Whole-genome sequencing of chronic lymphocytic leukemia identifies subgroups with distinct biological and clinical features

The value of genome-wide over targeted driver analyses for predicting clinical outcomes of cancer patients is debated. Here, we report the whole-genome sequencing of 485 chronic lymphocytic leukemia patients enrolled in clinical trials as part of the United Kingdom's 100,000 Genomes Project. We identify an extended catalog of recurrent coding and noncoding genetic mutations that represents a source for future studies and provide the most complete high-resolution map of structural variants, copy number changes and global genome features including telomere length, mutational signatures and genomic complexity. We demonstrate the relationship of these features with clinical outcome and show that integration of 186 distinct recurrent genomic alterations defines five genomic subgroups that associate with response to therapy, refining conventional outcome prediction. While requiring independent validation, our findings highlight the potential of whole-genome sequencing to inform future risk stratification in chronic lymphocytic leukemia

Characterisation of the mutational landscape of chronic lymphocytic leukaemia using genome-wide approaches

Chronic lymphocytic leukaemia (CLL) is characterised by a chronically relapsing course and clinical and biological heterogeneity, and none of the conventional treatment options are curative. CLL cases lack disease-defining mutations but they can be broadly classified into two prognostic groups by the immunoglobulin heavy chain variable (IgHV) gene mutational status, where CLL patients with IgHV unmutated status are associated with a more aggressive clinical course. This broad prognostic sub-classification was improved by the establishment of the Döhner five hierarchical classifications based on cytogenetic abnormalities, which do not entirely explain the genetic basis of the clinical heterogeneity of CLL patients. Recent genome-wide technologies have identified multiple additional recurrent alterations, some of which may have independent prognostic value. This increase in the number of potential CLL genomic markers necessitates the simultaneous screening of multiple genes in the clinic. Next Generation Sequencing (NGS) technologies offer significant advantages over other conventional molecular techniques in screening these genes, however, they have not been evaluated sufficiently, nor standardised for clinical implementation. Moreover, most CLL whole genome sequencing (WGS) and whole exome sequencing (WES) studies investigating the genetic heterogeneity of CLL have looked only at the coding regions, and data concerning the significance of recurrent mutations in regulatory elements is lacking. The elucidation of CLL genomic complexity and heterogeneity may contribute to our understanding about molecular pathogenesis in CLL, and may subsequently lead to an improved clinical management through specifically designed targeted therapies. Thus, the goal of this thesis was to characterise, using genome-wide technologies, the genomic landscape of CLL patients. This thesis also validated both the sensitivity and specificity of targeted sequencing panel (TSP) and WGS for clinical implementation in routine diagnostics, by investigating well-established and novel genomic markers of CLL. An integrated approach using TSP technologies with genome-wide high-density SNP array data was used to investigate candidate genes that are disrupted or mutated in CLL. This analysis was based on the hypothesis that minimally overlapping regions (MORs) would pinpoint genes that harboured recurrent acquired mutations driving CLL pathogenesis, and/or may also be enriched with common inherited single nucleotide SNPs that predispose for the development of CLL. Accordingly, this analysis identified recurrent somatic mutations affecting FBXW7 and SETD2, which was found to be consistent with findings of recent literature. Additionally, it identified a common SNP in TLR4 with a higher frequency in the CLL cohort compared to the normal population. The comparison between TSP and WGS highlighted the advantage of each technique and showed the importance of validating TSP bioinformatics pipelines before introducing them to clinical service and in the diagnostic services. Finally, functional annotation of WGS data of 46 CLL genomes revealed distinct genomic profiles in the two CLL IgHV subgroups, which may be explained, to some extent, by the prevalence of extracted mutational signatures in the two groups. Collectively, the data presented by this thesis provide a new insight into CLL genomic complexity and heterogeneity.</p

Preparation of 6-Mercaptopurine Loaded Liposomal Formulation for Enhanced Cytotoxic Response in Cancer Cells

6-Mercaptopurine (6-MP) is a well-known immunosuppressive medication with proven anti-proliferative activities. 6-MP possesses incomplete and highly variable oral absorption due to its poor water solubility, which might reduce its anti-cancer properties. To overcome these negative effects, we developed neutral and positively charged drug-loaded liposomal formulations utilizing the thin-film hydration technique. The prepared liposomal formulations were characterized for their size, polydispersity index (PDI), zeta potential, and entrapment efficiency. The average size of the prepared liposomes was between 574.67 ± 37.29 and 660.47 ± 44.32 nm. Positively charged liposomes (F1 and F3) exhibited a lower PDI than the corresponding neutrally charged ones (F2 and F4). Entrapment efficiency was higher in the neutral liposomes when compared to the charged formulation. F1 showed the lowest IC50 against HepG2, HCT116, and MCF-7 cancer cells. HepG2 cells treated with F1 showed the highest level of inhibition of cell proliferation with no evidence of apoptosis. Cell cycle analysis showed an increase in the G1/G0 and S phases, along with a decrease in the G2/M phases in the cell lines treated with drug loaded positively charged liposomes when compared to free positive liposomes, indicating arrest of cells in the S phase due to the stoppage of priming and DNA synthesis outside the mitotic phase. As a result, liposomes could be considered as an effective drug delivery system for treatment of a variety of cancers; they provide a chance that a nanoformulation of 6-MP will boost the cytotoxicity of the drug in a small pharmacological dose which provides a dosage advantage

Correction: Jamal et al. Preparation of 6-Mercaptopurine Loaded Liposomal Formulation for Enhanced Cytotoxic Response in Cancer Cells. <i>Nanomaterials</i> 2022, <i>12</i>, 4029

In the original publication [...

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

Whole-genome sequencing of chronic lymphocytic leukemia identifies subgroups with distinct biological and clinical features

The value of genome-wide over targeted driver analyses for predicting clinical outcomes of cancer patients is debated. Here, we report the whole-genome sequencing of 485 chronic lymphocytic leukemia patients enrolled in clinical trials as part of the United Kingdom's 100,000 Genomes Project. We identify an extended catalog of recurrent coding and noncoding genetic mutations that represents a source for future studies and provide the most complete high-resolution map of structural variants, copy number changes and global genome features including telomere length, mutational signatures and genomic complexity. We demonstrate the relationship of these features with clinical outcome and show that integration of 186 distinct recurrent genomic alterations defines five genomic subgroups that associate with response to therapy, refining conventional outcome prediction. While requiring independent validation, our findings highlight the potential of whole-genome sequencing to inform future risk stratification in chronic lymphocytic leukemia