POT1, BCL2, FBXW7, ZMYM3, MGA, BRAF and HIST1H1E genes. Interestingly, BCL2 and FBXW7 mutations were significantly associated with this subgroup and almost half of BCL2, IGLL5 and HISTH1E

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Background Several genetic alterations have been identified as driver events in chronic lymphocytic leukemia (CLL) pathogenesis and oncogenic evolution. Concurrent driver alterations usually coexist within the same tumoral clone, but how the cooperation of multiple genomic abnormalities contributes to disease progression remains poorly understood. Specifically, the biological and clinical consequences of concurrent high‐risk alterations such as del(11q)/ATM‐mutations and del(17p)/TP53‐mutations have not been established. Methods We integrated next‐generation sequencing (NGS) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 techniques to characterize the in vitro and in vivo effects of concurrent monoallelic or biallelic ATM and/or TP53 alterations in CLL prognosis, clonal evolution, and therapy response. Results Targeted sequencing analysis of the co‐occurrence of high‐risk alterations in 271 CLLs revealed that biallelic inactivation of both ATM and TP53 was mutually exclusive, whereas monoallelic del(11q) and TP53 alterations significantly co‐occurred in a subset of CLL patients with a highly adverse clinical outcome. We determined the biological effects of combined del(11q), ATM and/or TP53 mutations in CRISPR/Cas9‐edited CLL cell lines. Our results showed that the combination of monoallelic del(11q) and TP53 mutations in CLL cells led to a clonal advantage in vitro and in in vivo clonal competition experiments, whereas CLL cells harboring biallelic ATM and TP53 loss failed to compete in in vivo xenotransplants. Furthermore, we demonstrated that CLL cell lines harboring del(11q) and TP53 mutations show only partial responses to B cell receptor signaling inhibitors, but may potentially benefit from ATR inhibition. Conclusions Our work highlights that combined monoallelic del(11q) and TP53 alterations coordinately contribute to clonal advantage and shorter overall survival in CLL.

BIRC3 is monoallelically deleted in up to 80% of chronic lymphocytic leukemia (CLL) cases harboring del(11q). In addition, truncating mutations in the remaining allele of this gene can lead to BIRC3 biallelic inactivation, which has been shown to be a marker for reduced survival in CLL. Nevertheless, the biological mechanisms by which these lesions could contribute to del(11q) CLL pathogenesis and progression are partially unexplored. We implemented the CRISPR/Cas9-editing system to generate isogenic CLL cell lines harboring del(11q) and/or BIRC3 mutations, modeling monoallelic and biallelic BIRC3 loss. Our results reveal that monoallelic BIRC3 deletion in del(11q) cells promotes non-canonical NF-κB signaling activation via RelB-p52 nuclear translocation, being these effects allelic dose-dependent and therefore further enhanced in del(11q) cells with biallelic BIRC3 loss. Moreover, we demonstrate ex vivo in primary cells that del(11q) cases including BIRC3 within their deleted region show evidence of non-canonical NF-κB activation which correlates with high BCL2 levels and enhanced sensitivity to venetoclax. Furthermore, our results show that BIRC3 mutations in del(11q) cells promote clonal advantage in vitro and accelerate leukemic progression in an in vivo xenograft model. Altogether, this work highlights the biological bases underlying disease progression of del(11q) CLL patients harboring BIRC3 deletion and mutation.