Characterisation of genetic complexity in chronic lymphocytic leukaemia

Chronic lymphocytic leukaemia (CLL) is the most common adult leukaemia in the Western world, characterised by the accumulation of mature B-cells. The disease has a heterogeneous course whereby some patients do not require treatment for several years while others follow a more aggressive disease progression and require early therapeutic action. Although several prognostic markers have been described, it is still difficult to predict the prognosis of the disease at diagnosis or to explain the diversity associated with the disease. The goal of this thesis was therefore, to characterise the genomic complexity and molecular drivers of clonal expansion and maintenance of CLL using targeted next generation sequencing (NGS) and high resolution single nucleotide polymorphism (SNP) array. NGS analysis of the immunoglobulin variable heavy-chain (IgHV) in 497 pre-treatment CLL patients found IgHV subclones in 18.5%, far higher than previously reported. SNP array analysis in 411 CLL patients refined the minimally overlapping regions (MOR) on del(6q) to the ATG5 and PRDM1 genes. Further autophagy related genes were also found to be present in other MORs. Analysis of the autophagy pathway at the protein level showed higher autophagy activity in primary CLL cells compared with age-matched controls. NGS-IgHV enabled the existing Sanger-sequencing based prognostic system to be refined. Correlation to the clinical outcome data showed that this new classification was prognostically significant. Furthermore, the identification of IgHV subclones imply that the leukaemia initiating event in CLL takes place prior to recombination. Concurrently, the detection of altered autophagy levels suggests a role for autophagy in CLL. Collectively these data provide new insights into the pathogenesis of CLL

Mutational analysis of disease relapse in patients allografted for acute myeloid leukemia

Disease relapse is the major cause of treatment failure after allogeneic stem cell transplantation (allo-SCT) in acute myeloid leukemia (AML). To identify AML-associated genes prognostic of AML relapse post–allo-SCT, we resequenced 35 genes in 113 adults at diagnosis, 49 of whom relapsed. Two hundred sixty-two mutations were detected in 102/113 (90%) patients. An increased risk of relapse was observed in patients with mutations in WT1 (P = .018), DNMT3A (P = .045), FLT3 ITD (P = .071), and TP53 (P = .06), whereas mutations in IDH1 were associated with a reduced risk of disease relapse (P = .018). In 29 patients, we additionally compared mutational profiles in bone marrow at diagnosis and relapse to study changes in clonal structure at relapse. In 13/29 patients, mutational profiles altered at relapse. In 9 patients, mutations present at relapse were not detected at diagnosis. In 15 patients, additional available pre–allo-SCT samples demonstrated that mutations identified posttransplant but not at diagnosis were detectable immediately prior to transplant in 2 of 15 patients. Taken together, these observations, if confirmed in larger studies, have the potential to inform the design of novel strategies to reduce posttransplant relapse highlighting the potential importance of post–allo-SCT interventions with a broad antitumor specificity in contrast to targeted therapies based on mutational profile at diagnosis

New challenges in diagnosis of haemoglobinopathies: Migration of populations

The current influx of economic migrants and asylum seekers from countries with a high prevalence of haemoglobinopathies creates new challenges for health care systems and diagnostic laboratories. The migration of carriers introduces new and novel haemoglobinopathy mutations to the diagnostic repertoire of a laboratory, often creating new pressures to improve and update the carrier screening technology and diagnostic scope. For antenatal screening programmes, the marriage of partners from different ethnic groups can lead to the risk of compound heterozygote children being born novel mutation combinations, creating problems in the provision of accurate advice regarding the expected phenotype of the thalassaemia or haemoglobinopathy disorder. In the UK, the impact of immigration required the National Haemoglobinopathy Reference laboratory to change the strategy and techniques used for the molecular diagnosis of thalassaemia and the haemoglobinopathies. In 2005, due to the increasingly large range of β-thalassaemia mutations that needed to be diagnosed, the laboratory switched from a three-step screening procedure using ARMS-PCR to a simpler but more expensive one-step strategy of DNA sequencing of the beta and alpha globin genes for all referrals. After ten years of employing this strategy, a further 57 novel thalassaemia and haemoglobionpopthy alleles were discovered (11 new β-chain variants, 15 α-chain variants, 19 β-thalassaemia mutations and 12 α+-thalassaemia mutations), increasing further the extremely heterogeneous spectrum of globin gene mutations in the UK population

Targeted deep sequencing reveals clinically relevant subclonal IgHV rearrangements in chronic lymphocytic leukemia

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 (P397(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.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Whole-genome sequencing of chronic lymphocytic leukaemia reveals distinct differences in the mutational landscape between IgHVmut and IgHVunmut subgroups.

This corrects the article DOI: 10.1038/leu.2017.177.info:eu-repo/semantics/publishe

The light chain IgLV3-21 defines a new poor prognostic subgroup in Chronic Lymphocytic Leukemia: results of a multicenter study.

Unmutated (UM) immunoglobulin heavy chain variable region (IgHV) status or IgHV3-21 gene usage is associated with poor prognosis in chronic lymphocytic leukemia (CLL) patients. Interestingly, IgHV3-21 is often co-expressed with light chain IgLV3-21, which is potentially able to trigger cell-autonomous BCR-mediated signaling. However, this light chain has never been characterized independently of the heavy chain IgHV3-21.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Presence of multiple recurrent mutations confers poor trial outcome of relapsed/refractory CLL

International audienceAlthough 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 multiple-hit profile was associated with a median progression-free survival of 12 months compared with 22.5 months in the remaining patients (P = .003). Concurrent gene mutations are frequent in patients with relapsed/refractory CLL and are associated with worse outcom