Chronic lymphocytic leukemia is characterized by relapse after treatment and chemotherapy resistance. Similarly, in other malignancies leukemia cells accumulate mutations during growth, forming heterogeneous cell populations that are subject to Darwinian selection and may respond differentially to treatment. There is therefore a clinical need to monitor changes in the subclonal composition of cancers during disease progression. Here, we use whole-genome sequencing to track subclonal heterogeneity in 3 chronic lymphocytic leukemia patients subjected to repeated cycles of therapy. We reveal different somatic mutation profiles in each patient and use these to establish probable hierarchical patterns of subclonal evolution, to identify subclones that decline or expand over time, and to detect founder mutations. We show that clonal evolution patterns are heterogeneous in individual patients. We conclude that genome sequencing is a powerful and sensitive approach to monitor disease progression repeatedly at the molecular level. IntroductionDespite significant progress in the management of lymphomas and leukemias, relapse remains the major cause of death. Increased use of expensive targeted therapies and toxic chemotherapies (especially in the elderly) confronts us with an urgent need to improve response prediction for all cancer patients to reduce side effects and costs from ineffective treatment. Current diagnostic approaches to treatment selection, response monitoring, and relapse prediction are limited to single genes and apply only to a minority of hematologic cancers. This is at odds with modern concepts of tumor propagation and maintenance, which propose that every cell in an individual cancer is characterized by a combination of mutation events that comprise tumorigenic (driver) mutations, passive (passenger) mutations, and possibly predisposing germline risk variants. Cancer cells propagate and diversify during tumor growth, resulting in a heterogeneous population of genotypically and phenotypically distinct subclones that are related in a hierarchical lineage. As the composition of the local environment changes, for example as a consequence of drug treatment, tumor cell populations adapt and evolve by Darwinian selection. [1][2][3] Whole-genome sequencing (WGS) of a single tumor sample can be used to generate a comprehensive catalog of variants that provides a snapshot of the cell population en masse at a particular time point. 2,4-6 However, over time and with continued evolution of the cancer, this snapshot becomes progressively less representative of the disease. Recent reports have described whole-tumor genomes from single patients or cohorts of individuals mostly at single time points and irrespective of treatment. [7][8][9][10] This approach has enabled identification of mutations representative and in some cases highly predictive of histologic cancer type, outcome, and/or treatment response. [11][12][13][14][15] Comparison of sequence data from primary and metastatic tumor samples, or from multiple lo...
Key Points• Acquired pathogenic mutations in SAMHD1 are found in up to 11% of relapsed/refractory patients with CLL. • SAMHD1 is mobilized to sites of DNA damage.SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase and a nuclease that restricts HIV-1 in noncycling cells. Germ-line mutations in SAMHD1 have been described in patients with Aicardi-Goutières syndrome (AGS), a congenital autoimmune disease. In a previous longitudinal whole genome sequencing study of chronic lymphocytic leukemia (CLL), we revealed a SAMHD1 mutation as a potential founding event. Here, we describe an AGS patient carrying a pathogenic germ-line SAMHD1 mutation who developed CLL at 24 years of age. Using clinical trial samples, we show that acquired SAMHD1 mutations are associated with high variant allele frequency and reduced SAMHD1 expression and occur in 11% of relapsed/refractory CLL patients. We provide evidence that SAMHD1 regulates cell proliferation and survival and engages in specific protein interactions in response to DNA damage. We propose that SAMHD1 may have a function in DNA repair and that the presence of SAMHD1 mutations in CLL promotes leukemia development. (Blood. 2014;123(7):1021-1031)
Key Points• Targeted NGS of relapsed/ refractory CLL reveals a high incidence of concurrent mutations that mostly affect the TP53, ATM, and SF3B1 genes.• Concurrent mutations of the TP53, ATM, and/or SF3B1 genes confer short survival in patients with relapsed/ refractory CLL.Although TP53, NOTCH1, and SF3B1 mutations may impair prognosis of patients with chronic lymphocytic leukemia (CLL) receiving frontline therapy, the impact of these mutations or any other, alone or in combination, remains unclear at relapse. The genome of 114 relapsed/refractory patients included in prospective trials was screened using targeted next-generation sequencing of the TP53, SF3B1, ATM, NOTCH1, XPO1, SAMHD1, MED12, BIRC3, and MYD88 genes. We performed clustering according to both number and combinations of recurrent gene mutations. The number of genes affected by mutation was ‡2, 1, and 0 in 43 (38%), 49 (43%), and 22 (19%) respectively. Recurrent combinations of ‡2 mutations of TP53, SF3B1, and ATM were found in 22 (19%) patients. This multiplehit profile was associated with a median progression-free survival of 12 months compared with 22.5 months in the remaining patients (P 5 .003). Concurrent gene mutations are frequent in patients with relapsed/refractory CLL and are associated with worse outcome. (Blood. 2015;126(18):2110-2117 IntroductionChronic lymphocytic leukemia (CLL) is characterized by its unique immunophenotype of CD19sIg dim expressing clonal mature B cells and also by a highly variable clinical course. With the emergence of new classes of drugs such as the inhibitor of phosphatidylinositol 3-kinase, idelalisib, 1 and the irreversible inhibitor of Bruton tyrosine kinase, ibrutinib, 2 available treatment options have increased significantly and allow us to begin to develop models of treatment stratification. Over the last 3 years, the CLL genome has been thoroughly characterized by next-generation sequencing (NGS).3 Pioneer reports using this approach unraveled somatic mutations recurrently targeting multiple genes, among which TP53, SF3B1, NOTCH1, MYD88, and ATM were the most frequent. [4][5][6][7][8][9] Large retrospective studies in untreated patients from historical cohorts have recently shown the adverse prognostic impact of the TP53, NOTCH1, and SF3B1 mutations on time to treatment and overall survival (OS). [10][11][12] In addition, mutations in these genes may also be associated with poor progression-free survival (PFS) in frontline patients treated in prospective trials. 13,14 Conversely, the pejorative impact of TP53, SF3B1, and NOTCH1 mutations on the clinical outcome of patients with relapsed/ refractory (R/R) CLL is controversial. [15][16][17] Compared with untreated CLL, relapse, as advanced disease, may be associated with a high level of genomic diversification. 9,18 This process might result in For personal use only. on May 11, 2018. by guest www.bloodjournal.org From accumulation and co-occurrence of these or other genomic events leading to interactions that could be of prognostic relevance. Here, w...
Genome-wide array approaches and sequencing analyses are powerful tools for identifying genetic aberrations in cancers, including leukemias and lymphomas. However, the clinical and biological significance of such aberrations and their subclonal distribution are poorly understood. Here, we present the first genome-wide array based study of pre-treatment and relapse samples from patients with B-cell chronic lymphocytic leukemia (B-CLL) that uses the computational statistical tool OncoSNP. We show that quantification of the proportion of copy number alterations (CNAs) and copy neutral loss of heterozygosity regions (cnLOHs) in each sample is feasible. Furthermore, we (i) reveal complex changes in the subclonal architecture of paired samples at relapse compared with pre-treatment, (ii) provide evidence supporting an association between increased genomic complexity and poor clinical outcome (iii) report previously undefined, recurrent CNA/cnLOH regions that expand or newly occur at relapse and therefore might harbor candidate driver genes of relapse and/or chemotherapy resistance. Our findings are likely to impact on future therapeutic strategies aimed towards selecting effective and individually tailored targeted therapies.
Chronic lymphocytic leukaemia (CLL) consists of two biologically and clinically distinct subtypes defined by the abundance of somatic hypermutation (SHM) affecting the Ig variable heavy-chain locus (IgHV). The molecular mechanisms underlying these subtypes are incompletely understood. Here, we present a comprehensive whole-genome sequencing analysis of somatically acquired genetic events from 46 CLL patients, including a systematic comparison of coding and non-coding single-nucleotide variants, copy number variants and structural variants, regions of kataegis and mutation signatures between IgHVmut and IgHVunmut subtypes. We demonstrate that one-quarter of non-coding mutations in regions of kataegis outside the Ig loci are located in genes relevant to CLL. We show that non-coding mutations in ATM may negatively impact on ATM expression and find non-coding and regulatory region mutations in TCL1A, and in IgHVunmut CLL in IKZF3, SAMHD1,PAX5 and BIRC3. Finally, we show that IgHVunmut CLL is dominated by coding mutations in driver genes and an aging signature, whereas IgHVmut CLL has a high incidence of promoter and enhancer mutations caused by aberrant activation-induced cytidine deaminase activity. Taken together, our data support the hypothesis that differences in clinical outcome and biological characteristics between the two subgroups might reflect differences in mutation distribution, incidence and distinct underlying mutagenic mechanisms.
The immunoglobulin heavy-chain variable region gene (IgHV) mutational status is considered the gold standard of prognostication in chronic lymphocytic leukemia (CLL) and is currently determined by Sanger sequencing that allows the analysis of the major clone. Using next-generation sequencing (NGS), we sequenced the IgHV gene from two independent cohorts: (A) 270 consecutive patient samples obtained at diagnosis and (B) 227 patients from the UK ARCTIC-AdMIRe clinical trials. Using complementary DNA from purified CD19+CD5+ cells, we demonstrate the presence of multiple rearrangements in independent experiments and showed that 24.4% of CLL patients express multiple productive clonally unrelated IgHV rearrangements. On the basis of IgHV-NGS subclonal profiles, we defined five different categories: patients with (a) multiple hypermutated (M) clones, (b) 1 M clone, (c) a mix of M-unmutated (UM) clones, (d) 1 UM clone and (e) multiple UM clones. In population A, IgHV-NGS classification stratified patients into five different subgroups with median treatment-free survival (TFS) of >280(a), 131(b), 94(c), 29(d), 15(e) months (P<0.0001) and a median OS of >397(a), 292(b), 196(c), 137(d) and 100(e) months (P<0.0001). In population B, the poor prognosis of multiple UM patients was confirmed with a median TFS of 2 months (P=0.0038). In conclusion, IgHV-NGS highlighted one quarter of CLL patients with multiple productive IgHV subclones and improves disease stratification and raises important questions concerning the pre-leukemic cellular origin of CLL.
Key Points• Germline JAK2V617I mutation as a sole genetic event does not suppress hematopoietic stem cells.• JAK2V617I induces weaker constitutive activation than JAK2V617F but considerable cytokine hyperresponsiveness.The association between somatic JAK2 mutation and myeloproliferative neoplasms (MPNs) is now well established. However, because JAK2 mutations are associated with heterogeneous clinical phenotypes and often occur as secondary genetic events, some aspects of JAK2 mutation biology remain to be understood. We recently described a germline JAK2V617I mutation in a family with hereditary thrombocytosis and herein characterize the hematopoietic and signaling impact of JAK2V617I. Through targeted sequencing of MPN-associated mutations, exome sequencing, and clonality analysis, we demonstrate that JAK2V617I is likely to be the sole driver mutation in JAK2V617I-positive individuals with thrombocytosis. Phenotypic hematopoietic stem cells (HSCs) were increased in the blood and bone marrow of JAK2V617I-positive individuals and were sustained at higher levels than controls after xenotransplantation. In signaling and transcriptional assays, JAK2V617I demonstrated more activity than wild-type JAK2 but substantially less than JAK2V617F. After cytokine stimulation, JAK2V617I resulted in markedly increased downstream signaling compared with wild-type JAK2 and comparable with JAK2V617F. These findings demonstrate that JAK2V617I induces sufficient cytokine hyperresponsiveness in the absence of other molecular events to induce a homogeneous MPN-like phenotype. We also provide evidence that the JAK2V617I mutation may expand the HSC pool, providing insights into both JAK2 mutation biology and MPN disease pathogenesis. (Blood. 2013;121(20):4156-4165)
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