BCL3 rearrangements in B-cell lymphoid neoplasms occur in two breakpoint clusters associated with different diseases.

The t(14;19)(q32;q13) often juxtaposes BCL3 with immunoglobulin heavy chain (IGH) resulting in overexpression of the gene. In contrast to other oncogenic translocations, BCL3 rearrangement (BCL3-R) has been associated with a broad spectrum of lymphoid neoplasms. Here we report an integrative whole-genome sequence, transcriptomic, and DNA methylation analysis of 13 lymphoid neoplasms with BCL3-R. The resolution of the breakpoints at single base-pair revealed that they occur in two clusters at 5' (n=9) and 3' (n=4) regions of BCL3 associated with two different biological and clinical entities. Both breakpoints were mediated by aberrant class switch recombination of the IGH locus. However, the 5' breakpoints (upstream) juxtaposed BCL3 next to an IGH enhancer leading to overexpression of the gene whereas the 3' breakpoints (downstream) positioned BCL3 outside the influence of the IGH and were not associated with its expression. Upstream BCL3-R tumors had unmutated IGHV, trisomy 12, and mutated genes frequently seen in chronic lymphocytic leukemia (CLL) but had an atypical CLL morphology, immunophenotype, DNA methylome, and expression profile that differ from conventional CLL. In contrast, downstream BCL3-R neoplasms were atypical splenic or nodal marginal zone lymphomas (MZL) with mutated IGHV, complex karyotypes and mutated genes typical of MZL. Two of the latter four tumors transformed to a large B-cell lymphoma. We designed a novel fluorescence in situ hybridization assay that recognizes the two different breakpoints and validated these findings in 17 independent tumors. Overall, upstream or downstream breakpoints of BCL3-R are mainly associated with two subtypes of lymphoid neoplasms with different (epi)genomic, expression, and clinicopathological features resembling atypical CLL and MZL, respectively.

Chronic lymphocytic leukemia patient-derived xenografts recapitulate clonal evolution to Richter transformation.

Chronic lymphocytic leukemia (CLL) is a B-cell neoplasm with a heterogeneous clinical behavior. In 5-10% of patients the disease transforms into a diffuse large-B cell lymphoma known as Richter transformation (RT), which is associated with dismal prognosis. Here, we aimed to establish patient-derived xenograft (PDX) models to study the molecular features and evolution of CLL and RT. We generated two PDXs by injecting CLL (PDX12) and RT (PDX19) cells into immunocompromised NSG mice. Both PDXs were morphologically and phenotypically similar to RT. Whole-genome sequencing analysis at different time points of the PDX evolution revealed a genomic landscape similar to RT tumors from both patients and uncovered an unprecedented RT subclonal heterogeneity and clonal evolution during PDX generation. In PDX12, the transformed cells expanded from a very small subclone already present at the CLL stage. Transcriptomic analysis of PDXs showed a high oxidative phosphorylation (OXPHOS) and low B-cell receptor (BCR) signaling similar to the RT in the patients. IACS-010759, an OXPHOS inhibitor, reduced proliferation, and circumvented resistance to venetoclax. In summary, we have generated new RT-PDX models, one of them from CLL cells that mimicked the evolution of CLL to RT uncovering intrinsic features of RT cells of therapeutical value.

SF3B1 mutation-mediated sensitization to H3B-8800 splicing inhibitor in chronic lymphocytic leukemia.

Splicing factor 3B subunit 1 (SF3B1) is involved in pre-mRNA branch site recognition and is the target of antitumor-splicing inhibitors. Mutations in SF3B1 are observed in 15% of patients with chronic lymphocytic leukemia (CLL) and are associated with poor prognosis, but their pathogenic mechanisms remain poorly understood. Using deep RNA-sequencing data from 298 CLL tumor samples and isogenic SF3B1 WT and K700E-mutated CLL cell lines, we characterize targets and pre-mRNA sequence features associated with the selection of cryptic 3' splice sites upon SF3B1 mutation, including an event in the MAP3K7 gene relevant for activation of NF-κB signaling. Using the H3B-8800 splicing modulator, we show, for the first time in CLL, cytotoxic effects in vitro in primary CLL samples and in SF3B1-mutated isogenic CLL cell lines, accompanied by major splicing changes and delayed leukemic infiltration in a CLL xenotransplant mouse model. H3B-8800 displayed preferential lethality towards SF3B1-mutated cells and synergism with the BCL2 inhibitor venetoclax, supporting the potential use of SF3B1 inhibitors as a novel therapeutic strategy in CLL.

Detection of early seeding of Richter transformation in chronic lymphocytic leukemia.

Richter transformation (RT) is a paradigmatic evolution of chronic lymphocytic leukemia (CLL) into a very aggressive large B cell lymphoma conferring a dismal prognosis. The mechanisms driving RT remain largely unknown. We characterized the whole genome, epigenome and transcriptome, combined with single-cell DNA/RNA-sequencing analyses and functional experiments, of 19 cases of CLL developing RT. Studying 54 longitudinal samples covering up to 19 years of disease course, we uncovered minute subclones carrying genomic, immunogenetic and transcriptomic features of RT cells already at CLL diagnosis, which were dormant for up to 19 years before transformation. We also identified new driver alterations, discovered a new mutational signature (SBS-RT), recognized an oxidative phosphorylation (OXPHOS)high-B cell receptor (BCR)low-signaling transcriptional axis in RT and showed that OXPHOS inhibition reduces the proliferation of RT cells. These findings demonstrate the early seeding of subclones driving advanced stages of cancer evolution and uncover potential therapeutic targets for RT.

Serum soluble CD23 levels are an independent predictor of time to first treatment in chronic lymphocytic leukemia.

Serum soluble CD23 (sCD23) levels have been acknowledged as a prognostic factor in patients with chronic lymphocytic leukemia (CLL), but their potential relevance has not been analyzed in recent times. We retrospectively studied 338 CLL, small lymphocytic lymphoma, or CLL-type monoclonal B-cell lymphocytosis patients from a single institution, with available sCD23 levels at diagnosis. Baseline features and outcomes were compared between patients with sCD23 ≤/>1000 UI/L. The 140 patients (41%) who had sCD23 > 1000 UI/L showed adverse-risk clinical and biological characteristics. High sCD23 levels were predictive of a shorter time to first treatment (5-year probability of requiring treatment: 60 vs. 20%, p < 0.0001; hazard ratio (HR) = 1.72, p = 0.003 in a multivariable model also including the CLL International Prognostic Index and the absolute lymphocyte count), and a poorer 5-year overall survival (70 vs. 82%, p = 0.0009). These data suggest the potential of sCD23 to predict treatment-free survival and to shed light on mechanisms of activity and resistance to CD23-directed therapies.

Ibrutinib in Combination With Rituximab for Indolent Clinical Forms of Mantle Cell Lymphoma (IMCL-2015): A Multicenter, Open-Label, Single-Arm, Phase II Trial.

PURPOSE: The need for an individualized management of indolent clinical forms in mantle cell lymphoma (MCL) is increasingly recognized. We hypothesized that a tailored treatment with ibrutinib in combination with rituximab (IR) could obtain significant responses in these patients. METHODS: This is a multicenter single-arm, open-label, phase II study with a two-stage design conducted in 12 Spanish GELTAMO sites (ClinicalTrials.gov identifier: NCT02682641). Previously untreated MCL patients with indolent clinical forms defined by the following criteria were eligible: no disease-related symptoms, nonblastoid variants, Ki-67 < 30%, and largest tumor diameter ≤ 3 cm. Both leukemic non-nodal and nodal subtypes were recruited. Patients received ibrutinib 560 mg once daily and a total of eight doses of rituximab 375 mg/m2. Ibrutinib could be discontinued after 2 years in the case of sustained undetectable minimal residual disease (MRD). The primary end point was the complete response (CR) rate achieved after 12 cycles according to Lugano criteria. RESULTS: Fifty patients with MCL (male 66%; median age 65 years) were enrolled. After 12 cycles of treatment, 42 (84%; 95% CI, 74 to 94) patients had an overall response, including 40 (80%; 95% CI, 69 to 91) with CR. Moreover, undetectable MRD in peripheral blood was achieved in 87% (95% CI, 77 to 97) of cases. At 2 years, 24 of 35 evaluable patients (69%) could discontinue ibrutinib because of undetectable MRD. Four patients had disease progression; three were non-nodal MCL and carried high genomic complexity and TP53 mutations at enrollment. No unexpected toxicity was seen except one patient with severe aplastic anemia. CONCLUSION: Frontline IR combination achieves a high rate of CRs and undetectable MRD in indolent clinical forms of MCL. Discontinuation seems appropriate in cases with undetectable MRD, except for TP53-mutated cases.

Consumption of Ultra-Processed Food and Drinks and Chronic Lymphocytic Leukemia in the MCC-Spain Study.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in Western countries. Its etiology is largely unknown but increasing incidence rates observed worldwide suggest that lifestyle and environmental factors such as diet might play a role in the development of CLL. Hence, we hypothesized that the consumption of ultra-processed food and drinks (UPF) might be associated with CLL. Data from a Spanish population-based case-control study (MCC-Spain study) including 230 CLL cases (recruited within three years of diagnosis) and 1634 population-based controls were used. The usual diet during the previous year was collected through a validated food frequency questionnaire and food and drink consumption was categorized using the NOVA classification scheme. Logistic regression models adjusted for potential confounders were used. Overall, no association was reported between the consumption of UPF and CLL cases (OR per each 10% increase of the relative contribution of UPF to total dietary intake = 1.09 (95% CI: 0.94; 1.25)), independently of the Rai stage at diagnosis. However, when analyses were restricted to cases diagnosed within <1 year (incident), each 10% increment in the consumption of UPF was associated with a 22% higher odds ratio of CLL (95% CI: 1.02, 1.47) suggesting that the overall results might be affected by the inclusion of prevalent cases, who might have changed their dietary habits after cancer diagnosis. Given the low number of cases in the subgroup analyses and multiple tests performed, chance findings cannot totally be ruled out. Nonetheless, positive associations found in CLL incident cases merit further research, ideally in well-powered studies with a prospective design.

Mobile Health Strategies to Tackle Skin Neglected Tropical Diseases With Recommendations From Innovative Experiences: Systematic Review.

BACKGROUND: Neglected tropical diseases (NTDs) represent a diverse group of 20 communicable diseases that occur in tropical and subtropical areas in 149 countries, affecting over 1 billion people and costing developing economies billions of dollars every year. Within these diseases, those that present lesions on the skin surface are classified as skin NTDs (sNTDs). Mobile health interventions are currently being used worldwide to manage skin diseases and can be a good strategy in the epidemiological and clinical management of sNTDs. OBJECTIVE: We aimed to analyze existing evidence about mobile health interventions to control and manage sNTDs in low- and middle-income countries (LMICs) and make recommendations for what should be considered in future interventions. METHODS: A systematic review was conducted of the MEDLINE, Embase, and Scopus databases over 10 years up to April 30, 2020. All types of clinical studies were considered. Data were synthesized into evidence tables. Apps were selected through a comprehensive systematic search in the Google Play Store and Apple App Store conducted between March 20 and April 15, 2020. RESULTS: From 133 potentially relevant publications, 13 studies met our criteria (9.8%). These analyzed eight different interventions (three SMS text messaging interventions and five app interventions). Six of the 13 (46%) studies were community-based cross-sectional studies intended to epidemiologically map a specific disease, mainly lymphatic filariasis, but also cutaneous leishmaniasis, leprosy, and NTDs, as well as sNTDs in general. Most of the studies were considered to have a high (5/13, 39%) or moderate (4/13, 31%) risk of bias. Fifteen apps were identified in the Google Play Store, of which three were also in the Apple App Store. Most of the apps (11/15, 73%) were targeted at health care professionals, with only four targeted at patients. The apps focused on scabies (3/15, 20%), lymphatic filariasis (3/15, 20%), cutaneous leishmaniasis (1/15, 7%), leprosy (1/15, 7%), yaws and Buruli ulcer (1/15, 7%), tropical diseases including more than one sNTDs (3/15, 20%), and NTDs including sNTDs (2/15, 13%). Only 1 (7%) app focused on the clinical management of sNTDs. CONCLUSIONS: All mobile health interventions that were identified face technological, legal, final user, and organizational issues. There was a remarkable heterogeneity among studies, and the majority had methodological limitations that leave considerable room for improvement. Based on existing evidence, eight recommendations have been made for future interventions.

IgCaller for reconstructing immunoglobulin gene rearrangements and oncogenic translocations from whole-genome sequencing in lymphoid neoplasms.

Immunoglobulin (Ig) gene rearrangements and oncogenic translocations are routinely assessed during the characterization of B cell neoplasms and stratification of patients with distinct clinical and biological features, with the assessment done using Sanger sequencing, targeted next-generation sequencing, or fluorescence in situ hybridization (FISH). Currently, a complete Ig characterization cannot be extracted from whole-genome sequencing (WGS) data due to the inherent complexity of the Ig loci. Here, we introduce IgCaller, an algorithm designed to fully characterize Ig gene rearrangements and oncogenic translocations from short-read WGS data. Using a cohort of 404 patients comprising different subtypes of B cell neoplasms, we demonstrate that IgCaller identifies both heavy and light chain rearrangements to provide additional information on their functionality, somatic mutational status, class switch recombination, and oncogenic Ig translocations. Our data thus support IgCaller to be a reliable alternative to Sanger sequencing and FISH for studying the genetic properties of the Ig loci.

Occupational Exposure to Pesticides and Chronic Lymphocytic Leukaemia in the MCC-Spain Study.

We aimed to study the association between occupational exposure to pesticides and chronic lymphocytic leukemia (CLL) in Spain. Occupational exposure to pesticides (four insecticides, four herbicides and two fungicides) was evaluated using a job-exposure matrix for the Spanish population (MatEmESp) among 302 CLL cases and 1567 population controls in five regions of Spain, 2010-2013. Cumulative exposure scores (CES) were obtained by summing across the exposed jobs the product of prevalence, intensity and duration of exposure to each active substance. Principal components analysis (PCA) and logistic regression models adjusted for age, sex, region, education and occupational exposure to solvents were used. Around 20% of controls and 29% of cases were exposed to one or more pesticides. Compared to non-exposed, subjects in the highest tertile (3rd tertile) of CES of insecticides, herbicides, fungicides were more likely to have CLL [OR (95% CI), P-trend; 2.10 (1.38; 3.19), 0.002; 1.77 (1.12; 2.80), 0.12; and 1.67 (1.06; 2.64), 0.10, respectively). Following PCA, the first component (PC1, explaining 70% of the variation) equally led by seven active substances (the insecticide pyrethrin, all herbicides, all fungicides) was associated with a 26% higher odds of having CLL for 1-standard deviation increase in PC1 (95% CI: 1.14 to 1.40). These results confirm previous associations between CLL and exposure to pesticides and provide additional evidence by application groups and active substance. However, more research is needed to disentangle independent effects of individual active substances.

Genomic and epigenomic insights into the origin, pathogenesis, and clinical behavior of mantle cell lymphoma subtypes.

Mantle cell lymphoma (MCL) is a mature B-cell neoplasm initially driven by CCND1 rearrangement with 2 molecular subtypes, conventional MCL (cMCL) and leukemic non-nodal MCL (nnMCL), that differ in their clinicobiological behavior. To identify the genetic and epigenetic alterations determining this diversity, we used whole-genome (n = 61) and exome (n = 21) sequencing (74% cMCL, 26% nnMCL) combined with transcriptome and DNA methylation profiles in the context of 5 MCL reference epigenomes. We identified that open and active chromatin at the major translocation cluster locus might facilitate the t(11;14)(q13;32), which modifies the 3-dimensional structure of the involved regions. This translocation is mainly acquired in precursor B cells mediated by recombination-activating genes in both MCL subtypes, whereas in 8% of cases the translocation occurs in mature B cells mediated by activation-induced cytidine deaminase. We identified novel recurrent MCL drivers, including CDKN1B, SAMHD1, BCOR, SYNE1, HNRNPH1, SMARCB1, and DAZAP1. Complex structural alterations emerge as a relevant early oncogenic mechanism in MCL, targeting key driver genes. Breakage-fusion-bridge cycles and translocations activated oncogenes (BMI1, MIR17HG, TERT, MYC, and MYCN), generating gene amplifications and remodeling regulatory regions. cMCL carried significant higher numbers of structural variants, copy number alterations, and driver changes than nnMCL, with exclusive alterations of ATM in cMCL, whereas TP53 and TERT alterations were slightly enriched in nnMCL. Several drivers had prognostic impact, but only TP53 and MYC aberrations added value independently of genomic complexity. An increasing genomic complexity, together with the presence of breakage-fusion-bridge cycles and high DNA methylation changes related to the proliferative cell history, defines patients with different clinical evolution.

Sampling time-dependent artifacts in single-cell genomics studies.

Robust protocols and automation now enable large-scale single-cell RNA and ATAC sequencing experiments and their application on biobank and clinical cohorts. However, technical biases introduced during sample acquisition can hinder solid, reproducible results, and a systematic benchmarking is required before entering large-scale data production. Here, we report the existence and extent of gene expression and chromatin accessibility artifacts introduced during sampling and identify experimental and computational solutions for their prevention.

Targeting IRAK4 disrupts inflammatory pathways and delays tumor development in chronic lymphocytic leukemia.

Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in Toll-like receptor (TLR) signal transduction and innate immune responses. Recruitment and subsequent activation of IRAK4 upon TLR stimulation is mediated by the myeloid differentiation primary response 88 (MYD88) adaptor protein. Around 3% of chronic lymphocytic leukemia (CLL) patients have activating mutations of MYD88, a driver mutation in this disease. Here, we studied the effects of TLR activation and the pharmacological inhibition of IRAK4 with ND2158, an IRAK4 competitive inhibitor, as a therapeutic approach in CLL. Our in vitro studies demonstrated that ND2158 preferentially killed CLL cells in a dose-dependent manner. We further observed a decrease in NF-κB and STAT3 signaling, cytokine secretion, proliferation and migration of primary CLL cells from MYD88-mutated and -unmutated cases. In the Eµ-TCL1 adoptive transfer mouse model of CLL, ND2158 delayed tumor progression and modulated the activity of myeloid and T cells. Our findings show the importance of TLR signaling in CLL development and suggest IRAK4 as a therapeutic target for this disease.

Adherence to the 2018 WCRF/AICR cancer prevention guidelines and chronic lymphocytic leukemia in the MCC-Spain study.

INTRODUCTION: Preventable risk factors for chronic lymphocytic leukemia (CLL) remain largely unknown. The aim of this study was to evaluate the association between adherence to nutrition-based guidelines for cancer prevention and CLL, in the MCC-Spain case-control study. METHODS: A total of 318 CLL cases and 1293 population-based controls were included in the present study. The World Cancer Research Fund/American Institute for Cancer Research (WCRC/AICR) score based on the 2018 recommendations for cancer prevention (on body fatness, physical activity, and diet) was constructed. We used logistic regression analysis adjusting for potential confounders. RESULTS: Individuals in the highest tertile of the WCRF/AICR score had an odds ratio for CLL of 1.25 (95 % CI 0.91; 1.73) compared with individuals with low adherence (p-trend = 0.172). Each point increment in the score was associated with an OR for CLL of 1.06 (95 % CI 0.91; 1.23). Analyses by severity of disease did not show significant heterogeneity of effects. CONCLUSION: Overall, our results do not support an association between the WCRF/AICR score and CLL, yet we might have been limited by statistical power and study design to detect modest associations. Further research, ideally with a prospective design, long follow-up, and including additional lymphoma subtypes, is warranted to confirm the impact of composite healthy lifestyle behaviors on lymphoma risk.

Specific NOTCH1 antibody targets DLL4-induced proliferation, migration, and angiogenesis in NOTCH1-mutated CLL cells.

Targeting Notch signaling has emerged as a promising therapeutic strategy for chronic lymphocytic leukemia (CLL), particularly in NOTCH1-mutated patients. We provide first evidence that the Notch ligand DLL4 is a potent stimulator of Notch signaling in NOTCH1-mutated CLL cells while increases cell proliferation. Importantly, DLL4 is expressed in histiocytes from the lymph node, both in NOTCH1-mutated and -unmutated cases. We also show that the DLL4-induced activation of the Notch signaling pathway can be efficiently blocked with the specific anti-Notch1 antibody OMP-52M51. Accordingly, OMP-52M51 also reverses Notch-induced MYC, CCND1, and NPM1 gene expression as well as cell proliferation in NOTCH1-mutated CLL cells. In addition, DLL4 stimulation triggers the expression of protumor target genes, such as CXCR4, NRARP, and VEGFA, together with an increase in cell migration and angiogenesis. All these events can be antagonized by OMP-52M51. Collectively, our results emphasize the role of DLL4 stimulation in NOTCH1-mutated CLL and confirm the specific therapeutic targeting of Notch1 as a promising approach for this group of poor prognosis CLL patients.