A comparison of peripheral blood stem cell collection outcomes for multiple myeloma; mobilization matters in the era of IMiD induction.

Collection of peripheral blood stem cells (PBSCs) for autologous stem cell transplant (ASCT) requires mobilization from the bone marrow. There is variation in mobilization choice; during the COVID-19 pandemic BSBMT&CT guidelines recommended using granulocyte-colony stimulating factor (G-CSF) alone to minimize the use of chemotherapy. We report on the impact of mobilization regimen on stem cell collection, and whether IMiD-containing induction therapy impacts on mobilization and consequently transplant engraftment times for 83 patients undergoing ASCT at Leeds Teaching Hospitals. Cyclophosphamide plus G-CSF (cyclo-G) mobilization yielded more CD34+ cells (8.94 vs. 4.88 ×106/kg, p = < 0.0001) over fewer days (1.6 vs. 2.4 days, p = 0.007), and required fewer doses of salvage Plerixafor than G-CSF only (13.6% vs. 35%, p = 0.0407). IMiD-containing induction impaired all of these factors. CD34+ doses > 8×106/kg were more frequent with Cyclo-G (62% vs. 11%, p = 0.0001), including for those receiving IMiD 1st line induction (50% vs. 13.3%, p = 0.0381). Note that 92.6% of those receiving IMiD-free inductions were mobilized with Cyclo-G. The novel agents used in modern induction regimens (e.g Daratumumab) have been shown to impair yields, increasing the importance of optimizing mobilization regimens in the first instance. Furthermore, as cellular therapies become established in the management of multiple myeloma emerging data highlights the potential benefits of stem cell top up in the management of the haematological toxicities of these therapies. Our findings support re-adoption of Cyclo-G as the gold standard for mobilization to optimize PBSC harvesting and ensure sufficient cells for subsequent ASCTs.

Efficacy of coxsackievirus A21 against drug-resistant neoplastic B cells.

Primary drug resistance and minimal residual disease are major challenges in the treatment of B cell neoplasms. Therefore, this study aimed to identify a novel treatment capable of eradicating malignant B cells and drug-resistant disease. Oncolytic viruses eradicate malignant cells by direct oncolysis and activation of anti-tumor immunity, have proven anti-cancer efficacy, and are safe and well tolerated in clinical use. Here, we demonstrate that the oncolytic virus coxsackievirus A21 can kill a range of B cell neoplasms, irrespective of an anti-viral interferon response. Moreover, CVA21 retained its capacity to kill drug-resistant B cell neoplasms, where drug resistance was induced by co-culture with tumor microenvironment support. In some cases, CVA21 efficacy was actually enhanced, in accordance with increased expression of the viral entry receptor ICAM-1. Importantly, the data confirmed preferential killing of malignant B cells and CVA21 dependence on oncogenic B cell signaling pathways. Significantly, CVA21 also activated natural killer (NK) cells to kill neoplastic B cells and drug-resistant B cells remained susceptible to NK cell-mediated lysis. Overall, these data reveal a dual mode of action of CVA21 against drug-resistant B cells and support the development of CVA21 for the treatment of B cell neoplasms.

Delayed diagnosis resulting in increased disease burden in multiple myeloma: the legacy of the COVID-19 pandemic.

The COVID-19 pandemic has had global healthcare impacts, including high mortality from SARS-CoV-2 infection in cancer patients; individuals with multiple myeloma (MM) are especially susceptible to poor outcomes. However, even for MM patients who avoided severe infection, the ramifications of the pandemic have been considerable. The consequences of necessary socio-geographical behavior adaptation, including prolonged shielding and interruptions in delivery of non-pandemic medical services are yet to be fully understood. Using a real-world dataset of 323 consecutive newly diagnosed MM patients in England, we investigated the impact of the COVID-19 pandemic on routes to myeloma diagnosis, disease stage at presentation and relevant clinical outcomes. We demonstrate increasing MM presentations via emergency services and increased rates of bony and extra-medullary disease. Differences were seen in choice of induction therapy and the proportion of eligible patients undertaking autologous stem cell transplantation. Whilst survival was statistically inferior for emergency presentations, significant survival differences have yet to be demonstrated for the entire cohort diagnosed during the pandemic, making extended follow-up critical in this group. This dataset highlights wide-ranging issues facing MM patients consequent of the COVID-19 pandemic, with full impacts for clinicians and policy-makers yet to be elucidated.

Daratumumab, bortezomib and dexamethasone at first relapse for patients with multiple myeloma: A real-world multicentre UK retrospective analysis.

Daratumumab, bortezomib and dexamethasone (DVd) is approved for patients with relapsed multiple myeloma following the CASTOR phase 3 clinical trial. This retrospective multicentre analysis assesses the overall response rate (ORR) and progression-free survival (PFS) in routine clinical practice for patients at first relapse treated with DVd incorporating weekly bortezomib. Data were collected from 296 sequential patients treated across 15 UK centres. After a median follow-up of 21 months, the ORR was 82% (26% partial response, 56% very good partial response or better) and the median PFS was 16 months [95% confidence interval (CI) 12-20 months]. Results were similar regardless of prior lenalidomide exposure. The median time to next treatment was 20 months (95% CI 15-25 months) and the estimated overall survival at two years was 74%. Patients with high-risk features (by cytogenetics, International Staging System or extramedullary disease) and those treated within 18 months of initiation of progression-free treatment, or within 12 months of autologous stem cell transplant, had significantly inferior outcomes. The grade 2 and 3 peripheral neuropathy rate was 7%. DVd with weekly bortezomib was effective in a heterogenous real-world population at first relapse with a low rate of peripheral neuropathy. However, high-risk patients had inferior outcomes and should be considered for alternative treatments.

Shotgun-Based Mass Spectrometry Analysis of Phospholipid and Triacylglycerol Molecular Species and Eicosanoids in Salmon Muscle Tissue on Feeding Microbial Oil.

The continuous growth of aquaculture places a growing demand on alternative sources of fish oil (FO). Certain microorganisms provide a sustainable replacement for FO due to their content of EPA and DHA, which are essential for fish health. Appreciable evidence shows that changes in feeding sources may alter the nutritional components of salmon; however, the influence of diets on lipid species remains unclear. In this study, the identification and semi-quantification of lipid molecular species in salmon muscle during feeding with a microbial oil (MO) were carried out by focusing on triacylglycerol (TAG) and diacyl-phospholipid using shotgun-based mass spectrometry analysis. DHA in the MO diet was efficiently incorporated into phospholipid structures on feeding, followed by accumulation in salmon muscle. The MO diet elevated the level of certain EPA-containing TAGs, such as TAG C52:5 (16:0_16:0_20:5) and TAG C54:6 (16:0_18:1_20:5), indicating that the MO diet may be an excellent source for enhancement of the abundance of ω3 lipids. Further, prostaglandins (PGs) PGE2 and PGF3α were identified and quantified for the first time in salmonid tissue.

Risk and response adapted therapy following autologous stem cell transplant in patients with newly diagnosed multiple myeloma (RADAR (UK-MRA Myeloma XV Trial): study protocol for a phase II/III randomised controlled trial.

INTRODUCTION: Multiple myeloma is a plasma cell malignancy that accounts for 1%-2% of newly diagnosed cancers.At diagnosis, approximately 20% of patients can be identified, using cytogenetics, to have inferior survival (high-risk). Additionally, standard-risk patients, with detectable disease (minimal residual disease (MRD)-positive) postautologus stem cell transplant (ASCT), fare worse compared with those who do not (MRD-negative). Research is required to determine whether a risk-adapted approach post-ASCT could further improve patient outcomes. METHODS: RADAR is a UK, multicentre, risk-adapted, response-guided, open-label, randomised controlled trial for transplant-eligible newly diagnosed multiple myeloma patients, using combinations of lenalidomide (R), cyclophosphamide (Cy), bortezomib (Bor), dexamethasone (D) and isatuximab (Isa).Participants receive RCyBorD(x4) induction therapy, followed by high-dose melphalan and ASCT. Post-ASCT, there are three pathways as follows:A phase III discontinuation design to assess de-escalating therapy in standard-risk MRD-negative patients. Participants receive 12 cycles of Isa maintenance. Those who remain MRD-negative are randomised to either continue or stop treatment.A phase II/III multiarm multistage design to test treatment strategies for treatment escalation in standard-risk MRD-positive patients. Participants are randomised to either; R, RBorD(x4) +R, RIsa, or RBorIsaD(x4) + RIsa.A phase II design to assess the activity of intensive treatment strategies in high-risk patients. Participants are randomised to RBorD(x4) +R or RBorIsaD(x4) + RIsa.1400 participants will be registered to allow for 500, 450 and 172 participants in each pathway. Randomisations are equal and treatment is given until disease progression or intolerance. ETHICS AND DISSEMINATION: Ethical approval was granted by the London-Central Research Ethics Committee (20/LO/0238) and capacity and capability confirmed by the appropriate local research and development department for each participating centre prior to opening recruitment. Participant informed consent is required before trial registration and reconfirmed post-ASCT. Results will be disseminated by conference presentations and peer-reviewed publications. TRIAL REGISTRATION NUMBER: ISCRTN46841867.

Immune senescence in multiple myeloma-a role for mitochondrial dysfunction?

Age-related immune dysfunction is primarily mediated by immunosenescence which results in ineffective clearance of infective pathogens, poor vaccine responses and increased susceptibility to multi-morbidities. Immunosenescence-related immunometabolic abnormalities are associated with accelerated aging, an inflammatory immune response (inflammaging) and ultimately frailty syndromes. In addition, several conditions can accelerate the development of immunosenescence, including cancer. This is a bi-directional interaction since inflammaging may create a permissive environment for tumour development. Multiple myeloma (MM) is a mature B-cell malignancy that presents in the older population. MM exemplifies the interaction of age- (Host Response Biology; HRB) and disease-related immunological dysfunction, contributing to the development of a frailty syndrome which impairs the therapeutic impact of recent advances in treatment strategies. Understanding the mechanisms by which accelerated immunological aging is induced and the ways in which a tumour such as MM influences this process is key to overcoming therapeutic barriers. A link between cellular mitochondrial dysfunction and the acquisition of an abnormal immune phenotype has recently been described and has widespread physiological consequence beyond the impact on the immune system. Here we outline our current understanding of normal immune aging, describe the mechanism of immunometabolic dysfunction in accelerating this process, and propose the role these processes are playing in the pathogenesis of MM.

Frailty-adjusted therapy in Transplant Non-Eligible patients with newly diagnosed Multiple Myeloma (FiTNEss (UK-MRA Myeloma XIV Trial)): a study protocol for a randomised phase III trial.

INTRODUCTION: Multiple myeloma is a bone marrow cancer, which predominantly affects older people. The incidence is increasing in an ageing population.Over the last 10 years, patient outcomes have improved. However, this is less apparent in older, less fit patients, who are ineligible for stem cell transplant. Research is required in this patient group, taking into account frailty and aiming to improve: treatment tolerability, clinical outcomes and quality of life. METHODS AND ANALYSIS: Frailty-adjusted therapy in Transplant Non-Eligible patients with newly diagnosed Multiple Myeloma is a national, phase III, multicentre, randomised controlled trial comparing standard (reactive) and frailty-adjusted (adaptive) induction therapy delivery with ixazomib, lenalidomide and dexamethasone (IRD), and to compare maintenance lenalidomide to lenalidomide+ixazomib, in patients with newly diagnosed multiple myeloma not suitable for stem cell transplant. Overall, 740 participants will be registered into the trial to allow 720 and 478 to be randomised at induction and maintenance, respectively.All participants will receive IRD induction with the dosing strategy randomised (1:1) at trial entry. Patients randomised to the standard, reactive arm will commence at the full dose followed by toxicity dependent reactive modifications. Patients randomised to the adaptive arm will commence at a dose level determined by their International Myeloma Working Group frailty score. Following 12 cycles of induction treatment, participants alive and progression free will undergo a second (double-blind) randomisation on a 1:1 basis to maintenance treatment with lenalidomide+placebo versus lenalidomide+ixazomib until disease progression or intolerance. ETHICS AND DISSEMINATION: Ethical approval has been obtained from the North East-Tyne & Wear South Research Ethics Committee (19/NE/0125) and capacity and capability confirmed by local research and development departments for each participating centre prior to opening to recruitment. Participants are required to provide written informed consent prior to trial registration. Trial results will be disseminated by conference presentations and peer-reviewed publications. TRIAL REGISTRATION NUMBER: ISRCTN17973108, NCT03720041.

Ixazomib with cyclophosphamide and dexamethasone in relapsed or refractory myeloma: MUKeight phase II randomised controlled trial results.

The all-oral combination of ixazomib, cyclophosphamide, and dexamethasone (ICD) is well tolerated and effective in newly diagnosed and relapsed multiple myeloma (MM). We carried out MUKeight, a randomised, controlled, open, parallel group, multi-centre phase II trial in patients with relapsed MM after prior treatment with thalidomide, lenalidomide, and a proteasome inhibitor (ISRCTN58227268), with the primary objective to test whether ICD has improved clinical activity compared to cyclophosphamide and dexamethasone (CD) in terms of progression-free survival (PFS). Between January 2016 and December 2018, 112 participants were randomised between ICD (n = 58) and CD (n = 54) in 33 UK centres. Patients had a median age of 70 years and had received a median of four prior lines of therapy. 74% were classed as frail. Median PFS in the ICD arm was 5.6 months, compared to 6.7 months with CD (hazard ratio (HR) = 1.21, 80% CI 0.9-1.6, p = 0.3634). Response rates and overall survival were not significantly different between ICD and CD. Dose modifications or omissions, and serious adverse events (SAEs), occurred more often in the ICD arm. In summary, the addition of ixazomib to cyclophosphamide and dexamethasone did not improve outcomes in the comparatively frail patients enroled in the MUKeight trial.

Trends in autologous stem cell transplantation for newly diagnosed multiple myeloma: Changing demographics and outcomes in European Society for Blood and Marrow Transplantation centres from 1995 to 2019.

Multiple myeloma (MM) accounts for 10% of haematological malignancies. Overall survival (OS) has improved in recent years due to increased use of autologous stem cell transplantation (ASCT) in the treatment of newly diagnosed MM and the advent of novel agents, including proteasome inhibitors, immunomodulatory drugs and monoclonal antibodies. To assess trends in ASCT including patient selection, choice of induction regimen, depth of response and survival, we performed a retrospective analysis of all patients undergoing first ASCT for MM in European Society for Blood and Marrow Transplantation centres between 1995 and 2019. A total of 117 711 patients across 575 centres were included. The number of transplants performed increased sevenfold across the study period. The median age increased from 55 to 61 years, and the percentage of patients aged >65 years rose from 7% to 30%. Use of chemotherapy-based induction fell significantly, being largely replaced by bortezomib-based regimens. The two-year complete response rate increased from 22% to 42%. The five-year progression-free survival and OS rates increased from 28% to 31% and from 52% to 69%, respectively. Transplant mortality fell from 5.9% to 1.5%. Ongoing advances in MM treatment may challenge the future role of ASCT. However, at the current time, ASCT remains central to the MM treatment paradigm.

Defining Unmet Need Following Lenalidomide Refractoriness: Real-World Evidence of Outcomes in Patients With Multiple Myeloma.

BACKGROUND: The treatment paradigm for multiple myeloma (MM) continues to evolve with the development of novel therapies and the earlier adoption of continuous treatments into the treatment pathway. Lenalidomide-refractory patients now represent a challenge with inferior progression free survival (PFS) reported to subsequent treatments. We therefore sought to describe the natural history of MM patients following lenalidomide in the real world. METHODS: This was a retrospective cohort review of patients with relapsed MM who received lenalidomide-based treatments in the U.K. Data were collected for demographics, subsequent therapies, treatment responses, survival outcomes and clinical trial enrollment. RESULTS: 198 patients received lenalidomide-based treatments at a median of 2 prior lines of therapy at a median of 41 months (range 0.5-210) from diagnosis. 114 patients (72% of 158 evaluable) became refractory to lenalidomide. The overall survival (OS) after lenalidomide failure was 14.7 months having received between 0-6 subsequent lines of therapy. Few deep responses were observed with subsequent treatments and the PFS to each further line was < 7 months. There was a steep reduction in numbers of patients able to receive further treatment, with an associated increase in number of deaths. The OS of patients progressing on lenalidomide who did not enter a clinical trial incorporating novel agents was very poor (8.8 months versus 30 months, p 0.0002), although the trials group were a biologically fitter group. CONCLUSION: These data demonstrate the poor outcomes of patients failing lenalidomide-based treatments in the real world, the highlight need for more effective treatments.

Reovirus-induced cell-mediated immunity for the treatment of multiple myeloma within the resistant bone marrow niche.

BACKGROUND: Multiple myeloma (MM) remains an incurable disease and oncolytic viruses offer a well-tolerated addition to the therapeutic arsenal. Oncolytic reovirus has progressed to phase I clinical trials and its direct lytic potential has been extensively studied. However, to date, the role for reovirus-induced immunotherapy against MM, and the impact of the bone marrow (BM) niche, have not been reported. METHODS: This study used human peripheral blood mononuclear cells from healthy donors and in vitro co-culture of MM cells and BM stromal cells to recapitulate the resistant BM niche. Additionally, the 5TGM1-Kalw/RijHSD immunocompetent in vivo model was used to examine reovirus efficacy and characterize reovirus-induced immune responses in the BM and spleen following intravenous administration. Collectively, these in vitro and in vivo models were used to characterize the development of innate and adaptive antimyeloma immunity following reovirus treatment. RESULTS: Using the 5TGM1-Kalw/RijHSD immunocompetent in vivo model we have demonstrated that reovirus reduces both MM tumor burden and myeloma-induced bone disease. Furthermore, detailed immune characterization revealed that reovirus: (i) increased natural killer (NK) cell and CD8+ T cell numbers; (ii) activated NK cells and CD8+ T cells and (iii) upregulated effector-memory CD8+ T cells. Moreover, increased effector-memory CD8+ T cells correlated with decreased tumor burden. Next, we explored the potential for reovirus-induced immunotherapy using human co-culture models to mimic the myeloma-supportive BM niche. MM cells co-cultured with BM stromal cells displayed resistance to reovirus-induced oncolysis and bystander cytokine-killing but remained susceptible to killing by reovirus-activated NK cells and MM-specific cytotoxic T lymphocytes. CONCLUSION: These data highlight the importance of reovirus-induced immunotherapy for targeting MM cells within the BM niche and suggest that combination with agents which boost antitumor immune responses should be a priority.

Plasmacytoid dendritic cells orchestrate innate and adaptive anti-tumor immunity induced by oncolytic coxsackievirus A21.

BACKGROUND: The oncolytic virus, coxsackievirus A21 (CVA21), has shown promise as a single agent in several clinical trials and is now being tested in combination with immune checkpoint blockade. Combination therapies offer the best chance of disease control; however, the design of successful combination strategies requires a deeper understanding of the mechanisms underpinning CVA21 efficacy, in particular, the role of CVA21 anti-tumor immunity. Therefore, this study aimed to examine the ability of CVA21 to induce human anti-tumor immunity, and identify the cellular mechanism responsible. METHODS: This study utilized peripheral blood mononuclear cells from i) healthy donors, ii) Acute Myeloid Leukemia (AML) patients, and iii) patients taking part in the STORM clinical trial, who received intravenous CVA21; patients receiving intravenous CVA21 were consented separately in accordance with local institutional ethics review and approval. Collectively, these blood samples were used to characterize the development of innate and adaptive anti-tumor immune responses following CVA21 treatment. RESULTS: An Initial characterization of peripheral blood mononuclear cells, collected from cancer patients following intravenous infusion of CVA21, confirmed that CVA21 activated immune effector cells in patients. Next, using hematological disease models which were sensitive (Multiple Myeloma; MM) or resistant (AML) to CVA21-direct oncolysis, we demonstrated that CVA21 stimulated potent anti-tumor immune responses, including: 1) cytokine-mediated bystander killing; 2) enhanced natural killer cell-mediated cellular cytotoxicity; and 3) priming of tumor-specific cytotoxic T lymphocytes, with specificity towards known tumor-associated antigens. Importantly, immune-mediated killing of both MM and AML, despite AML cells being resistant to CVA21-direct oncolysis, was observed. Upon further examination of the cellular mechanisms responsible for CVA21-induced anti-tumor immunity we have identified the importance of type I IFN for NK cell activation, and demonstrated that both ICAM-1 and plasmacytoid dendritic cells were key mediators of this response. CONCLUSION: This work supports the development of CVA21 as an immunotherapeutic agent for the treatment of both AML and MM. Additionally, the data presented provides an important insight into the mechanisms of CVA21-mediated immunotherapy to aid the development of clinical biomarkers to predict response and rationalize future drug combinations.

Multiple myeloma with multiple neurological presentations.

Multiple myeloma is a haematological malignancy with clonal plasma cell proliferation and production of monoclonal immunoglobulins. Its neurological complications are relatively common, caused by both the disease and the treatment. Neurologists should therefore be familiar with its neurological manifestations and complications. We describe a 40-year-old woman who presented with lower cranial neuropathies mimicking variant Guillain-Barré syndrome, with normal brain and spinal cord imaging and cerebrospinal fluid (CSF) albuminocytological dissociation, and subsequently diagnosed with IgD myeloma. She relapsed repeatedly with differing neurological presentations: numb chin syndrome and twice with impaired vision, first from cerebral venous sinus thrombosis and later from leptomeningeal infiltration of the optic chiasm. We discuss the neurological complications of myeloma, emphasising the need to consider it in a wide variety of neurological presentations and repeatedly to reassess its associated neurological diagnoses. We also highlight the complexity of myeloma treatment.

Patient-Reported Outcome Results From the Open-Label, Randomized Phase III Myeloma X Trial Evaluating Salvage Autologous Stem-Cell Transplantation in Relapsed Multiple Myeloma.

PURPOSE: Salvage autologous stem-cell transplantation (sASCT) in patients with multiple myeloma (MM) relapsing after a prior autologous stem-cell transplantation leads to increased remission duration and overall survival. We report a comprehensive study on patient-reported outcomes, including quality of life (QoL) and pain in sASCT. METHODS: Patients were randomly assigned to either sASCT or nontransplantation consolidation (NTC). Pain and QoL were assessed as secondary outcomes using validated QoL instruments (European Organisation for Research and Treatment of Cancer QLQ-C30 and myeloma-specific module, QLQ-MY20; the Brief Pain Inventory [Short Form]; and the Leeds Assessment of Neuropathic Symptoms and Signs [Self-Assessment] scale). RESULTS: A total of 288 patients (> 96%) consented to the QoL substudy. The median follow-up was 52 months. The European Organisation for Research and Treatment of Cancer QLQ-C30 Global health status scores were higher (better) in the NTC group at 100 days after random assignment (P = .0496), but not at later time points. Pain interference was higher (worse) in the sASCT group than in the NTC group at 6 months after random assignment (P = .0267), with patients with sASCT reporting higher scores for Pain interference with daily living for up to 2 years after random assignment. Patients reporting lower concerns about adverse effects of treatment after sASCT had a time to progression advantage. CONCLUSION: Patients with sASCT with relapsed MM demonstrated a comparative reduction in QoL and greater impact of treatment adverse effects lasting for 6 months and up to 2 years for pain, after which patients who had received sASCT reported better outcomes. Patients who experienced lower adverse effects after sASCT had longer time to progression and overall survival, showing the need to improve symptom management peritransplantation. To our knowledge, this study provides the most comprehensive picture of QoL before and after sASCT in patients with relapsed MM.

The impact of cytogenetics on duration of response and overall survival in patients with relapsed multiple myeloma (long-term follow-up results from BSBMT/UKMF Myeloma X Relapse [Intensive]): a randomised, open-label, phase 3 trial.

The Myeloma X trial (ISCRTN60123120) registered patients with relapsed multiple myeloma. Participants were randomised between salvage autologous stem cell transplantation (ASCT) or weekly cyclophosphamide following re-induction therapy. Cytogenetic analysis performed at trial registration defined t(4;14), t(14;16) and del(17p) as high-risk. The effect of cytogenetics on time to progression (TTP) and overall survival was investigated. At 76 months median follow-up, ASCT improved TTP compared to cyclophosphamide (19 months (95% confidence interval [95% CI] 16-26) vs. 11 months (9-12), hazard ratio [HR]: 0·40, 95% CI: 0·29-0·56, P < 0·001), on which the presence of any single high-risk lesion had a detrimental impact [likelihood ratio test (LRT): P = 0·011]. ASCT also improved OS [67 months (95% CI 59-not reached) vs. 55 months (44-67), HR: 0·64, 95% CI: 0·42-0·99, P = 0·0435], with evidence of a detrimental impact with MYC rearrangement (LRT: P = 0·021). Twenty-one (24·7%) cyclophosphamide patients received an ASCT post-trial, median OS was not reached (95% CI: 39-not reached) for these participants compared to 31 months (22-39), in those who did not receive a post-trial ASCT. The analysis further supports the benefit of salvage ASCT, which may still be beneficial after second relapse in surviving patients. There is evidence that this benefit reduces in cytogenetic high-risk patients, highlighting the need for targeted study in this patient group.

Changes in the liver transcriptome of farmed Atlantic salmon (Salmo salar) fed experimental diets based on terrestrial alternatives to fish meal and fish oil.

BACKGROUND: Dependence on marine natural resources threatens the sustainability of Atlantic salmon aquaculture. In the present study, Atlantic salmon fed for 14 weeks with an experimental diet based on animal by-products and vegetable oil (ABP) exhibited reduced growth performance compared with others fed a fish meal/fish oil based experimental diet (MAR) and a plant protein/vegetable oil-based experimental diet (VEG). To characterize the molecular changes underlying the differences in growth performance, we conducted a 44 K microarray study of the liver transcriptome of the three dietary groups. RESULTS: The microarray experiment identified 122 differentially expressed features (Rank Products, PFP < 10%). Based on their associated Gene Ontology terms, 46 probes were classified as metabolic and growth-relevant genes, 25 as immune-related, and 12 as related to oxidation-reduction processes. The microarray results were validated by qPCR analysis of 29 microarray-identified transcripts. Diets significantly modulated the transcription of genes involved in carbohydrate metabolism (gck and pfkfb4), cell growth and proliferation (sgk2 and htra1), apoptosis (gadd45b), lipid metabolism (fabp3, idi1, sqs), and immunity (igd, mx, ifit5, and mhcI). Hierarchical clustering and linear correlation analyses were performed to find gene expression patterns among the qPCR-analyzed transcripts, and connections between them and muscle and liver lipid composition. Overall, our results indicate that changes in the liver transcriptome and tissue lipid composition were driven by cholesterol synthesis up-regulation by ABP and VEG diets, and the lower carbohydrate intake in the ABP group. Two of the microarray-identified genes (sgk2 and htra1) might be key to explaining glucose metabolism regulation and the dietary-modulation of the immune system in fish. To evaluate the potential of these genes as predictive biomarkers, we subjected the qPCR data to a stepwise discriminant analysis. Three sets of no more than four genes were found to be able to predict, with high accuracy (67-94%), salmon growth and fatty acid composition. CONCLUSIONS: This study provides new findings on the impact of terrestrial animal and plant products on the nutrition and health of farmed Atlantic salmon, and a new method based on gene biomarkers for potentially predicting desired phenotypes, which could help formulate superior feeds for the Atlantic salmon aquaculture industry.

Transcriptome profiling of antiviral immune and dietary fatty acid dependent responses of Atlantic salmon macrophage-like cells.

BACKGROUND: Due to the limited availability and high cost of fish oil in the face of increasing aquaculture production, there is a need to reduce usage of fish oil in aquafeeds without compromising farm fish health. Therefore, the present study was conducted to determine if different levels of vegetable and fish oils can alter antiviral responses of salmon macrophage-like cells (MLCs). Atlantic salmon (Salmo salar) were fed diets containing 7.4% (FO7) or 5.1% (FO5) fish oil. These diets were designed to be relatively low in EPA + DHA (i.e. FO7: 1.41% and FO5: 1%), but near the requirement level, and resulting in comparable growth. Vegetable oil (i.e. rapeseed oil) was used to balance fish oil in experimental diets. After a 16-week feeding trial, MLCs isolated from fish in these dietary groups were stimulated by a viral mimic (dsRNA: pIC) for 6 h (qPCR assay) and 24 h (microarray and qPCR assays). RESULTS: The fatty acid composition of head kidney leukocytes varied between the two dietary groups (e.g. higher 20:5n-3 in the FO7 group). Following microarray assays using a 44K salmonid platform, Rank Products (RP) analysis showed 14 and 54 differentially expressed probes (DEP) (PFP < 0.05) between the two diets in control and pIC groups (FO5 vs. FO7), respectively. Nonetheless, Significance Analysis of Microarrays (SAM, FDR < 0.05) identified only one DEP between pIC groups of the two diets. Moreover, we identified a large number (i.e. 890 DEP in FO7 and 1128 DEP in FO5 overlapping between SAM and RP) of pIC-responsive transcripts, and several of them were involved in TLR-/RLR-dependent and cytokine-mediated pathways. The microarray results were validated as significantly differentially expressed by qPCR assays for 2 out of 9 diet-responsive transcripts and for all of the 35 selected pIC-responsive transcripts. CONCLUSION: Fatty acid-binding protein adipocyte (fabp4) and proteasome subunit beta type-8 (psmb8) were significantly up- and down-regulated, respectively, in the MLCs of fish fed the diet with a lower level of fish oil, suggesting that they are important diet-responsive, immune-related biomarkers for future studies. Although the different levels of dietary fish and vegetable oils involved in this study affected the expression of some transcripts, the immune-related pathways and functions activated by the antiviral response of salmon MLCs in both groups were comparable overall. Moreover, the qPCR revealed transcripts responding early to pIC (e.g. lgp2, map3k8, socs1, dusp5 and cflar) and time-responsive transcripts (e.g. scarb1-a, csf1r, traf5a, cd80 and ctsf) in salmon MLCs. The present study provides a comprehensive picture of the putative molecular pathways (e.g. RLR-, TLR-, MAPK- and IFN-associated pathways) activated by the antiviral response of salmon MLCs.

The effect of salvage autologous stem-cell transplantation on overall survival in patients with relapsed multiple myeloma (final results from BSBMT/UKMF Myeloma X Relapse [Intensive]): a randomised, open-label, phase 3 trial.

BACKGROUND: The Myeloma X trial previously reported improved durability of response (time to disease progression) in patients with relapsed multiple myeloma with salvage autologous stem-cell transplantation (ASCT) compared with oral cyclophosphamide in patients with multiple myeloma relapsing after a first ASCT. We report the final overall survival results of the trial. METHODS: BSBMT/UKMF Myeloma X was a multicentre, randomised, open-label, phase 3 trial done at 51 centres in the UK. Eligible patients with multiple myeloma relapsing after a previous ASCT were re-induced with intravenous bortezomib (1·3 mg/m(2) on days 1, 4, 8, 11), intravenous doxorubicin (9 mg/m(2) per day on days 1-4), and oral dexamethasone (40 mg/day on days 1-4, 8-11, and 15-18 during cycle 1 and days 1-4 during cycles 2-4), with supportive care as per local institutional protocols before randomisation in a 1:1 ratio to either high-dose melphalan (200 mg/m(2)) and salvage ASCT or weekly oral cyclophosphamide (400 mg/m(2) per week for 12 weeks). Randomisation was by permuted blocks stratified by length of first remission and response to re-induction treatment. The primary endpoint was time to disease progression; the study was also powered to detect a difference in the secondary endpoint, overall survival. Further secondary endpoints were the proportion of patients achieving an objective response, progression-free survival, overall survival, toxic effects and safety, pain, and quality of life. Prespecified exploratory endpoints included time to second objective disease progression (PFS2). Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00747877, and the European Clinical Trials Database, number 2006-005890-24, and is now in long-term follow-up. FINDINGS: Between April 16, 2008, and Nov 19, 2012, 297 patients were registered into the study and 174 were randomly assigned to receive either high-dose melphalan and salvage ASCT (n=89) or oral weekly cyclophosphamide (n=85). 173 (58%) of 297 patients relapsed after more than 24 months from first ASCT. 75 (43%) of 174 randomised patients had died at follow-up: salvage ASCT (n=31 [35%]) versus oral weekly cyclophosphamide (n=44 [52%]). Updated time to disease progression shows continued advantage in the salvage ASCT group compared with the weekly cyclophosphamide group (19 months [95% CI 16-26] vs 11 months [9-12]; hazard ratio [HR] 0·45 [95% CI 0·31-0·64] log-rank p<0·0001). Median overall survival was superior in the salvage ASCT group compared with weekly cyclophosphamide group (67 months [95% CI 55-not estimable] vs 52 months [42-60]; log-rank p=0·022; HR 0·56 [0·35-0·90], p=0·0169). Time to second objective disease progression was superior in the salvage ASCT group compared with the weekly cyclophosphamide group (67 months [52-not estimable] vs 35 months [31-43]; HR 0·37 [0·24-0·57], log-rank p<0·0001). During extended follow-up, no further treatment-related or treatment-unrelated adverse events were reported. 15 second primary malignancies were reported in 12 patients (salvage ASCT [n=7] vs oral weekly cyclophosphamide [n=5]). The cumulative incidence of second primary malignancies at 60 months after trial entry was 5·2% (2·1-8·2). INTERPRETATION: Salvage ASCT increases overall survival during consolidation of re-induction treatment in patients with multiple myeloma at first relapse after a first ASCT. The delay of salvage ASCT to third-line treatment or later might not confer the same degree of advantage as seen with salvage ASCT at first relapse. FUNDING: Cancer Research UK, Janssen-Cilag, and Chugai Pharma UK.

Stem Cell Harvesting after Bortezomib-Based Reinduction for Myeloma Relapsing after Autologous Transplantation: Results from the British Society of Blood and Marrow Transplantation/United Kingdom Myeloma Forum Myeloma X (Intensive) Trial.

The phase III British Society of Blood and Marrow Transplantation/United Kingdom Myeloma Forum Myeloma X trial (MMX) demonstrated prospectively, for the first time, superiority of salvage autologous stem cell transplantation over chemotherapy maintenance for multiple myeloma (MM) in first relapse after previous ASCT. However, many patients have stored insufficient stem cells (PBSC) for second ASCT and robust evidence for remobilization after first ASCT is lacking. We report the feasibility, safety, and efficacy of remobilization after bortezomib-doxorubicin-dexamethasone reinduction in MMX and outcomes of second ASCT with these cells. One hundred ten patients underwent ≥1 remobilization with 32 and 4, undergoing second and third attempts, respectively. Toxicities of remobilization were similar to those seen in first-line mobilization. After all attempts, 52% of those with insufficient previously stored PBSC had harvested a sufficient quantity to proceed to second ASCT. Median PBSC doses infused, neutrophil engraftment, and time to discharge after second ASCT were similar regardless of stem cell source, as were the toxicities of second ASCT. No significant differences between PBSC sources were noted in depth of response to ASCT or time to progression. Harvesting after bortezomib-doxorubicin-dexamethasone reinduction for MM at first relapse is safe and feasible and yields a reliable cell product for second ASCT. The study is registered with ClinicalTrials.gov (NCT00747877) and EudraCT (2006-005890-24).