Outcome of elderly patients with diffuse large B-cell lymphoma treated with R-CHOP: results from the UK NCRI R-CHOP14v21 trial with combined analysis of molecular characteristics with the DSHNHL RICOVER-60 trial.

BACKGROUND: There is an on-going debate whether 2- or 3-weekly administration of R-CHOP is the preferred first-line treatment for elderly patients with diffuse large B-cell lymphoma (DLBCL). The UK NCRI R-CHOP14v21 randomized phase 3 trial did not demonstrate a difference in outcomes between R-CHOP-14 and R-CHOP-21 in newly diagnosed DLBCL patients aged 19-88 years, but data on elderly patients have not been reported in detail so far. Here, we provide a subgroup analysis of patients ≥60 years treated on the R-CHOP14v21 trial with extended follow-up. PATIENTS AND METHODS: Six hundred and four R-CHOP14v21 patients ≥60 years were included in this subgroup analysis, with a median follow-up of 77.7 months. To assess the impact of MYC rearrangements (MYC-R) and double-hit-lymphoma (DHL) on outcome in elderly patients, we performed a joint analysis of cases with available molecular data from the R-CHOP14v21 (N = 217) and RICOVER-60 (N = 204) trials. RESULTS: Elderly DLBCL patients received high dose intensities with median total doses of ≥98% for all agents. Toxicities were similar in both arms with the exception of more grade ≥3 neutropenia (P < 0.0001) and fewer grade ≥3 thrombocytopenia (P = 0.05) in R-CHOP-21 versus R-CHOP-14. The elderly patient population had a favorable 5-year overall survival (OS) of 69% (95% CI: 65-73). We did not identify any subgroup of patients that showed differential response to either regimen. In multivariable analysis including individual factors of the IPI, gender, bulk, B2M and albumin levels, only age and B2M were of independent prognostic significance for OS. Molecular analyses demonstrated a significant impact of MYC-R (HR = 1.96; 95% CI: 1.22-3.16; P = 0.01) and DHL (HR = 2.21; 95% CI: 1.18-4.11; P = 0.01) on OS in the combined trial cohorts, independent of other prognostic factors. CONCLUSIONS: Our data support equivalence of both R-CHOP application forms in elderly DLBCL patients. Elderly MYC-R and DHL patients have inferior prognosis and should be considered for alternative treatment approaches. TRIAL NUMBERS: ISCRTN 16017947 (R-CHOP14v21); NCT00052936 (RICOVER-60).

IL-10 production by CLL cells is enhanced in the anergic IGHV mutated subset and associates with reduced DNA methylation of the IL10 locus.

Chronic lymphocytic leukemias (CLLs) with unmutated (U-CLL) or mutated (M-CLL) IGHV have variable features of immunosuppression, possibly influenced by those CLL cells activated to produce interleukin 10 (IL-10). The two subsets differ in their levels of anergy, defined by low surface immunoglobulin M levels/signaling capacity, and in their DNA methylation profile, particularly variable in M-CLL. We have now found that levels of IL-10 produced by activated CLL cells were highly variable. Levels were higher in M-CLL than in U-CLL and correlated with anergy. DNA methylation analysis of IL10 locus revealed two previously uncharacterized 'variably methylated regions' (CLL-VMRs1/2) in the gene body, but similarly low methylation in the promoter of both U-CLL and M-CLL. CLL-VMR1/2 methylation was lower in M-CLL than in U-CLL and inversely correlated with IL-10 induction. A functional signal transducer and activator of transcription 3 (STAT3) binding site in CLL-VMR2 was confirmed by proximity ligation and luciferase assays, whereas inhibition of SYK-mediated STAT3 activation resulted in suppression of IL10. The data suggest epigenetic control of IL-10 production. Higher tumor load may compensate the reduced IL-10 production in U-CLL, accounting for clinical immunosuppression in both subsets. The observation that SYK inhibition also suppresses IL-10 provides a potential new rationale for therapeutic targeting and immunological rescue by SYK inhibitors in CLL.

Ex vivo assays of dendritic cell activation and cytokine profiles as predictors of in vivo effects in an anti-human CD40 monoclonal antibody ChiLob 7/4 phase I trial.

Immunostimulatory antibodies entering the clinic create challenge in terms of not only pharmacodynamics for monitoring anticipated mechanisms but also predetermining cytotoxicity. We show the use of ex vivo whole-blood samples to predict the activation requirements, cytokine signature, and adverse events of an anti-human-CD40 chimeric IgG1 antibody, ChiLob 7/4. Assessments were initially undertaken on human myeloid (mDC1) and plasmacytoid (pDC) dendritic cells, in which an absolute need for cross-linking was shown through the upregulation of activation markers CD83 and CCR7. Subsequent cytokine secretion evaluations of ex vivo whole blood showed the cross-linked antibody-induced increases in MIP1ß, interleukin (IL)-8, IL-12, TNFα, and IL-6. This cytokine signature compared favorably with the Toll-like receptor (TLR) ligand lipopolysaccharide (LPS), in which levels of TNFα and IL-6 were significantly higher, suggesting a less intense proinflammatory response and possible modified cytokine release syndrome when used in human trials. Following first-in-human use of this agent within a dose escalation study, in vivo evaluations of dendritic cell activation and secreted cytokines closely matched the predetermined immunomonitoring endpoints. Patients showed a comparable pattern of MIP1ß, IL-8, and IL-12 secretion, but no TNFα and IL-6 were identified. Mild symptoms relating to a cytokine release syndrome were seen at an equivalent dosage to that observed for dendritic cell activation and cytokine release. In summary, ChiLob 7/4 induces a distinctive pattern of dendritic cell activation and cytokine secretion in ex vivo assays that can be predictive of in vivo responses. Such preclinical approaches to monoclonal antibody evaluation may inform both the starting dosages and the anticipated cytokine release events that could occur, providing a valuable adjunct for future first-in-human assessments of immunostimulatory antibodies.

Whole-body vibration exposure in metropolitan bus drivers.

BACKGROUND: Back injuries are common in transit drivers, and can result in substantial direct and indirect cost to the employer and employee. Whole-body vibration (WBV) is one risk factor for drivers. Standards have been adopted (ISO 2631-1) to guide researchers in measuring and analysing WBV levels. Lately, a new standard has been added (ISO 2631-5) that takes impulsive exposures into account. AIMS: The aims of this study were to determine the levels of vibration for bus drivers using both ISO 2631-1 and 2631-5 standards, and whether there are differences in vibration levels and seat transmissibility between different road types. METHODS: Thirteen bus drivers drove a 7-year-old bus, instrumented to measure WBV in the seat and floor. The 52 km long test route included freeway, city streets and speed humps. Additionally, for comparison, a subset of five drivers also drove a car over the same route. RESULTS: Road type had a significant effect on all the vibration parameters. Based on exposure limit values in the standards, the continuous z-A (w)(8) exposures exceeded the limit value on freeways, and the impulsive z-VDV(8) and S (ed) exposures were above limit values in city streets and speed humps. Bus WBV exposures were about twice as high relative to the car and the bus seat amplified rather than attenuated WBV exposures. CONCLUSIONS: Bus drivers are potentially being exposed to daily vibration levels higher than recommended especially on certain road types. The current seat in this study does not attenuate the vibration.

Defects in lymphocyte subsets and serological memory persist a median of 10 years after high-dose therapy and autologous progenitor cell rescue for malignant lymphoma.

The number of survivors having undergone high-dose therapy (HDT) followed by auto-SCT continues to increase, although some of the long-term sequelae remain incompletely understood. The immunological status and quality of life of 37 HDT/auto-SCT survivors with lymphoma in continuous remission of ≥3 years were assessed alongside 14 age-matched controls. At a median follow-up of 10.5 years (range 2.2-20.2) following HDT/auto-SCT, the proportion of CD4(+) cells remained significantly reduced in patients compared with controls (median 43.4% vs 62.5%, respectively; P = < 0.001), predominantly a result of sustained reduction in the naive CD4(+) component (P < 0.001). Naive CD8(+) lymphocytes (P = 0.014) and transitional B cells (P = 0.008) were also significantly reduced, but differences in other lymphocyte subsets were not observed. Uptake of revaccination following HDT/auto-SCT was sporadic; between 11% and 33% of patients had serological titres outside the protective ranges for five of six routinely used vaccines. In the main, patients were found to have a good quality of life, although their EORTC QLQ-C30 questionnaire scores were significantly lower for the physical and social functioning domains compared with controls. Ten years after HDT/auto-SCT immunological deficits persist; to avoid excess risk of preventable disease, serological immunity should be assessed post HDT/auto-SCT followed by appropriate revaccination.

Observed differences in upper extremity forces, muscle efforts, postures, velocities and accelerations across computer activities in a field study of office workers.

This study, a part of the PRedicting Occupational biomechanics in OFfice workers (PROOF) study, investigated whether there are differences in field-measured forces, muscle efforts, postures, velocities and accelerations across computer activities. These parameters were measured continuously for 120 office workers performing their own work for two hours each. There were differences in nearly all forces, muscle efforts, postures, velocities and accelerations across keyboard, mouse and idle activities. Keyboard activities showed a 50% increase in the median right trapezius muscle effort when compared to mouse activities. Median shoulder rotation changed from 25 degrees internal rotation during keyboard use to 15 degrees external rotation during mouse use. Only keyboard use was associated with median ulnar deviations greater than 5 degrees. Idle activities led to the greatest variability observed in all muscle efforts and postures measured. In future studies, measurements of computer activities could be used to provide information on the physical exposures experienced during computer use. Practitioner Summary: Computer users may develop musculoskeletal disorders due to their force, muscle effort, posture and wrist velocity and acceleration exposures during computer use. We report that many physical exposures are different across computer activities. This information may be used to estimate physical exposures based on patterns of computer activities over time.

Developing a framework for assessing muscle effort and postures during computer work in the field: the effect of computer activities on neck/shoulder muscle effort and postures.

The present study, a part of the PROOF (PRedicting Occupational biomechanics in OFfice workers) study, aimed to determine whether trapezius muscle effort was different across computer activities in a field study of computer workers, and also investigated whether head and shoulder postures were different across computer activities. One hundred twenty participants were measured continuously for two hours each while performing their own computer work. Keyboard activities were associated with the highest intensity of left and right trapezius muscle efforts, and mouse activities were associated with the smallest variability in left and right trapezius muscle efforts. Corresponding trends in head and shoulder postures included that the greatest head flexion and left and right shoulder internal rotation was observed during keyboard activities, and that the smallest variability in head flexion, head lateral tilt, and right shoulder internal rotation was observed during mouse activities. Identifying which muscle efforts and postures are different across computer activities is the first essential step for developing prediction rules for muscle efforts and postures, which can be used to link muscle efforts and postures to musculoskeletal symptoms in epidemiological studies.

Development, validation and application of ELISAs for pharmacokinetic and HACA assessment of a chimeric anti-CD40 monoclonal antibody in human serum.

As part of a Phase I chimeric anti-CD40 monoclonal antibody clinical trial, two enzyme-linked immunosorbent assays (ELISAs) were developed for secondary endpoints: 1) for the pharmacokinetic (PK) monitoring of serum antibody levels and 2) for immunogenic screening of human anti-chimeric antibody (HACA) responses. The ELISA is a well established immunoassay, with clear guidelines for validation when used as a quantitative assay. However, these parameters may not always be relevant for a semi-quantitative assay used to assess whether a sample is positive or negative for a novel marker such as an antibody developed against a therapeutic antibody. We report here the development of a quantitative PK ELISA and a semi-quantitative HACA ELISA, and the different approaches of validation to prove each assay are 'fit for purpose.' The parameters of linearity (R²>0.99), accuracy (±30%), lowest level of detection (4 µg/ml), intra-assay (coefficient of variation (CV) <20%) and inter-assay (CV<20%) variability were assessed for the quantitative PK assay. For the semi-quantitative HACA assay, parameters of linearity (R²>0.99), lowest level of detection, intra (CV<10%) and inter-assay (CV<30%) variability were assessed using a surrogate positive control. The validation outcome showed that each assay was robust, reliable and accurate to meet the requirements of the intended analytical application, that being to 1) quantitatively determine the concentration of antibody in the serum and 2) determine whether a sample is positive or negative for human anti-chimeric antibodies. Each assay has been successfully translated for use in a clinical trial with adequate quality controls and acceptance criteria set for monitoring consistency and performance.

Initial dose intensity has limited impact on the outcome of ABVD chemotherapy for advanced Hodgkin lymphoma (HL): data from UKLG LY09 (ISRCTN97144519).

BACKGROUND: This analysis was undertaken to assess the relationship between the dose intensity (DI) of initial chemotherapy and outcome in a large cohort of patients with advanced Hodgkin lymphoma treated in a randomised controlled trial, in which detailed dose data were collected prospectively. PATIENTS AND METHODS: Three-hundred and eighty patients randomly assigned to receive standard doxorubicin, bleomycin, vinblastine and dacarbazine who underwent at least two cycles of treatment were studied. With a median follow-up of 6.9 years, progression-free survival (PFS) from the end of cycle 2 was analysed according to DI during those cycles. RESULTS: During the first two cycles, 25% of patients received >97% of planned DI, 37% received between 86% and 97% and 38% received <86%. DI during the first two cycles was correlated with DI during the remainder of the course, but there was no evidence that early DI influenced PFS (hazard ratio 0.87, 95% confidence interval 0.67-1.11; P = 0.265). Multivariate analysis also failed to confirm the influence of early DI on PFS or overall survival. CONCLUSIONS: At the range of DI delivered in a multicentre trial using conventional therapy, there is no clear evidence that early DI influences outcome. This should be tested in a prospective study.

Low mean level sustained and intermittent grip exertions: influence of age on fatigue and recovery.

The goal of this study was to quantify localised muscle fatigue resulting from low mean levels of exertion in younger (< 40 years) and older (> 50 years) adults. Fatigue, elicited in the finger flexor muscles by intermittent (10% mean maximum voluntary contraction (MVC)) and sustained (8% MVC) handgrip exercises, was quantified by a muscle twitch force response before, immediately after and during 3 h following exercise. Despite greater mean loads, recovery time was shorter following intermittent than sustained contractions, which suggests that recovery from fatigue is more sensitive to rest within the work cycle than mean work. The more pronounced effects for younger than older individuals following the sustained exertion indicate that changes in muscle fibre type composition might predispose older individuals to be more resistant to fatigue resulting from sustained contractions of low level. Performing hand exertion tasks requiring low mean force levels contributes to similar long-lasting fatigue effects regardless of gender and age. Intermittent periods of complete rest reduce muscle fatigue. Since fatigue was not perceived during recovery from the tested sustained and intermittent contractions, subjective evaluations may not be a reliable indicator of localised muscle fatigue.

Will histone deacetylase inhibitors require combination with other agents to fulfil their therapeutic potential?

Histone deacetylase inhibitors have progressed rapidly from the laboratory to clinical testing. This review highlights the promising data for their combination with a wide range of established and novel anticancer agents and discusses the mechanisms that underpin these interactions.

Primary mediastinal B-cell lymphoma.

Primary mediastinal B-cell lymphoma (PMBCL) is a sub-type of the heterogeneous diffuse large B-cell lymphoma category, and comprises approximately 5% of all non-Hodgkin's lymphomas (NHL). It was first recognized as a distinct clinico-pathologic entity 20 years ago, and recent work has further characterized specific molecular features. Gene expression profiling has suggested a partial overlap with nodular sclerosing Hodgkin lymphoma (HL), with which it shares some clinical features. The optimal management remains a matter of debate. There is uncertainty as to whether weekly alternating chemotherapy regimens may be more effective than CHOP, whether consolidation radiotherapy (RT) to the mediastinum is always required, whether PET scanning can be used to determine this, and whether the use of rituximab as part of initial therapy will change the answers to these questions. The International Extranodal Lymphoma Study Group (IELSG) 26 clinicopathologic study of PMBCL, which has recently opened, represents a first attempt to gather data prospectively on some of these issues.

Low-dose lenograstim is as effective as standard dose in shortening neutrophil engraftment time following myeloablative chemotherapy and peripheral blood progenitor cell rescue.

Granulocyte colony-stimulating factor (G-CSF) is widely used following myeloablative chemotherapy (high-dose therapy; HDT) and peripheral blood progenitor cell rescue (PBPCR) to reduce neutrophil engraftment time. The dose and duration required to gain maximum clinical and economic benefit has not been fully investigated. This double blind placebo-controlled randomised trial was performed to determine whether short course low-dose or standard-dose Lenograstim (L) would influence recovery of haematopoiesis following HDT and PBPCR. Sixty-one patients were randomised between May 1999 and November 2004, to receive standard-dose lenograstim (263 microg/d), low-dose lenograstim (105 microg/d) or placebo injections. These commenced on day +5 following PBPCR and continued until neutrophil engraftment [absolute neutrophil count (ANC)] > or = 0.5 x 10(9)/l. Patients received standard supportive care until haemopoietic recovery. Both standard- and low-dose lenograstim resulted in a significantly shorter median time to neutrophil recovery (ANC > or = 0.1 x 10(9)/l:10.0 vs. 11.0 d, P = 0.025; ANC > or = 0.5 x 10(9)/l:11.0 vs. 14.0 d, P = 0.0002) compared with placebo. There was no significant difference in blood product support, antibiotic usage, documented infection, overall survival or relapse-free survival between the groups. Short course low-dose lenograstim is as effective as standard-dose in reducing neutrophil engraftment time following HDT and PBPCR.

Pharmacological inhibitors of NF-kappaB accelerate apoptosis in chronic lymphocytic leukaemia cells.

Nuclear factor-kappaB (NF-kappaB) is a transcription factor that plays a critical role in the inappropriate survival of various types of malignant cells. Chronic lymphocytic leukaemia (CLL) is the most common B-cell malignancy in the Western world. Although overexpression and regulation of NF-kappaB has been described in CLL, its function remains unclear. Exposure of CLL cells to BAY117082 or Kamebakaurin, potent pharmacological inhibitors of the NF-kappaB pathway, accelerated apoptosis in approximately 70% of cases. Sensitivity to NF-kappaB pathway inhibitors was not related to the prognostic markers VH status, CD38 or Zap70 expression, or to the levels of nuclear NF-kappaB. Normal peripheral B cells were resistant to the apoptosis-inducing effects of these compounds. Cell death induced by the inhibitors was associated with activation of caspase-9 and -3, and loss of mitochondrial membrane polarization, but did not involve changes in the expression of Bcl-2 or Mcl-1. Inhibitors caused an increase in c-jun NH2-terminal kinase activity in CLL, but this did not appear to be important for apoptosis. Microarray analysis identified some potential novel NF-kappaB target genes, including interleukin-16- and the Bcl-2- related survival protein Bcl-w. These results demonstrate that a substantial proportion of CLL are dependent on NF-kappaB for enhanced survival and suggest that inhibition of NF-kappaB may have therapeutic potential.

Distinct promoters mediate constitutive and inducible Bcl-XL expression in malignant lymphocytes.

Bcl-X(L) is a Bcl-2-related survival protein that is essential for normal development. Bcl-X(L) expression is rapidly induced by a wide range of survival signals and many cancer cells constitutively express high levels. The Bcl-X gene has a complex organization with multiple promoters giving rise to RNAs with alternate 5' non-coding exons. Here we have investigated the mechanisms that control basal and induced expression of Bcl-X(L) in B-lymphoma cells. Antisense experiments demonstrated that Bcl-X(L) was essential for survival of Akata6 B-lymphoma cells. The levels of RNAs containing the IB Bcl-X non-coding exon, derived from the distal 1B promoter, correlated with basal expression of Bcl-X(L) in primary malignant B cells and this promoter was highly active in B-cell lines. The activity of this promoter was largely dependent on a single Ets binding site and Ets family proteins were bound at this promoter in intact cells. CD40 ligand (CD40L)-induced cell survival was associated with increased Bcl-X(L) expression and accumulation of exon IA-containing RNAs, derived from the proximal 1A promoter. Nuclear factor-kappaB (NF-kappaB) inhibition prevented induction of Bcl-X(L) protein and exon IA-containing RNAs by CD40L. Therefore, the distal Bcl-X 1B promoter plays a critical role in driving constitutive expression-mediated via Ets family proteins in malignant B cells, whereas NF-kappaB plays a central role in the induction of Bcl-X(L) in response to CD40 signalling via the proximal 1A promoter.