A low total metabolic tumor volume independently predicts for a longer time to first treatment in initially observed, low tumor burden follicular lymphoma.

Watchful waiting is an acceptable management strategy for advanced-stage, low tumor burden (LTB) patients with follicular lymphoma (FL). However, the prediction of how long this treatment-free observation period will last remains imperfect. We explored whether total metabolic tumor volume (TMTV) and other positron emission tomography parameters were predictive of time to first treatment (TTFT). We analyzed 97 grade 1-3A advanced-stage LTB FL patients and found that a high TMTV was associated with other tumor burden features at diagnosis. Patients with a TMTV above our established cutoff of 50 mL had a significantly shorter median duration of observation (2.6 vs. 8.8 years; p = 0.001). At 5 years, 77% of patients with a high TMTV and 46% of patients with a low TMTV required treatment. In the multivariable analysis, a high TMTV was the only independent factor predicting TTFT (hazard ratio = 2.09; p = 0.017). Overall, TMTV is a strong predictor of the duration of observation in LTB FL patients. Upon validation of our cutoff in external series and standardization of the methodology, the TMTV could become an additional factor to consider deferring or initiating treatment in otherwise LTB patients.

Genomic landscape of follicular lymphoma across a wide spectrum of clinical behaviors.

While some follicular lymphoma (FL) patients do not require treatment or experience prolonged responses, others relapse early, and little is known about genetic alterations specific to patients with a particular clinical behavior. We selected 56 grade 1-3A FL patients according to their need of treatment or timing of relapse: never treated (n = 7), non-relapsed (19), late relapse (14), early relapse or POD24 (11), and primary refractory (5). We analyzed 56 diagnostic and 12 paired relapse lymphoid tissue biopsies and performed copy number alteration (CNA) analysis and next generation sequencing (NGS). We identified six focal driver losses (1p36.32, 6p21.32, 6q14.1, 6q23.3, 9p21.3, 10q23.33) and 1p36.33 copy-neutral loss of heterozygosity (CN-LOH). By integrating CNA and NGS results, the most frequently altered genes/regions were KMT2D (79%), CREBBP (67%), TNFRSF14 (46%) and BCL2 (40%). Although we found that mutations in PIM1, FOXO1 and TMEM30A were associated with an adverse clinical behavior, definitive conclusions cannot be drawn, due to the small sample size. We identified common precursor cells harboring early oncogenic alterations of the KMT2D, CREBBP, TNFRSF14 and EP300 genes and 16p13.3-p13.2 CN-LOH. Finally, we established the functional consequences of mutations by means of protein modeling (CD79B, PLCG2, PIM1, MCL1 and IRF8). These data expand the knowledge on the genomics behind the heterogeneous FL population and, upon replication in larger cohorts, could contribute to risk stratification and the development of targeted therapies.

Clinico-biological features and outcome of patients with splenic marginal zone lymphoma with histological transformation.

We describe 36 patients with splenic marginal zone lymphoma (SMZL) with transformation (SMZL-T), including 15 from a series of 84 patients with SMZL diagnosed at the Hospital Clinic of Barcelona (HCB) and 21 diagnosed with SMZL-T in other centres. In the HCB cohort, the cumulative incidence of transformation at 5 years was 15%. Predictors for transformation were cytopenias, hypoalbuminaemia, complex karyotype (CK) and both the Intergruppo Italiano Linfomi (ILL) and simplified Haemoglobin, Platelet count, lactate dehydrogenase (LDH) and extrahilar Lymphadenopathy (HPLL)/ABC scores (P < 0·05). The only independent predictor for transformation in multivariate analysis was CK [hazard ratio (HR) 4·025, P = 0·05]. Patients with SMZL-T had a significantly higher risk of death than the remainder (HR 3·89, P < 0·001). Of the 36 patients with SMZL-T, one developed Hodgkin lymphoma and 35 a diffuse large B-cell lymphoma, 71% with a non-germinal centre phenotype. The main features were B symptoms, lymphadenopathy, and high serum LDH. CK was observed in 12/22 (55%) SMZL-T and fluorescence in situ hybridisation detected abnormalities of MYC proto-oncogene, basic helix-loop-helix transcription factor (MYC), B-cell leukaemia/lymphoma 2 (BCL2) and/or BCL6 in six of 14 (43%). In all, 21 patients received immunochemotherapy, six chemotherapy, one radiotherapy and three splenectomy. The complete response (CR) rate was 61% and the median survival from transformation was 4·92 years. Predictors for a worse survival in multivariate analysis were high-risk International Prognostic Index (HR 5·294, P = 0·016) and lack of CR (HR 2·67, P < 0·001).

First external validation of the FLIPI-L score in a single-center series of patients with follicular lymphoma.

The FLIPI-L has recently been proposed as a novel prognostic index in follicular lymphoma (FL), combining FLIPI and the presence of lymphopenia. In our single-center validation in 381 FL patients, lymphopenia was less frequent than in the original publication and thus the distribution of risk categories was different. Although it was not able to properly predict time to first treatment, FLIPI-L performed slightly better than FLIPI alone in the prediction of response, early relapse, progression-free and overall survival, and histological transformation. This new tool or others encompassing parameters from the microenvironment might improve upon the prognostic ability of classical scores.

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.

CART19-BE-01: A Multicenter Trial of ARI-0001 Cell Therapy in Patients with CD19+ Relapsed/Refractory Malignancies.

We evaluated the administration of ARI-0001 cells (chimeric antigen receptor T cells targeting CD19) in adult and pediatric patients with relapsed/refractory CD19+ malignancies. Patients received cyclophosphamide and fludarabine followed by ARI-0001 cells at a dose of 0.4-5 × 106 ARI-0001 cells/kg, initially as a single dose and later split into 3 fractions (10%, 30%, and 60%) with full administration depending on the absence of cytokine release syndrome (CRS). 58 patients were included, of which 47 received therapy: 38 with acute lymphoblastic leukemia (ALL), 8 with non-Hodgkin's lymphoma, and 1 with chronic lymphocytic leukemia. In patients with ALL, grade ≥3 CRS was observed in 13.2% (26.7% before versus 4.3% after the amendment), grade ≥3 neurotoxicity was observed in 2.6%, and the procedure-related mortality was 7.9% at day +100, with no procedure-related deaths after the amendment. The measurable residual disease-negative complete response rate was 71.1% at day +100. Progression-free survival was 47% (95% IC 27%-67%) at 1 year: 51.3% before versus 39.5% after the amendment. Overall survival was 68.6% (95% IC 49.2%-88%) at 1 year. In conclusion, the administration of ARI-0001 cells provided safety and efficacy results that are comparable with other academic or commercially available products. This trial was registered as ClinicalTrials.gov: NCT03144583.

Baseline correlations and prognostic impact of serum monoclonal proteins in follicular lymphoma.

The presence of a serum monoclonal component has been associated with poor outcomes in some lymphomas. However, data in follicular lymphoma (FL) are scarce. We studied 311 FL patients diagnosed at a single institution, for whom information on serum immunofixation electrophoresis (sIFE) at diagnosis was available. Baseline characteristics and outcomes were compared between patients with a positive (+sIFE) and a negative sIFE (-sIFE). sIFE was positive in 82 patients (26%). Baseline features were comparable between both groups, except for an older age and higher proportion of elevated ß2 -microglobulin levels in the +sIFE group. With a median follow-up of 4.6 years, a +sIFE was associated with a higher risk of early relapse (POD24, 27% vs. 15%, P = 0·02), shorter progression-free survival (PFS; 42% vs. 52% at 5 years, P = 0·008), and shorter overall survival (OS; 59% vs. 77% at 10 years, P = 0·046). In patients >60 years, a +sIFE was an independent predictor of OS [hazard ratio (HR) = 2·4, 95% confidence interval (CI): 1·2-5·0; P = 0·02]. Approximately one quarter of patients with FL has a +sIFE at diagnosis, which is a predictor of poor outcome. These findings encourage further investigation of its relationship with B-cell biology and the tumour microenvironment.

Prognostic ability of five clinical risk scores in follicular lymphoma: A single-center evaluation.

With the intention of identifying follicular lymphoma (FL) patients at higher risk of progression, early relapse (POD24), histological transformation (HT) or death, multiple risk scores (RS) have been proposed. However, it has not yet been established whether any of them globally outperforms the others. We evaluated the clinical utility and statistical performance of the five most widely used clinical scores (IPI, ILI, FLIPI, FLIPI2, PRIMA-PI) in a single-center series of 414 grade 1-3A FL patients diagnosed in the rituximab era. Overall concordance (proportion of patients allocated to the same risk category by all five RS) was 24%. FLIPI and FLIPI2 were predictive of time to first treatment. All five scores were predictive of response, POD24, progression-free, and OS, while only FLIPI predicted HT. IPI identified a small subset (7%) of truly high-risk patients (10-year OS of 16%). In subgroup analyses, we showed that ILI is useful in the prognostication of limited-disease patients, and PRIMA-PI is an age-independent score that can identify a high-risk subset of older patients. Performance metrics were slightly better for IPI in terms of calibration (Harrell's c-index 0.73), without major differences among RS regarding other parameters. Although the incorporation of molecular and imaging data will continue to refine the stratification of FL patients, FLIPI remains the most powerful clinical prognostic index in the rituximab era, predicting the greatest number of endpoints.

Age and comorbidity are determining factors in the overall and relative survival of patients with follicular lymphoma.

Frailty and concurrent medical conditions are crucial factors in the management of follicular lymphoma (FL). We evaluated the impact of age and comorbidity on survival, causes of death, histological transformation (HT), and second malignancies (SM) in a large single-center series of grade 1-3A FL. We studied 414 patients diagnosed in the rituximab era, categorized into three age groups (≤60, 61-70, >70 years) and two comorbidity groups (Charlson Comorbidity Index, CCI, 0-1 and ≥2). Despite a similar cumulative incidence of relapse, older and comorbid patients had a lower 10-year overall survival (OS, 88, 65, and 41% for patients ≤60 years, 61-70 years, and >70 years, P<0.0001; and 76 vs. 51% for CCI 0-1 and ≥2, P<0.0001). In a multivariate analysis for OS, comorbidity retained its prognostic impact (HR=2.5, P=0.0003). The proportion of patients dying due to FL was higher among those ≤60 years (74%) and those with a CCI 0-1 (67%). Furthermore, 10-year excess mortality (survival reduction) was more prominent for patients >70 years (30%) and those with a CCI ≥2 (32%). Patients with a CCI ≥2 also had a higher incidence of SM. These data encourage a comprehensive pre-treatment evaluation and a tailored therapeutic approach for all FL patients.

Non-coding recurrent mutations in chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is a frequent disease in which the genetic alterations determining the clinicobiological behaviour are not fully understood. Here we describe a comprehensive evaluation of the genomic landscape of 452 CLL cases and 54 patients with monoclonal B-lymphocytosis, a precursor disorder. We extend the number of CLL driver alterations, including changes in ZNF292, ZMYM3, ARID1A and PTPN11. We also identify novel recurrent mutations in non-coding regions, including the 3' region of NOTCH1, which cause aberrant splicing events, increase NOTCH1 activity and result in a more aggressive disease. In addition, mutations in an enhancer located on chromosome 9p13 result in reduced expression of the B-cell-specific transcription factor PAX5. The accumulative number of driver alterations (0 to ≥4) discriminated between patients with differences in clinical behaviour. This study provides an integrated portrait of the CLL genomic landscape, identifies new recurrent driver mutations of the disease, and suggests clinical interventions that may improve the management of this neoplasia.

Altered patterns of global protein synthesis and translational fidelity in RPS15-mutated chronic lymphocytic leukemia.

Genomic studies have recently identified RPS15 as a new driver gene in aggressive and chemorefractory cases of chronic lymphocytic leukemia (CLL). RPS15 encodes a ribosomal protein whose conserved C-terminal domain extends into the decoding center of the ribosome. We demonstrate that mutations in highly conserved residues of this domain affect protein stability, by increasing its ubiquitin-mediated degradation, and cell-proliferation rates. On the other hand, we show that mutated RPS15 can be loaded into the ribosomes, directly impacting on global protein synthesis and/or translational fidelity in a mutation-specific manner. Quantitative mass spectrometry analyses suggest that RPS15 variants may induce additional alterations in the translational machinery, as well as a metabolic shift at the proteome level in HEK293T and MEC-1 cells. These results indicate that CLL-related RPS15 mutations might act following patterns known for other ribosomal diseases, likely switching from a hypo- to a hyperproliferative phenotype driven by mutated ribosomes. In this scenario, loss of translational fidelity causing altered cell proteostasis can be proposed as a new molecular mechanism involved in CLL pathobiology.

Study of Biocompatibility of Membranes in Online Hemodiafiltration.

BACKGROUND: The biocompatibility of dialysis membranes is a determining factor in avoiding chronic microinflammation in patients under haemodialysis. Lower biocompatibility has been related to increased inflammatory status, which is known to be associated with cardiovascular events. Classically, cellulose membranes have been considered bioincompatible. A new-generation of asymmetric cellulose triacetate (CTA) membranes allows the performance of high convective transport techniques, but there have been no studies of their biocompatibility. The aim of the present study was to analyze and compare the biocompatibility characteristics of 4 membranes, including CTA, in online hemodiafiltration (OL-HDF) patients. METHODS: We included 15 patients in -OL-HDF. After a 2-week washout period with helixone membrane, each patient was treated with the 4 membranes (polyamide, polynephron, helixone and CTA) for 4 weeks in a randomized order. The other dialysis parameters were kept stable throughout the study. We studied changes in markers of the activation of the complement system, monocytes, platelets, and adhesion molecules with the 4 membranes, as well as inflammatory parameters. RESULTS: Biocompatibility was similar among the membranes. There were no sustained differences in complement activation, measured by C3a and C5a levels, or in platelet activation, determined by levels of P-selectin and platelet-derived microparticles (CD41a+). No differences were observed in activated monocyte levels (CD14+/CD16+) or in plasma levels of interleukin (IL)-1, IL-6, IL-10 or high-sensitivity C-reactive protein, although tumour necrosis factor-α levels decreased when the patients were dialyzed with CTA. No significant differences were found in markers of endothelial damage, assessed by levels of plasminogen activator inhibitor-1 and adhesion molecules (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1). CONCLUSION: The 4 membranes evaluated in this study in stable patients on OL-HDF, including the new-generation CTA, show similar biocompatibility with the methods applied.

Selective BTK inhibition improves bendamustine therapy response and normalizes immune effector functions in chronic lymphocytic leukemia.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has been shown to be highly effective in patients with chronic lymphocytic leukemia (CLL) and is approved for CLL treatment. Unfortunately, resistance and intolerance to ibrutinib has been observed in several studies, opening the door for more specific BTK inhibitors. CC-292 (spebrutinib) is a BTK inhibitor with increased specificity for BTK and less inhibition of other kinases. Our in vitro studies showed that CC-292 potently inhibited B-cell receptor signaling, activation, proliferation and chemotaxis of CLL cells. In in vivo studies using the adoptive transfer TCL1 mouse model of CLL, CC-292 reduced tumor load and normalized tumor-associated expansion of T cells and monocytes, while not affecting T cell function. Importantly, the combination of CC-292 and bendamustine impaired CLL cell proliferation in vivo and enhanced the control of CLL progression. Our results demonstrate that CC-292 is a specific BTK inhibitor with promising performance in combination with bendamustine in CLL. Further clinical trials are warranted to investigate the therapeutic efficacy of this combination regimen.

Response duration and survival shorten after each relapse in patients with follicular lymphoma treated in the rituximab era.

Follicular lymphoma (FL) is an indolent disease characterized by long survival but frequent relapses. Before the introduction of rituximab, the clinical course of these patients showed a shorter response duration (RD) after each relapse. In this study, we analysed if this pattern of shortened responses remains in patients treated in the rituximab era. We selected 348 patients newly diagnosed with FL in two institutions between 2001 and 2014 that received chemoimmunotherapy. After a median follow-up of 6·3 years, 10-year progression-free and overall survivals were 53% and 72%, respectively. All patients received first-line, 111 second-line and 41 third-line treatments, with a 5-year RD of 62%, 39% and 24%, respectively (P < 0·0001). Variables predicting longer RD after first-line treatment were normal ß2microglobulin, complete remission achievement and maintenance with rituximab. Patients with longer RD after first-line showed significantly longer RD after second-line therapy. Autologous stem-cell transplantation after second-line therapy did not significantly impact RD. Median survival after first, second and third therapies was not reached, 7·6 and 4·8 years, respectively, whereas relative survival with respect to a sex- and age-matched Spanish population, the decrease in the life expectancy at 10 years was 17%, 45% and 79%, respectively. Thus, RD still shortens after each relapse in patients with FL treated in first line with rituximab combinations.

Mutations in the RAS-BRAF-MAPK-ERK pathway define a specific subgroup of patients with adverse clinical features and provide new therapeutic options in chronic lymphocytic leukemia.

Mutations in genes of the RAS-BRAF-MAPK-ERK pathway have not been fully explored in patients with chronic lymphocytic leukemia. We, therefore, analyzed the clinical and biological characteristics of chronic lymphocytic leukemia patients with mutations in this pathway and investigated the in vitro response of primary cells to BRAF and ERK inhibitors. Putative damaging mutations were found in 25 of 452 patients (5.5%). Among these, BRAF was mutated in nine patients (2.0%), genes upstream of BRAF (KITLG, KIT, PTPN11, GNB1, KRAS and NRAS) were mutated in 12 patients (2.6%), and genes downstream of BRAF (MAPK2K1, MAPK2K2, and MAPK1) were mutated in five patients (1.1%). The most frequent mutations were missense, subclonal and mutually exclusive. Patients with these mutations more frequently had increased lactate dehydrogenase levels, high expression of ZAP-70, CD49d, CD38, trisomy 12 and unmutated immunoglobulin heavy-chain variable region genes and had a worse 5-year time to first treatment (hazard ratio 1.8, P=0.025). Gene expression analysis showed upregulation of genes of the MAPK pathway in the group carrying RAS-BRAF-MAPK-ERK pathway mutations. The BRAF inhibitors vemurafenib and dabrafenib were not able to inhibit phosphorylation of ERK, the downstream effector of the pathway, in primary cells. In contrast, ulixertinib, a pan-ERK inhibitor, decreased phospho-ERK levels. In conclusion, although larger series of patients are needed to corroborate these findings, our results suggest that the RAS-BRAF-MAPK-ERK pathway is one of the core cellular processes affected by novel mutations in chronic lymphocytic leukemia, is associated with adverse clinical features and could be pharmacologically inhibited.

Tailored approaches grounded on immunogenetic features for refined prognostication in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) patients with differential somatic hypermutation status of the immunoglobulin heavy variable genes, namely mutated or unmutated, display fundamental clinico-biological differences. Considering this, we assessed prognosis separately within mutated (M-CLL) and unmutated (U-CLL) CLL in 3015 patients, hypothesizing that the relative significance of relevant indicators may differ between these two categories. Within Binet A M-CLL patients, besides TP53 abnormalities, trisomy 12 and stereotyped subset #2 membership were equivalently associated with the shortest time-to-first-treatment and a treatment probability at five and ten years after diagnosis of 40% and 55%, respectively; the remaining cases exhibited 5-year and 10-year treatment probability of 12% and 25%, respectively. Within Binet A U-CLL patients, besides TP53 abnormalities, del(11q) and/or SF3B1 mutations were associated with the shortest time-to-first-treatment (5- and 10-year treatment probability: 78% and 98%, respectively); in the remaining cases, males had a significantly worse prognosis than females. In conclusion, the relative weight of indicators that can accurately risk stratify early-stage CLL patients differs depending on the somatic hypermutation status of the immunoglobulin heavy variable genes of each patient. This finding highlights the fact that compartmentalized approaches based on immunogenetic features are necessary to refine and tailor prognostication in CLL.

Deleterious Effect of Steroids on Cytomegalovirus Infection Rate after Allogeneic Stem Cell Transplantation Depends on Pretransplant Cytomegalovirus Serostatus of Donors and Recipients.

This study examined the impact of prednisone (PDN) on cytomegalovirus (CMV) infection after allogeneic stem cell transplantation (allo-SCT) according to donor and recipient CMV serostatus. Seventy-five patients underwent allo-SCT from June 2010 to July 2012. The risk of CMV infection according to donor and recipient serostatus was defined as follows: high risk (HR; D-/R+), intermediate risk (IR; D+/R+ and D+/R-), and low risk (D-/R-). Forty-five patients (60%) developed CMV infection, and 46 patients (61%) received steroids (PDN ≥ 1 mg/kg/day) to treat acute graft-versus-host disease. CMV infection was more common in those treated with steroids than in those not treated with steroids (70% versus 44%, respectively, P < .05). Overall, 40% of patients had recurrent CMV infection (50% PDN versus 24% no PDN, P < .05). Steroids had no impact on the incidence of CMV infection or its recurrence in HR patients; however, steroids did prolong the need for antiviral treatment. The incidence of CMV infection in IR patients was higher in those receiving PDN (80% PDN versus 41% no PDN, P = .01); recurrence rates were also higher (55% PDN versus 18% no PDN, P = .02). We analyzed CMV-specific immune reconstitution in the first 22 patients of the series and observed that patients on steroids had lower levels of CMV-specific lymphocytes TCD8 (P < .05 on days +60, +100, and +180) and that CMV-specific immune reconstitution (defined as lymphocytes CD8/IFN ≥ 1 cell/µL) was achieved later (after day +100 post-SCT) in the steroid group.

Clinical impact of clonal and subclonal TP53, SF3B1, BIRC3, NOTCH1, and ATM mutations in chronic lymphocytic leukemia.

Genomic studies have revealed the complex clonal heterogeneity of chronic lymphocytic leukemia (CLL). The acquisition and selection of genomic aberrations may be critical to understanding the progression of this disease. In this study, we have extensively characterized the mutational status of TP53, SF3B1, BIRC3, NOTCH1, and ATM in 406 untreated CLL cases by ultra-deep next-generation sequencing, which detected subclonal mutations down to 0.3% allele frequency. Clonal dynamics were examined in longitudinal samples of 48 CLL patients. We identified a high proportion of subclonal mutations, isolated or associated with clonal aberrations. TP53 mutations were present in 10.6% of patients (6.4% clonal, 4.2% subclonal), ATM mutations in 11.1% (7.8% clonal, 1.3% subclonal, 2% germ line mutations considered pathogenic), SF3B1 mutations in 12.6% (7.4% clonal, 5.2% subclonal), NOTCH1 mutations in 21.8% (14.2% clonal, 7.6% subclonal), and BIRC3 mutations in 4.2% (2% clonal, 2.2% subclonal). ATM mutations, clonal SF3B1, and both clonal and subclonal NOTCH1 mutations predicted for shorter time to first treatment irrespective of the immunoglobulin heavy-chain variable-region gene (IGHV) mutational status. Clonal and subclonal TP53 and clonal NOTCH1 mutations predicted for shorter overall survival together with the IGHV mutational status. Clonal evolution in longitudinal samples mainly occurred in cases with mutations in the initial samples and was observed not only after chemotherapy but also in untreated patients. These findings suggest that the characterization of the subclonal architecture and its dynamics in the evolution of the disease may be relevant for the management of CLL patients.

Transcriptome characterization by RNA sequencing identifies a major molecular and clinical subdivision in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) has heterogeneous clinical and biological behavior. Whole-genome and -exome sequencing has contributed to the characterization of the mutational spectrum of the disease, but the underlying transcriptional profile is still poorly understood. We have performed deep RNA sequencing in different subpopulations of normal B-lymphocytes and CLL cells from a cohort of 98 patients, and characterized the CLL transcriptional landscape with unprecedented resolution. We detected thousands of transcriptional elements differentially expressed between the CLL and normal B cells, including protein-coding genes, noncoding RNAs, and pseudogenes. Transposable elements are globally derepressed in CLL cells. In addition, two thousand genes-most of which are not differentially expressed-exhibit CLL-specific splicing patterns. Genes involved in metabolic pathways showed higher expression in CLL, while genes related to spliceosome, proteasome, and ribosome were among the most down-regulated in CLL. Clustering of the CLL samples according to RNA-seq derived gene expression levels unveiled two robust molecular subgroups, C1 and C2. C1/C2 subgroups and the mutational status of the immunoglobulin heavy variable (IGHV) region were the only independent variables in predicting time to treatment in a multivariate analysis with main clinico-biological features. This subdivision was validated in an independent cohort of patients monitored through DNA microarrays. Further analysis shows that B-cell receptor (BCR) activation in the microenvironment of the lymph node may be at the origin of the C1/C2 differences.

Mutations in CHD2 cause defective association with active chromatin in chronic lymphocytic leukemia.

Great progress has recently been achieved in the understanding of the genomic alterations driving chronic lymphocytic leukemia (CLL). Nevertheless, the specific molecular mechanisms governing chromatin remodeling in CLL are unknown. Here we report the genetic and functional characterization of somatic mutations affecting the chromatin remodeler CHD2, one of the most frequently mutated genes in CLL (5.3%) and in monoclonal B lymphocytosis (MBL, 7%), a B-cell expansion that can evolve to CLL. Most of the mutations affecting CHD2, identified by whole-exome sequencing of 456 CLL and 43 MBL patients, are either truncating or affect conserved residues in functional domains, thus supporting a putative role for CHD2 as a tumor suppressor gene. CHD2 mutants show altered nuclear distribution, and a chromodomain helicase DNA binding protein 2 (CHD2) mutant affected in its DNA-binding domain exhibits defective association with active chromatin. Clinicobiological analyses show that most CLL patients carrying CHD2 mutations also present mutated immunoglobulin heavy chain variable region genes (IGHVs), being the most frequently mutated gene in this prognostic subgroup. This is the first study providing functional evidence supporting CHD2 as a cancer driver and opens the way to further studies of the role of this chromatin remodeler in CLL.