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

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

MicroRNA profiling and identification of let-7a as a target to prevent chemotherapy-induced primordial follicles apoptosis in mouse ovaries.

Cancer treatments as cyclophosphamide and its active metabolites are highly gonadotoxic leading to follicle apoptosis and depletion. Considering the risk of subsequent infertility, fertility preservation is recommended. Beside the germ cells and gametes cryopreservation options, ovarian pharmacological protection during treatment appears to be very attractive. Meanwhile, the advances in the field of oncology have brought microRNAs into spotlight as a potential feature of cancer treatment. Herein, we investigated miRNAs expressions in response to chemotherapy using postnatal-day-3 (PND3) mouse ovaries. Our results revealed that several miRNAs are differently expressed during chemotherapy exposure. Amongst them, let-7a was the most profoundly downregulated and targets genes involved in crucial cellular processes including apoptosis. Thus we developed a liposome-based system to deliver the let-7a mimic in whole PND3 ovaries in vitro. We showed that let-7a mimic prevented the upregulation of genes involved in cell death and reduced the chemotherapy-induced ovarian apoptosis, suggesting that it can be an interesting target to preserve ovarian function. However, its impact on subsequent follicular development has to be further elucidated in vivo using an appropriate delivery system. In this study, we demonstrated that miRNA replacement approaches can be a useful tool to reduce chemotherapy-induced ovarian damage in the future.

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.

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

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.

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 (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.

Antileukemic activity of valproic acid in chronic lymphocytic leukemia B cells defined by microarray analysis.

Epigenetic code modifications by histone deacetylase inhibitors have recently been proposed as potential new therapies for hematological malignancies. Chronic lymphocytic leukemia (CLL) remains incurable despite the introduction of new treatments. CLL B cells are characterized by an apoptosis defect rather than excessive proliferation, but proliferation centers have been found in organs such as the bone marrow and lymph nodes. In this study, we analyzed gene expression modifications in CLL B cells after treatment with valproic acid (VPA), a well-tolerated anti-epileptic drug with HDAC inhibitory activity. CLL B cells obtained from 14 patients were treated in vitro with a concentration of 1 mM VPA for 4 h. VPA effects on gene expression were thereafter studied using Affymetrix technology, and some identified genes were validated by real-time PCR and western blot. We observed that VPA induced apoptosis by downregulating several anti-apoptotic genes and by upregulating pro-apoptotic genes. Furthermore, VPA significantly increased chemosensitivity to fludarabine, flavopiridol, bortezomib, thalidomide and lenalidomide. VPA inhibited the proliferation of CpG/IL2-stimulated CLL B cells and modulated many cell cycle messenger RNAs. In conclusion, exposure of CLL B cells to VPA induced apoptosis, potentiated chemotherapeutic agent effects and inhibited proliferation. These data strongly suggest the use of VPA in CLL treatment, particularly in combination with antileukemia agents.

Doubling time of soluble CD23: a powerful prognostic factor for newly diagnosed and untreated stage A chronic lymphocytic leukemia patients.

Soluble CD23 (sCD23) levels correlate with the stage, prognosis and overall survival (OS) of patients with chronic lymphocytic leukemia (CLL). Therefore, we prospectively evaluated sCD23 doubling time (sCD23DT) as a prognostic factor for time to treatment (TTT) and OS in 56 newly diagnosed and untreated CLL patients at Binet stage A, and compared it to the most commonly used biological prognostic factors: lymphocyte doubling time, immunoglobulin variable heavy chain (IgVH) mutational status and zeta-associated protein-70 (ZAP-70), CD38, and lipoprotein lipase (LPL) expression. In patients with sCD23DT <1 year, the median TTT and OS were 20 and 83 months compared to 141 and 177 months in patients with sCD23DT >1 year (P<0.0001). Among patients with poor prognostic factors (ZAP-70+, LPL+ and CD38+), an sCD23DT <1 year identified a subpopulation with a shorter TTT. Patients with unmutated IgVH and an sCD23DT <1 year had a median TTT and OS of 14 and 83 months, respectively, whereas these values were 70 and >177 months when sCD23DT was >1 year (P<0.0001 and P=0.0219, respectively). Finally, in a Cox multivariate analysis, sCD23DT was the sole independent prognostic factor for TTT (P=0.0027). Furthermore, sCD23DT refines the prognosis given by other classical prognostic factors. These observations support the introduction of sCD23 evaluation into the routine assessment of CLL patients.