Profiling of volatile organic compounds produced by clinical Aspergillus isolates using gas chromatography-mass spectrometry.

Volatile organic compounds (VOCs) in exhaled breath may identify the presence of invasive pulmonary aspergillosis. We aimed to detect VOC profiles emitted by in vitro cultured, clinical Aspergillus isolates using gas chromatography-mass spectrometry (GC-MS). Three clinical Aspergillus isolates and a reference strain were cultured while conidiation was prevented. Headspace samples were analyzed using a standardized method. Breath samples of patients from which the cultures were obtained were checked for the presence of the VOCs found in vitro. Each Aspergillus isolate produced a distinct VOC profile. These profiles could not be confirmed in exhaled breath in vivo.

International Working Group consensus response evaluation criteria in lymphoma (RECIL 2017).

In recent years, the number of approved and investigational agents that can be safely administered for the treatment of lymphoma patients for a prolonged period of time has substantially increased. Many of these novel agents are evaluated in early-phase clinical trials in patients with a wide range of malignancies, including solid tumors and lymphoma. Furthermore, with the advances in genome sequencing, new "basket" clinical trial designs have emerged that select patients based on the presence of specific genetic alterations across different types of solid tumors and lymphoma. The standard response criteria currently in use for lymphoma are the Lugano Criteria which are based on [18F]2-fluoro-2-deoxy-D-glucose positron emission tomography or bidimensional tumor measurements on computerized tomography scans. These differ from the RECIST criteria used in solid tumors, which use unidimensional measurements. The RECIL group hypothesized that single-dimension measurement could be used to assess response to therapy in lymphoma patients, producing results similar to the standard criteria. We tested this hypothesis by analyzing 47 828 imaging measurements from 2983 individual adult and pediatric lymphoma patients enrolled on 10 multicenter clinical trials and developed new lymphoma response criteria (RECIL 2017). We demonstrate that assessment of tumor burden in lymphoma clinical trials can use the sum of longest diameters of a maximum of three target lesions. Furthermore, we introduced a new provisional category of a minor response. We also clarified response assessment in patients receiving novel immune therapy and targeted agents that generate unique imaging situations.

eNose technology can detect and classify human pathogenic molds in vitro: a proof-of-concept study of Aspergillus fumigatus and Rhizopus oryzae.

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.

Efficacy of cisplatin-based immunochemotherapy plus alloSCT in high-risk chronic lymphocytic leukemia: final results of a prospective multicenter phase 2 HOVON study.

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.

The pan phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor SAR245409 (voxtalisib/XL765) blocks survival, adhesion and proliferation of primary chronic lymphocytic leukemia cells.

The phosphoinositide 3-kinases (PI3Ks) are critical components of the B-cell receptor (BCR) pathway and have an important role in the pathobiology of chronic lymphocytic leukemia (CLL). Inhibitors of PI3Kδ block BCR-mediated cross-talk between CLL cells and the lymph node microenvironment and provide significant clinical benefit to CLL patients. However, the PI3Kδ inhibitors applied thus far have limited direct impact on leukemia cell survival and thus are unlikely to eradicate the disease. The use of inhibitors of multiple isoforms of PI3K might lead to deeper remissions. Here we demonstrate that the pan-PI3K/mammalian target of rapamycin inhibitor SAR245409 (voxtalisib/XL765) was more pro-apoptotic to CLL cells--irrespective of their ATM/p53 status--than PI3Kα or PI3Kδ isoform selective inhibitors. Furthermore, SAR245409 blocked CLL survival, adhesion and proliferation. Moreover, SAR245409 was a more potent inhibitor of T-cell-mediated production of cytokines, which support CLL survival. Taken together, our in vitro data provide a rationale for the evaluation of a pan-PI3K inhibitor in CLL patients.

Detection of Airway Colonization by Aspergillus fumigatus by Use of Electronic Nose Technology in Patients with Cystic Fibrosis.

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.

Rituximab and risk of second primary malignancies in patients with non-Hodgkin lymphoma: a systematic review and meta-analysis.

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.

Assessment of p53 and ATM functionality in chronic lymphocytic leukemia by multiplex ligation-dependent probe amplification.

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.

Quality of life of patients with chronic lymphocytic leukaemia in the Netherlands: results of a longitudinal multicentre study.

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.

The impact of SF3B1 mutations in CLL on the DNA-damage response.

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.

Real-world costs of chronic lymphocytic leukaemia in the Netherlands.

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.

Monoclonal B-cell lymphocytosis: recommendations from the Dutch Working Group on CLL for daily practice.

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.

Dutch guidelines for diagnosis and treatment of chronic lymphocytic leukaemia 2011.

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.

CD40 stimulation sensitizes CLL cells to rituximab-induced cell death.

In vitro CD40-stimulated chronic lymphocytic leukemia (CLL) cells are resistant to cytotoxic drugs. In sharp contrast, we here show that CD40 stimulation sensitizes CLL cells to rituximab-mediated cell death. This increased sensitivity is specific for anti-CD20 treatment. Rituximab-mediated death in CD40-stimulated CLL cells shows rapid kinetics (within hours), and is caspase and p53 independent, but depends on extracellular Ca(2+) and reactive oxygen species (ROS) production. By increasing basal ROS production, CD40 stimulation sensitizes CLL cells to rituximab-mediated death. Our findings provide a rationale for combination treatment of CLL with cytotoxic drugs and anti-CD20 monoclonal antibodies.

ROS-mediated upregulation of Noxa overcomes chemoresistance in chronic lymphocytic leukemia.

In recent years considerable progress has been made in treatment strategies for chronic lymphocytic leukemia (CLL). However, the disease remains incurable because of the development of chemoresistance. Strategies to overcome resistance mechanisms are therefore highly needed. At least two mechanisms contribute to the development of resistance to drugs; acquired mutations resulting in a dysfunctional p53 response and shifts in the balance between apoptosis-regulating proteins. Platinum-based compounds have been successfully applied in relapsed lymphoma and recently also in high-risk CLL. In this study we investigated the efficacy and mechanism of action of cisplatinum (CDDP) in chemorefractory CLL. Independent of p53-functional status, CDDP acted synergistically with fludarabine (F-ara-A). The response involved generation of reactive oxygen species (ROS), which led to specific upregulation of the proapoptotic BH3-only protein Noxa. Induction of Noxa resulted in cell death by apoptosis as inhibition of caspase activation completely abrogated cell death. Furthermore, drug-resistance upon CD40-ligand stimulation, a model for the protective stimuli provided in lymph nodes, could also be overcome by CDDP/F-ara-A. ROS accumulation resulted in Noxa upregulation mainly at the transcriptional level and this was, at least in part, mediated by the mitogen-activated protein kinase p38. Finally, Noxa RNA-interference markedly decreased sensitivity to CDDP/F-ara-A, supporting a key role for Noxa as mediator between ROS signaling and apoptosis induction. Our data indicate that interference in the cellular redox balance can be exploited to overcome chemoresistance in CLL.

Dichotomy in NF-kappaB signaling and chemoresistance in immunoglobulin variable heavy-chain-mutated versus unmutated CLL cells upon CD40/TLR9 triggering.

Chronic lymphocytic leukemia (CLL) cells circulating in peripheral blood (PB) differ from the leukemic fraction in lymph nodes (LNs) with respect to cell division and drug sensitivity. CD40 stimulation of PB CLL cells in vitro results in chemoresistance and provides a partial model for the LN microenvironment. The TLR9 ligand CpG induces proliferation in immunoglobulin variable heavy-chain-unmutated CLL, but apoptosis in immunoglobulin variable heavy-chain-mutated CLL. To juxtapose proliferative with antiapoptotic signals, we investigated the effects of CpG in the context of CD40 ligation in mutated versus unmutated CLL cells in this study. Prolonged CD40 ligation induced classical, followed by alternative nuclear factor-kappaB (NF-kappaB), activity in both subgroups, correlating with enhanced Bfl-1 and Bcl-X(L) levels, respectively. A dichotomy in NF-kappaB signaling occurred on combined CD40/TLR9 triggering. This induced declining p52 and Bcl-X(L) levels, and reversed chemoresistance only in mutated cells, whereas unmutated cells proliferated, maintained p52 and Bcl-X(L) and remained chemoresistant. The pivotal contribution of Bcl-X(L) to chemoresistance was shown by the BH3 mimetic ABT-737 and RNA interference. Finally, in ex vivo LN samples, p52, p65 and Bcl-X(L) levels were highly expressed, corroborating the in vitro findings. Thus, a distinction in NF-kappaB activation and drug susceptibility in mutated versus unmutated (LN-like) CLL cells was uncovered, which was causally linked to Bcl-X(L) levels.