T. C. M. Morris

Variation in dimethyl sulfoxide use in stem cell transplantation: a survey of EBMT centres

Summary:The cryoprotectant dimethyl sulfoxide (DMSO) is known to have toxic side effects, yet guidelines for its use in stem cell transplantation do not exist. To assess current practice in the use of DMSO and the incidence of DMSO-related complications, a single page questionnaire was mailed to 444 EBMT centres involved in autologous transplantation. The responses from 97 centres showed a wide variation in practice between transplant units regarding the concentration of DMSO used, daily DMSO dose restriction and the use of cell washing. The overall incidence of DMSO toxicity was approximately one in 70 transplants and most cases were cardiovascular and respiratory in nature. There was a trend to reduced complication rates in centres using lower concentrations of DMSO or washing cells prior to return. A large-scale prospective study of the strategies for reduction in exposure to DMSO and reduction in toxic effects is required before guidelines in the use of DMSO in stem cell cryopreservation can be promulgated.

Comparative Selectivity and Specificity of the Proteasome Inhibitors BzLLLCOCHO, PS-341, and MG-132

The 26S proteasome is a multicatalytic protease responsible for regulated intracellular protein degradation. Its function is mediated by three main catalytic activities: (a) chymotrypsin-like (CT-L), (b) trypsin-like, and (c) peptidylglutamyl peptide hydrolysing (PGPH). Proteasome inhibition is an emerging therapy for many cancers and is a novel treatment for multiple myeloma. Here, we profile the contributions of the three catalytic activities in multiple myeloma cell lines and compare the specificity and cytotoxicity of the novel proteasome inhibitor BzLLLCOCHO and inhibitors PS-341 (Velcade, bortezomib) and MG-132. Using fluorogenic substrates and an active site-directed probe specific for proteasome catalytic subunits, we show differential subunit specificity for each of the inhibitors. Addition of BzLLLCOCHO strongly inhibited all three catalytic activities, treatment with PS-341 completely inhibited CT-L and PGPH activities, and treatment with MG-132 resulted in weak inhibition of the CT-L and PGPH activities. Multiple myeloma cells were more sensitive to induction of apoptosis by PS-341 and MG-132 than BzLLLCOCHO. This study emphasizes the need for further investigation of the effects of these compounds on gene and protein expression in the cell to allow for the development of more specific and targeted inhibitors.

High-dose chemotherapy plus autologous stem-cell transplantation as consolidation therapy in patients with relapsed multiple myeloma after previous autologous stem-cell transplantation (NCRI Myeloma X Relapse [Intensive trial]): a randomised, open-label, phase 3 trial

BACKGROUND Relapsed multiple myeloma has no standard treatment, and the role of autologous stem-cell transplantation (ASCT) has not been fully defined. We aimed to compare high-dose melphalan plus salvage ASCT with cyclophosphamide in patients with relapsed multiple myeloma who had previously undergone ASCT. METHODS This multicentre, randomised, open-label, phase 3 study recruited patients aged at least 18 years with multiple myeloma who needed treatment for first progressive or relapsed disease at least 18 months after a previous ASCT from 51 centres across the UK. Before randomisation, eligible patients received bortezomib, doxorubicin, and dexamethasone (PAD) induction therapy and then underwent peripheral blood stem-cell mobilisation and harvesting if applicable. Eligible patients (with adequate stem-cell harvest) were randomly assigned (1:1), using an automated telephone randomisation line, to either high-dose melphalan 200 mg/m(2) plus salvage ASCT or oral cyclophosphamide (400mg/m(2) per week for 12 weeks). Randomisation was stratified by length of first remission or plateau and response to PAD re-induction therapy. The primary endpoint was time to disease progression, analysed by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00747877, and EudraCT, number 2006-005890-24. FINDINGS Between April 16, 2008, and Nov 19, 2012, 297 patients were registered, of whom 293 received PAD re-induction therapy. Between Aug 26, 2008, and Nov 16, 2012, 174 patients with sufficient PBSCs were randomised to salvage ASCT (n=89) or cyclophosphamide (n=85). After a median follow-up of 31 months (IQR 19-42), median time to progression was significantly longer in the salvage ASCT than in the cyclophosphamide group (19 months [95% CI 16-25] vs 11 months [9-12]; hazard ratio 0·36 [95% CI 0·25-0·53]; p<0·0001). Frequently reported (in >10% of patients) grade 3-4 adverse events with PAD induction, salvage ASCT, and cyclophosphamide were: neutropenia (125 [43%] of 293 patients after PAD, and 63 [76%] of 83 patients in the salvage ASCT group vs 11 [13%] of 84 patients in the cyclophosphamide group), thrombocytopenia (150 [51%] after PAD, and 60 [72%] vs four [5%], respectively), and peripheral neuropathy (35 [12%] after PAD, and none vs none, respectively). INTERPRETATION This study provides evidence for the improved efficacy of high-dose melphalan plus salvage ASCT when compared with cyclophosphamide in patients with relapsed multiple myeloma eligible for intensive therapy, which might help to guide clinical decisions regarding the management of such patients. FUNDING Cancer Research UK.

Analysis of the effect of prior therapy on progenitor cell yield: use of a chemotherapy scoring system.

A quantitative analysis of peripheral blood stem cell (PBSC) yield, measuring absolute numbers of CD34+ cells × 106/kg and CFU‐C × 104/kg was performed in 74 consecutive patients. The interval or ‘gap’ from the end of previous chemotherapy to the date of priming was recorded in weeks.

Changes in natural killer cells, the CD57CD8 subset, and related cytokines in healthy aging.

Aging has been shown to be accompanied by various changes in the lymphocyte subset distribution in the elderly. We have investigated more fully, and in a large number of subjects, age-related changes within several subpopulations bearing natural killer (NK) cell-associated surface antigens and changes in several cytokines involved in NK cell expansion. A total of 229 healthy subjects from all decades of life from 20 to 98 years of age was included in this cross-sectional study. A significant increase with age was found in both the absolute counts and the proportions of CD3−CD(16+56)+, CD3+CD(16+56)+, CD57+CD8+, CD57+CD8(low)+, and CD57+CD8− cells, whereas the CD57+CD8(high)+ subset, which may represent the cytolytic T cell population more precisely, showed less change with age. Some evidence is also provided to suggest that these expanded NK cell populations are in an activated state. Soluble IL-2 receptor levels were also found to increase significantly with age and correlated with certain NK cell subsets. Although the functions of some of these subsets remain to be elucidated, their expansion in the elderly may represent a remodeling of the immune system with increasing age, with an increase in non-MHC-restricted cells perhaps compensating for the previously reported decline in T and B cells in the elderly. Alternatively, increased numbers of these cells may be a direct result of cytokine dysregulation or increased antigenic or neoplastic cell challenge.

Severe neurotoxicity because of dimethyl sulphoxide following peripheral blood stem cell transplantation.

Severe neurotoxicity because of dimethyl sulphoxide following peripheral blood stem cell transplantation

Adiponectin is produced by lymphocytes and is a negative regulator of granulopoiesis

Lymphocytes have long been established to play an important role in the regulation of hematopoiesis and produce many cytokines that act on hematopoietic progenitor cells. Previous studies by our group have shown that normal, unstimulated lymphocytes produce a protein that inhibits normal bone marrow GM colony formation. Adiponectin is an adipokine that has been demonstrated to act as a negative regulator of hematopoiesis and immune function. This study aimed to determine if the inhibitory molecule that we described previously was adiponectin. Here, we show transcription, translation, and secretion of adiponectin from lymphocytes and demonstrate that its receptors, AdipoR1 and AdipoR2, are expressed by bone marrow MNCs. We show that although the adiponectin expression is low in lymphocytes, it is sufficient to induce a significant inhibitory effect on GM precursors (CFU‐GM) and activate the AMPK pathway in these cells. The regulation of adiponectin production by lymphocytes and its detailed function in suppressing GM colony formation need to be elucidated now. Our findings suggest a functional role for adiponectin as a negative regulator of granulopoiesis.

Bortezomib is effective in primary plasma cell leukemia

The ubiquitin proteasome pathway plays a critical role in regulating a number of cellular processes crucial to tumorigenesis and has recently emerged as a new molecular target for cancer therapy [1]. Sensitivity to proteasome inhibitors has been demonstrated in a number of malignancies, particularly multiple myeloma. The first proteasome inhibitor to enter clinical trials, bortezomib (Velcade) has demonstrated marked anti-myeloma activity and has been approved for the treatment of relapsed and refractory myeloma. Plasma cell leukemia (PCL) has been defined as circulating peripheral blood plasma cells exceeding 2610/l or 20% of peripheral blood cells [2]. If observed at the time of diagnosis, it is known as primary PCL and has a poor outcome from both conventional therapy and autologous or allogeneic transplantation [3,4].

ZAP-70 mRNA quantification in B-cell chronic lymphocytic leukaemia

Abstract:  Objective: The mutational status of the immunoglobulin (Ig) VH gene in B‐cell chronic lymphocytic leukaemia (B‐CLL) identifies two subgroups of patients with significantly different outcomes. We investigated the association of ZAP‐70 expression with IgVH mutational status in B‐CLL by quantifying ZAP‐70 mRNA, to evaluate its use as a surrogate marker for mutational status. The aim of this study was to develop a quantitative reverse transcriptase‐polymerase chain reaction (RQ‐PCR) assay for the detection of ZAP‐70 expression in a group of patients whose mutational status and cytogenetics had been determined previously. Methods: RQ‐PCR was used to analyse ZAP‐70 expression from 42 B‐CLL patients. B cells were purified using CD19 magnetic bead system and total RNA was isolated. RQ‐PCR was performed using Taqman PCR. Results: Twenty‐five patients (60%) had mutated and 17 (40%) had unmutated IgVH genes; 94% (16/17) of patients with unmutated IgVH gene were ZAP‐70 positive as assessed by RQ‐PCR and 92% (23/25) of patients with mutated IgVH gene were ZAP‐70 negative. In three patients, ZAP‐70 expression and IgVH mutational status were discordant. Conclusion: This paper describes an RQ‐PCR assay for the detection of ZAP‐70 expression and confirms that IgVH unmutated CLL cells have a high expression of ZAP‐70 in comparison with IgVH mutated CLL. This robust method acts as a surrogate marker for IgVH mutational status albeit with <100% concordance. However, it does provide better concordance with mutational status than that reported using flow cytometry.

Bone Marrow Necrosis

Bone marrow necrosis is most frequently diagnosed at postmortem examination. Antemortem diagnosis is still uncommon. In a recent review of world literature, we have found 133 cases of bone marrow necrosis diagnosed during life. It has been observed during the course of a wide variety of diseases, most commonly in association with acute and chronic leukemia, carcinoma, malignant lymphoma, infections, and sickle cell disease.