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Background: Randomised trials of rituximab maintenance (MR) for patients with follicular lymphoma support improved progression-free survival (PFS), but the effect on overall survival has been inconclusive. To evaluate the effect of MR on overall survival according to patient and disease characteristics, and to explore certain adverse events, we performed an individual patient data (IPD) meta-analysis., Methods: All investigators of randomised controlled trials that compared MR therapy with observation or treatment only at relapse (no MR) for patients with follicular lymphoma were invited to participate in an IPD meta-analysis. We obtained baseline patient and disease characteristics and time to progression and death for each patient. All analyses took into account the trial and original randomised treatment group. We analysed data in two ways: a two-stage analysis and a multivariate model including patient and disease characteristics., Findings: Seven trials including 2315 patients were analysed. Overall survival of patients improved with MR compared with no MR (hazard ratio [HR] 0.79, 95% CI 0.66-0.96). We could not detect any patient or disease characteristics that were associated with a survival benefit with MR. In all of the models, MR had a beneficial effect on overall survival compared with observation for all types of patients, which was not shown in a particular subgroup in which the patient had already received rituximab in the induction phase and received first-line therapy. MR improved PFS compared with observation (HR 0.57, 95% CI 0.51-0.64). The risk of adverse events was higher with MR, specifically infection of any grade and grade 3-4 infections., Interpretation: Based on IPD from randomised controlled trials, MR improves overall survival consistently in all patients, regardless of patient and disease characteristics when compared with observation, and should be prescribed after a successful induction with R-CVP or R-CHOP for patients with follicular lymphoma. It is still uncertain if that holds when the patient has already received rituximab in his/hers first induction. The effect of MR after bendamustine-rituximab induction compared with rituximab at progression should be further explored., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In the HOVON68 CLL trial, patients 65 to 75 years of age had no survival benefit from the addition of low-dose alemtuzumab to fludarabine and cyclophosphamide (FC) in contrast to younger patients. The reasons are explored in this 5-year trial update using both survival analysis and competing risk analysis on non-CLL-related mortality. Elderly FCA patients died more frequently from causes not related to CLL, and more often related to comorbidity (mostly cardiovascular) than to infection. In a Cox multivariate analysis, del(17p), performance status >0, and comorbidity were associated with a higher non-CLL-related mortality in the elderly independent of the treatment modality. Thus, while the 'fit' elderly with no comorbidity or performance status of 0 might potentially benefit from chemo-immunotherapy with FC, caution is warranted, when considering alemtuzumab treatment in elderly patients with cardiovascular comorbidity.

Invasive pulmonary mold disease (IPMD) is often fatal in neutropenic patients. This is because IPMD is difficult to diagnose timely, especially when non-Aspergillus molds are the causative agent, as they are usually not associated with a positive galactomannan assay. In 2013 we showed that exhaled breath analysis might be used to diagnose invasive aspergillosis through profiling of patterns in exhaled volatile organic compounds (VOCs) by electronic nose (eNose) technology. The current study aimed to determine (1) whether molds can be discriminated from other microorganisms (using two mold species: Aspergillus fumigatus and a pathogenic mold not associated with a positive galactomannan assay, i.c. Rhizopus oryzae) and (2) whether both molds can be discriminated from each other. First, we cultured strains of Streptococcus pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, A. fumigatus and R. oryzae in separate airtight bottles. We examined whether an eNose (Cyranose 320) could discriminate the headspaces of bottles with molds from those with bacteria/yeasts. Second, we examined whether an eNose could discriminate A. fumigatus and R. oryzae. Diagnostic algorithms were created using canonical discriminant analysis after principle component analysis. Primary outcome parameter was the validated accuracy. The eNose discriminated A. fumigatus from bacteria/yeasts with a cross-validated accuracy of 92.9% (sensitivity 95.2%, specificity 91.9%). The eNose had an accuracy (validated using split-half analysis) of 100% in discriminating A. fumigatus from R. oryzae. Our study suggests that an eNose can identify and classify molds in vitro. This warrants prospective in vivo studies aimed at detecting and classifying IPMD using exhaled breath.

Allogeneic stem cell transplantation (alloSCT) remains the only curative option for CLL patients. Whereas active disease at the time of alloSCT predicts poor outcome, no standard remission-induction regimen exists. We prospectively assessed outcome after cisplatin-containing immune-chemotherapy (R-DHAP) followed by alloSCT in 46 patients (median age 58 years) fulfilling modified European Society for Blood and Marrow Transplantation (EBMT) CLL Transplant Consensus criteria being refractory to or relapsed (R/R) <1 year after fludarabine or <2 years after fludarabine-based immunochemotherapy or R/R with del(17p). Twenty-nine patients received ⩾3 cycles of R-DHAP and sixteen <3 cycles (4 because of disease progression, 8 for toxicity and 4 toxic deaths). Overall rate of response to R-DHAP was 58%, 31 (67%) proceeded to alloSCT after conditioning with fludarabine and 2 Gy TBI. Twenty (65%) remained free from progression at 2 years after alloSCT, including 17 without minimal residual disease. Intention-to-treat 2-year PFS and overall survival of the 46 patients were 42 and 51% (35.5 months median follow-up); del(17p) or fludarabine refractoriness had no impact. R-DHAP followed by alloSCT is a reasonable treatment to be considered for high-risk CLL patients without access or resistance to targeted therapies.

Currently, there is no noninvasive test that can reliably diagnose early invasive pulmonary aspergillosis (IA). An electronic nose (eNose) can discriminate various lung diseases through an analysis of exhaled volatile organic compounds. We recently published a proof-of-principle study showing that patients with prolonged chemotherapy-induced neutropenia and IA have a distinct exhaled breath profile (or breathprint) that can be discriminated with an eNose. An eNose is cheap and noninvasive, and it yields results within minutes. We determined whether Aspergillus fumigatus colonization may also be detected with an eNose in cystic fibrosis (CF) patients. Exhaled breath samples of 27 CF patients were analyzed with a Cyranose 320. Culture of sputum samples defined the A. fumigatus colonization status. eNose data were classified using canonical discriminant analysis after principal component reduction. Our primary outcome was cross-validated accuracy, defined as the percentage of correctly classified subjects using the leave-one-out method. The P value was calculated by the generation of 100,000 random alternative classifications. Nine of the 27 subjects were colonized by A. fumigatus. In total, 3 subjects were misclassified, resulting in a cross-validated accuracy of the Cyranose detecting IA of 89% (P = 0.004; sensitivity, 78%; specificity, 94%). Receiver operating characteristic (ROC) curve analysis showed an area under the curve (AUC) of 0.89. The results indicate that A. fumigatus colonization leads to a distinctive breathprint in CF patients. The present proof-of-concept data merit external validation and monitoring studies., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Background: Addition of the anti-CD20 monoclonal antibody rituximab to chemotherapy improves response rates and survival in patients with B-cell non-Hodgkin lymphoma (NHL). However, rituximab induces a transient B-cell depletion and a dose-dependent T-cell inactivation that could impair T-cell immunosurveillance. The impact of rituximab on second primary malignancy (SPM) risk remains unclear so far. We thus carried out a systematic review to compare SPM risk among patients treated or not with rituximab., Patients and Methods: We retrieved trials from MEDLINE and EMBASE and updated data presented at American Society of Hematology and American Society of Clinical Oncology meetings from 1998 to 2013. We selected randomized, controlled trials addressing newly or relapsed/progressive B-cell NHL in which randomization arms differed only from rituximab administration. Two authors extracted data and assessed the study quality., Results: We analyzed nine trials involving 4621 patients. At a median follow-up of 73 months, a total of 169 SPMs were observed in patients randomized to rituximab compared with 165 SPMs in patients not randomized to rituximab (OR = 0.88; 95% CI 0.66-1.19). The proportion of females, histology subtypes, use of rituximab in first line or in maintenance did not influence SPM risk (P = 0.94, P = 0.80, P = 0.87, P = 0.87, respectively). Cumulative exposure through prolonged administration in trials with rituximab maintenance did not contribute to an increased risk of SPM (P = 0.86)., Conclusion: Our meta-analysis suggests no SPM predisposition among NHL survivors exposed to rituximab at a median follow-up of 6 years., (© The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Purpose: To describe the health-related quality of life (HRQoL) of an unselected population of patients with chronic lymphocytic leukaemia (CLL) including untreated patients., Methods: HRQoL was measured by the EORTC QLQ-C30 including the CLL16 module, EQ-5D, and VAS in an observational study over multiple years. All HRQoL measurements per patient were connected and analysed using area under the curve analysis over the entire study duration. The total patient group was compared with the general population, and three groups of CLL patients were described separately, i.e. patients without any active treatment ("watch and wait"), chlorambucil treatment only, and patients with other treatment(s)., Results: HRQoL in the total group of CLL patients was compromised when compared with age- and gender-matched norm scores of the general population. CLL patients scored statistically worse on the VAS and utility score of the EQ-5D, all functioning scales of the EORTC QLQ-C30, and the symptoms of fatigue, dyspnoea, sleeping disturbance, appetite loss, and financial difficulties. In untreated patients, the HRQoL was slightly reduced. In all treatment stages, HRQoL was compromised considerably. Patients treated with chlorambucil only scored worse on the EORTC QLQ-C30 than patients who were treated with other treatments with regard to emotional functioning, cognitive functioning, bruises, uncomfortable stomach, and apathy., Conclusions: CLL patients differ most from the general population on role functioning, fatigue, concerns about future health, and having not enough energy. Once treatment is indicated, HRQoL becomes considerably compromised. This applies to all treatments, including chlorambucil, which is considered to be a mild treatment.

Background: Ofatumumab is a human anti-CD20 monoclonal antibody that has proven efficacy as monotherapy in refractory chronic lymphocytic leukaemia. We assessed the efficacy and safety of ofatumumab maintenance treatment versus observation for patients in remission after re-induction treatment for relapsed chronic lymphocytic leukaemia., Methods: This open-label, multicentre, randomised phase 3 study enrolled patients aged 18 years or older from 130 centres in 24 countries who had chronic lymphocytic leukaemia in complete or partial remission after second-line or third-line treatment. Eligible patients had a WHO performance status of 0-2, had a response assessment within the previous 3 months, did not have refractory disease, autoimmune haemolytic anaemia requiring treatment, chronic or active infection requiring treatment, and had not previously received maintenance treatment or autologous or allogeneic stem-cell transplant. Using a randomisation list generated by a central computerised system and an interactive voice recognition system, we randomly assigned (1:1) patients to receive ofatumumab (300 mg followed by 1000 mg 1 week later and every 8 weeks for up to 2 years) or undergo observation. Randomisation was stratified by number and type of previous treatment and remission status after induction treatment (block size of four). Treatment assignment was open label. The primary endpoint was investigator-assessed progression-free survival in the intention-to-treat population. We report the results of a prespecified interim analysis after two-thirds of the planned study events (disease progression or death) had happened. This trial is closed to accrual but follow-up is ongoing. This trial is registered with ClinicalTrials.gov, number NCT00802737., Findings: Between May 6, 2010, and June 19, 2014, we enrolled 474 patients: 238 patients were randomly assigned to receive ofatumumab maintenance treatment and 236 to undergo observation. One (<1%) patient in the ofatumumab group did not receive the allocated intervention (withdrawal of consent). The median follow-up was 19·1 months (IQR 10·3-28·8). Progression-free survival was improved in patients assigned to the ofatumumab group (29·4 months, 95% CI 26·2-34·2) compared with those assigned to observation (15·2 months, 11·8-18·8; hazard ratio 0·50, 95% CI 0·38-0·66; p<0·0001). The most common grade 3 or higher adverse events up to 60 days after last treatment were neutropenia (56 [24%] of 237 patients in the ofatumumab group vs 23 [10%] of 237 in the observation group) and infections (31 [13%] vs 20 [8%]). 20 (8%) of 237 patients in the ofatumumab group and three (1%) of 237 patients in the observation group had adverse events that led to permanent discontinuation of treatment. Up to 60 days after last treatment, two deaths related to adverse events occurred in the ofatumumab treatment group and five deaths related to adverse events occurred in the observation group; no deaths were attributed to the study drug., Interpretation: These data are important for the development of optimum maintenance strategies in patients with relapsed chronic lymphocytic leukaemia, notably in the present era of targeted drugs, many of which are to be used until progression., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The ATM-p53 DNA-damage response (DDR) pathway has a crucial role in chemoresistance in CLL, as indicated by the adverse prognostic impact of genetic aberrations of TP53 and ATM. Identifying and distinguishing TP53 and ATM functional defects has become relevant as epigenetic and posttranscriptional dysregulation of the ATM/p53 axis is increasingly being recognized as the underlying cause of chemoresistance. Also, specific treatments sensitizing TP53- or ATM-deficient CLL cells are emerging. We therefore developed a new ATM-p53 functional assay with the aim to (i) identify and (ii) distinguish abnormalities of TP53 versus ATM and (iii) enable the identification of additional defects in the ATM-p53 pathway. Reversed transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) was used to measure ATM and/or p53-dependent genes at the RNA level following DNA damage using irradiation. Here, we showed that this assay is able to identify and distinguish three subgroups of CLL tumors (i.e., TP53-defective, ATM-defective and WT) and is also able to detect additional samples with a defective DDR, without molecular aberrations in TP53 and/or ATM. These findings make the ATM-p53 RT-MLPA functional assay a promising prognostic tool for predicting treatment responses in CLL.

A single-center, prospective, non-randomized clinical study was performed to examine the safety and feasibility of early discharge in patients undergoing consolidation chemotherapy for acute leukemia, or autologous stem cell transplant for lymphoma or multiple myeloma. Patients were discharged into ambulatory care the day after the last chemotherapy administration and were subsequently seen at the ambulatory care unit three times a week. One hundred and one of 224 patients were ineligible for the program, mostly because of their medical situation, the lack of a caregiver or the travel time to the hospital. The remaining 123 patients were able to spend more than 70% of the time at home. In 44% of cycles they were never readmitted. This study demonstrates the safety, feasibility and benefits of managing carefully selected patients. Patients and their caregivers felt safe and comfortable at home, and the vast majority preferred home care to in-hospital treatment.

Mutations or deletions in TP53 or ATM are well-known determinants of poor prognosis in chronic lymphocytic leukemia (CLL), but only account for approximately 40% of chemo-resistant patients. Genome-wide sequencing has uncovered novel mutations in the splicing factor sf3b1, that were in part associated with ATM aberrations, suggesting functional synergy. We first performed detailed genetic analyses in a CLL cohort (n=110) containing ATM, SF3B1 and TP53 gene defects. Next, we applied a newly developed multiplex assay for p53/ATM target gene induction and measured apoptotic responses to DNA damage. Interestingly, SF3B1 mutated samples without concurrent ATM and TP53 aberrations (sole SF3B1) displayed partially defective ATM/p53 transcriptional and apoptotic responses to various DNA-damaging regimens. In contrast, NOTCH1 or K/N-RAS mutated CLL displayed normal responses in p53/ATM target gene induction and apoptosis. In sole SF3B1 mutated cases, ATM kinase function remained intact, and γH2AX formation, a marker for DNA damage, was increased at baseline and upon irradiation. Our data demonstrate that single mutations in sf3b1 are associated with increased DNA damage and/or an aberrant response to DNA damage. Together, our observations may offer an explanation for the poor prognosis associated with SF3B1 mutations.

In a prospective multicenter phase II study, we evaluated the effect of three courses of vincristine, doxorubicin and dexamethasone followed by high-dose melphalan and autologous stem cell transplantation on an intention-to-treat basis. Sixty-nine newly diagnosed patients with amyloid light chain amyloidosis were included between November 2000 and January 2006: 37 men and 32 women with a median age of 56 years, including 46% of patients with cardiac and 22% of patients with involvement of 3 or 4 organs. Initial results presented in 2008 showed a 4-year overall survival rate of 62% among all the patients, while the 4-year survival rate after transplantation was 78%. Here we report the long-term follow-up data after a median follow up of 115 months of the patients still alive. Median survival of all patients was 96 months from registration and for the transplanted patients ten years from the date of transplantation. Twelve (12%) patients died during induction therapy with vincristine, doxorubicin and dexamethasone, including 8 patients (12%) due to treatment-related mortality. Two patients died within one month following high-dose melphalan. We conclude that vincristine, doxorubicin and dexamethasone should not be applied as induction therapy for intensification in amyloid light chain amyloidosis. However, a 2-step approach consisting of a non-intensive less toxic induction therapy followed by high-dose melphalan and autologous stem cell transplantation may result in extended survival in newly diagnosed patients with amyloid light chain amyloidosis (clinicaltrials.gov identifier: 01207094)., (Copyright© Ferrata Storti Foundation.)

In chronic lymphocytic leukemia (CLL), strategies to overcome drug resistance due to p53 dysfunction are highly needed. Platinum-based compounds such as cisplatinum (CDDP) are active in fludarabine-refractory CLL through a largely unknown mechanism. We analyzed the mechanism of action of CDDP in the context of p53 dysfunctionality. In vitro treatment with CDDP did not induce death in quiescent CLL cells, but did induce apoptosis in CD40-ligand (and CpG) stimulated and proliferating cells, irrespective of p53 function. In the p53 dysfunctional prolymphocytic cell-line MEC1, CDDP treatment resulted in apoptosis, cell cycle arrest and ABL1-dependent expression of TAp73, CDKN1A, PUMA and BID. TAp73 RNA-interference decreased sensitivity to CDDP. Finally, both in vitro stimulated CLL cells and lymph node (LN) derived CLL cells showed increased TAp73 expression in comparison with quiescent peripheral blood derived cells. Activity of CDDP may therefore be mediated by TAp73, especially in the context of activation such as occurs in the LN microenvironment.

Allogeneic hematopoietic stem cell transplantation (HSCT) is widely used to treat hematopoietic cell disorders but is often complicated by graft-versus-host disease (GVHD), which causes severe epithelial damage. Here we have investigated longitudinally the effects of induction chemotherapy, conditioning radiochemotherapy, and allogeneic HSCT on composition, phenotype, and recovery of circulating innate lymphoid cells (ILCs) in 51 acute leukemia patients. We found that reconstitution of ILC1, ILC2, and NCR(-)ILC3 was slow compared with that of neutrophils and monocytes. NCR(+) ILC3 cells, which are not present in the circulation of healthy persons, appeared both after induction chemotherapy and after allogeneic HSCT. Circulating patient ILCs before transplantation, as well as donor ILCs after transplantation, expressed activation (CD69), proliferation (Ki-67), and tissue homing markers for gut (α4β7, CCR6) and skin (CCR10 and CLA). The proportion of ILCs expressing these markers was associated with a decreased susceptibility to therapy-induced mucositis and acute GVHD. Taken together, these data suggest that ILC recovery and treatment-related tissue damage are interrelated and affect the development of GVHD., (© 2014 by The American Society of Hematology.)

The randomized Haemato Oncology Foundation for Adults in The Netherlands 68 phase 3 trial compared front-line chemotherapy with chemotherapy plus the CD52 monoclonal antibody alemtuzumab for high-risk chronic lymphocytic leukemia, defined as at least 1 of the following: unmutated immunoglobulin heavy chain genes, deletion 17p or 11q, or trisomy 12. Fit patients were randomized to receive either 6 28-day cycles of oral FC chemotherapy (days 1 through 3: fludarabine 40 mg/m(2) per day and cyclophosphamide 250 mg/m(2) per day: n = 139) or FC plus subcutaneous alemtuzumab 30 mg day 1 (FCA, n = 133). FCA prolonged the primary end point, progression-free survival (3-year progression-free survival 53 vs 37%, P = .01), but not the secondary end point, overall survival (OS). However, a post hoc analysis showed that FCA increased OS in patients younger than 65 years (3-year OS 85% vs 76%, P = .035). FCA also increased the overall response rate (88 vs 78%, P = .036), and the bone marrow minimal residual disease-negative complete remission rate (64% vs 43%, P = .016). Opportunistic infections were more frequent following FCA, but without an increase in treatment related mortality (FCA: 3.8%, FC: 4.3%). FCA improves progression-free survival in high-risk chronic lymphocytic leukemia. As anticipated, FCA is more immunosuppressive than FC, but with due vigilance, does not lead to a higher treatment-related mortality. This study was registered at www.trialregister.nl as trial no. NTR529., (© 2014 by The American Society of Hematology.)

In chronic lymphocytic leukemia (CLL), CD8(+) T cells exhibit features of exhaustion and impaired functionality. Yet, reactivations of latent viruses such as cytomegalovirus (CMV) are uncommon in untreated CLL, suggesting that antiviral responses are uncompromised. We analyzed phenotypical and functional characteristics of CMV-specific CD8(+) T cells in CLL patients in comparison with age-matched healthy controls (HCs). Despite increased expression of the inhibitory receptors PD1, CD160, and CD244 on total CD8(+) T cells in CLL, expression levels of these markers were decreased on CMV-tetramer(+)CD8(+) T cells. Second, cytokine production upon stimulation with both phorbol 12-myristate 13-acetate/ionomycin and CMV-peptide-loaded antigen-presenting cells was intact in CMV-tetramer(+)CD8(+) T cells. Third, CMV-tetramer(+)CD8(+) T cells of CLL patients and HCs were equally effective in killing CMV-peptide-loaded target cells. Finally, quantitative imaging flow cytometry revealed that the proportion of CD8(+) T cells forming immunologic synapses with CMV-peptide-loaded B cells was intact. In conclusion, despite evidence for global T-cell dysfunction in CLL, we show here that CLL-derived CMV-specific CD8(+) T cells display lower expression of exhaustion markers and are functionally intact. These data indicate that the changes in the T-cell compartment in CLL may be more heterogeneous than presently assumed.

Resistance to chemotherapy-induced apoptosis in CLL is associated with overexpression of antiapoptotic proteins induced by signals from the microenvironment. In vitro, dasatinib effectively inhibits expression of anti-apoptotic regulators and restores fludarabine sensitivity in activated CLL. The aim of this study was to evaluate efficacy of one cycle of dasatinib monotherapy (100mg/day, days 1-28) followed by combination of dasatinib with fludarabine (40mg/m²/day, days 1-3 every 28 day) for a total of 6 cycles in fludarabine-refractory CLL. The primary endpoint was overall response rate according to the IWCLL'08 criteria. 20 patients were enrolled: 18 completed at least one cycle of treatment of which 67% finished at least 2 cycles of combination treatment. 3 of these 18 patients reached a formal PR (16.7%). Majority of patients obtained some reduction in lymph node (LN) size. Most frequent toxicity was related to myelosuppression. NF-κB RNA expression levels of circulating CLL cells decreased whereas the levels of pro-apoptotic NOXA increased during treatment. In conclusion, dasatinib/fludarabine combination has modest clinical efficacy in fludarabine-refractory patients., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We performed a comprehensive cost calculation identifying the main cost drivers of treatment of chronic lymphocytic leukaemia in daily practice. In our observational study 160 patient charts were reviewed repeatedly to assess the treatment strategies from diagnosis till the study end. Ninety-seven patients (61%) received ≥1 treatment lines during an average follow-up time of 6.4 years. The average total costs per patient were €41,417 (€539 per month). The costs varied considerably between treatment groups and between treatment lines. Although patients were treated with expensive chemo(immuno-)therapy, the main cost driver was inpatient days for other reasons than administration of chemo(immuno-)therapy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Although in vitro studies pointed to the tumor necrosis factor family member APRIL (a proliferation-inducing ligand) in mediating survival of chronic lymphocytic leukemia (CLL) cells, clear evidence for a role in leukemogenesis and progression in CLL is lacking. APRIL significantly prolonged in vitro survival of CD5(+)B220(dull) leukemic cells derived from the murine Eμ-TCL1-Tg (TCL1-Tg [transgenic]) model for CLL. APRIL-TCL1 double-Tg mice showed a significantly earlier onset of leukemia and disruption of splenic architecture, and survival was significantly reduced. Interestingly, clonal evolution of CD5(+)B220(dull) cells (judged by BCR clonality) did not seem to be accelerated by APRIL; both mouse strains were oligoclonal at 4 months. Although APRIL binds different receptors, APRIL-mediated leukemic cell survival depended on tumor necrosis factor receptor superfamily member 13B (TACI) ligation. These findings indicate that APRIL has an important role in CLL and that the APRIL-TACI interaction might be a selective novel therapeutic target for human CLL.

Chronic lymphocytic leukemia (CLL) cells multiply in secondary lymphoid tissue, but the mechanisms leading to their proliferation are still uncertain. In addition to B-cell receptor (BCR)-triggered signals, other microenvironmental factors might well be involved. In proliferation centers, leukemic B cells are in close contact with CD4(+)CD40L(+) T cells. Therefore, we here dissected the signals provided by autologous activated T cells (Tact) to CLL cells. Although the gene expression profile induced by Tact was highly similar to that induced by sole CD40 signaling, an obvious difference was that Tact induced proliferation of CLL cells. We determined that stimulation with only CD40L+IL-21 was sufficient to induce robust proliferation in CLL cells. We then defined an interleukin (IL)-21-induced gene signature in CLL, containing components of Janus kinase/signal transducer and activator of transcription and apoptosis pathways, and this signature could be detected in lymph node (LN) samples from patients. Finally, we could detect IL-21 RNA and protein in LN, and IL-21 production ex vivo by LN CD4(+)CXCR5(+) follicular helper T cells. These results indicate that in addition to BCR signaling, activated T cells might contribute to CLL cell proliferation via CD40 and IL-21. Targeting these signaling pathways might offer new venues for treatment of CLL.

In the lymph node (LN) environment, chronic lymphocytic leukemia (CLL) cells display increased NF-κB activity compared with peripheral blood CLL cells, which contributes to chemoresistance. Antagonists of cellular inhibitor of apoptosis proteins (cIAPs) can induce apoptosis in various cancer cells in a tumor necrosis factor-α (TNFα)-dependent manner and are in preclinical development. Smac-mimetics promote degradation of cIAP1 and cIAP2, which results in TNFR-mediated apoptosis via formation of a ripoptosome complex, comprising RIPK1, Fas-associated protein with death domain, FLICE-like inhibitory protein and caspase-8. CD40 stimulation of CLL cells in vitro is used as a model to mimic the LN microenvironment and results in NF-κB activation and TNFα production. In this study, we investigated the response of CLL cells to smac-mimetics in the context of CD40 stimulation. We found that treatment with smac-mimetics results in cIAP1 and cIAP2 degradation, yet although TNFα is produced, this did not induce apoptosis. Despite the presence of all components, the ripoptosome complex did not form upon smac-mimetic treatment in CLL cells. Thus, CLL cells seem to possess aberrant upstream NF-κB regulation that prevents ripoptosome formation upon IAP degradation. Unraveling the exact molecular mechanisms of disturbed ripoptosome formation may offer novel targets for treatment in CLL.

The ATM-p53 DNA damage response pathway plays a crucial role in chemoresistance in chronic lymphocytic leukemia, as indicated by the adverse prognostic impact of deletions of 17p (locus of TP53) and 11q (locus of ATM) detected by fluorescence in situ hybridization (FISH) analysis. In addition to deletions, mutations in these respective genes are also associated with chemoresistance, and add to the prognostic information provided by FISH. In order to explore the possibility that dysfunction of the ATM-p53 pathway might also result from mechanisms other than ATM/TP53 deletion/mutation, assays have been developed that probe the functional integrity of the ATM-p53 pathway. Currently, four different p53 function assays have been developed that are based on the measurement of p53 and p53-dependent genes at the RNA (real-time polymerase chain reaction [RT-PCR]p21; RT-PCRmiR34a; reverse transcription-multiplex ligation-dependent probe amplification assay [RT-MLPA]p21, bax, puma and CD95) or protein (fluorescence activated cell sorting [FACS]p53-p21) level in untreated cells or following irradiation or drug treatment. Here we provide an overview of these assays based on the available literature.

Chronic lymphocytic leukemia (CLL) is still incurable, with considerable resistance to the standard therapy. CLL cells receive anti-apoptotic and pro-proliferation stimuli in lymph nodes and bone marrow, mainly through B cell receptor activation and TNF-receptor family ligation. In recent years, the focus for finding new drugs has shifted to blocking signals from the microenvironment. Novel therapeutical agents interfere with these microenvironmental interactions, and include inhibitors of kinases Syk, Btk and PI3Kδ. In this review we will focus on the microenvironmental interactions of CLL and the role of tyrosine kinases. Furthermore, early results from clinical trials with kinase inhibitors are discussed., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In patients with chronic lymphocytic leukemia (CLL), effector and memory CD4 + and CD8 + T cells are expanded. We investigated whether these CLL specific T-cell expansions also occur in monoclonal B lymphocytosis (MBL), the pre-leukemic state of CLL, which is currently distinguished from CLL by an arbitrarily chosen cut-off value of CD19 of 5.0 × 10(9)/L. Whereas an increase in effector and memory CD4 + and CD8 + T cells was found in CLL, these expansions could not be found in MBL. Although a significant correlation was found between absolute B cell count (CD19) and T cell numbers, correlation coefficients were rather low. Therefore, we analyzed whether an optimal threshold for CD19 number could be defined which best related to an expansion of T cells. The B-cell threshold that best predicted expansion of CD3 +, CD4 + and CD8 + T cells, respectively, was 10 × 10(9)/L. Our study indicates that a higher cut-off value than the current 5.0 × 10(9)/L relates better to the biological impact of CLL.

In patients with chronic lymphocytic leukemia (CLL), numbers of CD8 + CD45RA +/- CD27- effector T cells are expanded. We investigated whether this expansion is also present in other B cell malignancies and the possible mechanism underlying these changes. Whereas an increase in total CD4+and CD8+ T cell numbers was found only in CLL, numbers of CD4+ and CD8+ effector T cells were significantly increased in both CLL and indolent lymphoma, but not aggressive lymphoma and myeloma. Interestingly, PD-1 expression was decreased on effector T cells and inversely correlated with effector T cell numbers, suggesting a functional role for PD-1 in regulating T cell homeostasis. In vitro experiments revealed impaired up-regulation of PD-1 upon T cell activation in the presence of malignant but also healthy B cells. Our data suggest that in CLL and indolent lymphoma, the malignant B cells affect PD-1 expression on effector T cells, resulting in an expansion of these subsets.

Monoclonal B-cell lymphocytosis (MBL) is defined by the presence of small B-cell clones in asymptomatic individuals. Usually, MBL cells are characterised by a chronic lymphocytic leukaemia (CLL) phenotype ('CLL phenotype MBL'); however, an atypical phenotype ('atypical-CLL phenotype MBL') or non-Hodgkin lymphoma phenotype ('non-CLL phenotype MBL') can be found as well. The prevalence of MBL in the general population with an age over 40 years is 3 to 5%. Subjects with MBL develop CLL requiring treatment at a rate of 1 to 2% per year. At the moment official guidelines with respect to MBL are not available in the Netherlands. On the basis of the available data, we will discuss the definitions of MBL , highlight clinical consequences and offer recommendations for daily practice. Individuals with clinically suspected MBL should undergo a complete evaluation by a haematologist. In case of CLL phenotype MBL , further annual follow-up can take place by the general practitioner. If signs of progression occur patients should be referred to a haematologist.

Purpose: Chronic lymphocytic leukemia (CLL) cells in lymph nodes (LN), from which relapses are postulated to originate, display an antiapoptotic profile in contrast to CLL cells from peripheral blood (PB). The BH3 mimetic ABT-737 antagonizes the antiapoptotic proteins Bcl-X(L) and Bcl-2 but not Mcl-1 or Bfl-1. Previously, it was shown that CD40-stimulated CLL cells were resistant to ABT-737. We aimed to define which antiapoptotic proteins determine resistance to ABT-737 in CLL and whether combination of known antileukemia drugs and ABT-737 was able to induce apoptosis of CD40-stimulated CLL cells., Experimental Design: To mimic the LN microenvironment, PB lymphocytes of CLL patients were cultured on feeder cells expressing CD40L and treated with ABT-737 with or without various drugs. In addition, we carried out overexpression or knockdown of pro- and antiapoptotic proteins in immortalized primary B cells., Results: Upon CD40 stimulation patient-specific variations in ABT-737 sensitivity correlated with differences in levels of Mcl-1 and its antagonist Noxa. Knockdown of Noxa, as well as Mcl-1 overexpression, corroborated the importance of the Noxa/Mcl-1 ratio in determining the response to ABT-737. Inhibition of NF-κB resulted in increased Noxa levels and enhanced sensitivity to ABT-737. Interestingly, increasing the Noxa/Mcl-1 ratio, by decreasing Mcl-1 (dasatinib and roscovitine) or increasing Noxa levels (fludarabine and bortezomib), resulted in synergy with ABT-737., Conclusions: Thus, the Noxa/Mcl-1 balance determines sensitivity to ABT-737 in CD40-stimulated CLL cells. These data provide a rationale to investigate the combination of drugs which enhance the Noxa/Mcl-1 balance with ABT-737 to eradicate CLL in chemoresistant niches., (©2011 AACR.)

Background: Studies in cardiac surgery have reported increased postoperative morbidity and mortality after allogeneic red blood cell (RBC) transfusions. Whether platelet (PLT) and/or plasma transfusions are a marker for more concomitant RBC transfusions or are independently associated with complications after cardiac surgery is unknown., Study Design and Methods: Data from two randomized controlled studies were combined to analyze the effects of PLT and/or plasma transfusions on postoperative infections, length of stay in the intensive care unit (ICU), all-cause mortality, and mortality in the presence or absence of infections in the postoperative period., Results: After adjusting for confounding factors, plasma units and not RBC transfusions were associated with all-cause mortality. White blood cell (WBC)-containing RBC transfusions and PLT transfusions were associated with mortality occurring in the presence of or after infections. The number of (WBC-containing) RBC transfusions was also significantly associated with postoperative infections and with ICU stay for 4 or more days., Conclusion: Although it is difficult to separate the effects of blood components, we found that in cardiac surgery, perioperative plasma transfusions are independently associated with all-cause mortality. WBC-containing RBC transfusions and PLT transfusions are independently associated with mortality in the presence of infections in the postoperative period. Future transfusion studies in cardiac surgery should concomitantly consider the possible adverse effects of all the various transfused blood components., (© 2011 American Association of Blood Banks.)

Background: Although new therapeutic strategies have been developed to control Crohn's disease, medical treatment for refractory cases is not able to prevent extensive and/or repeat surgery. Recently, several cases have been reported of successful remission induction in Crohn's disease patients by means of hematopoietic stem cell transplantation (HSCT). Here we report our long-term (4 to 6 years) outcome in three patients., Patients: Three patients (two male, one female) with active severe Crohn's disease were planned to undergo autologous HSCT. All patients were intolerant or refractory to conventional therapies, including anti-TNFα antibodies. Patients either refused surgery or surgery was considered not to be a feasible alternative due to the extensive disease involvement of the small intestine., Methods: Peripheral blood stem cells were mobilized using a single infusion of cyclophosphamide 4 g/m(2), followed on day 4 by subcutaneous injections with G-CSF 5 μg/kg twice daily until leukapheresis. CD34+ cells were isolated after leukapheresis by magnetic cell sorting. In two of the three patients a second round of stem cell mobilization using G-CSF only was required, either because of low yield or because of insufficient recovery after CD34 selection. Prior to transplantation, immune ablation was achieved using cyclophosphamide 50mg/kg/day (4 days), antithymocyte globulin 30 mg/kg/day (3 days) and prednisolone 500 mg (3 days). Endoscopy, barium small bowel enteroclysis and MRI enterography were performed., Results: All three patients successfully completed stem cell mobilization, and two of them subsequently underwent conditioning and autologous HSCT with CD34+ cell selection. Treatment was well tolerated, with acceptable toxicity. Now, 5 and 6 years post-transplantation, these patients are in remission under treatment. The third patient went into remission after mobilization and therefore she decided not to undergo conditioning and HSCT transplantation. After a successful pregnancy she relapsed two years later. Since then, she suffers from refractory Crohn's disease for which we are now reconsidering conditioning and transplantation., Conclusion: Autologous HSCT appears to be safe and can be an alternative strategy for Crohn's disease patients with severe and therapy resistant disease., (Copyright © 2011 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.)

Sensitivity of chronic lymphocytic leukemia (CLL) cells to anti-CD20 mAbs is low and, therefore, the efficacy of monotherapy with current anti-CD20 mAbs is limited. At present, it is not known whether sensitivity of CLL cells to CD20 mAbs is modulated by microenvironmental stimuli. We have shown previously that in vitro CD40 stimulation of peripheral blood-derived CLL cells results in resistance to cytotoxic drugs. In the present study, we show that, in contrast, CD40 stimulation sensitizes CLL cells to the recently described novel type II anti-CD20 mAb GA101. Cell death occurred without cross-linking of GA101 and involved a lysosome-dependent mechanism. Combining GA101 with various cytotoxic drugs resulted in additive cell death, not only in CD40-stimulated CLL cells, but also in p53-dysfunctional CLL cells. Our findings indicate that GA101 has efficacy against chemoresistant CLL, and provide a rationale for combining cytotoxic drugs with anti-CD20 mAbs.

One of the principal responsibilities of the Chronic Lymphocytic Leukaemia (CLL) Working Party of the Dutch/Belgium Haemato-Oncology Foundation for Adults in the Netherlands (HOVON) is to create up-to-date guidelines for CLL . In this article, the revised guidelines for diagnosis and treatment are summarised. Despite recent expansion in treatment options for patients with CLL , the disease remains incurable in most cases and the optimal treatment approach for several subgroups of patients is still unclear. Therefore, it remains highly important to treat patients within clinical studies as much as possible. In this article, the current studies initiated by the HOVON CLL working party are emphasised.