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Supplementary Figure 1: Consort flow diagram of patient flow through TT4 arms Supplementary Figure 2: Cumulative Incidence of Death and Post-Relapse Survival (P-values for S-TT4 vs L-TT4; TRM, p = 0.61; MRM, p = 0.70; OIM, p = 0.25). Supplementary Figure 3: Clinical outcomes according to the presence of PETidentified focal lesions (FL; =3) by TT4 arm Supplementary Figure 4: Ingenuity pathways identified a network of 8 interrelated genes.

Supplementary Table 1: Total Therapy 4 (TT4) protocol schema Supplementary Table 2: Adverse Events by Arm and Maximum NCI CTC Grade Supplementary Table 3: Top 51 gene probes distinguishing multiple myeloma with and without cytogenetic abnormalities (CA)

Despite the dramatic improvement in multiple myeloma (MM) treatment, high-dose therapy and autologous stem cell transplantation (ASCT) continues to offer superior progression free and overall survi...

Autologous stem cell transplantation (ASCT) is standard of care in biologically fit, newly diagnosed multiple myeloma (MM) patients, offering better therapeutic outcomes and improved quality of life (QoL). However, with the UK’s 1st national lockdown on 23/03/2020, several guidelines recommended deferring ASCT due to risks of infection, with resource limitations forcing some units to suspend ASCT entirely. Such changes to patients’ treatment plans inevitably altered their lived experience during these uncertain times with expected impact on QoL. We conducted a qualitative study using semi-structured interviews to gain insight into MM patients’ understanding of their disease, initial therapy and ASCT, and their response to therapy changes. A clinical snapshot of how COVID-19 affected the MM ASCT service in a single UK institution is also provided, including changes to chemotherapy treatment plans, timing, and prioritisation of ASCT. Framework analysis identified 6 overarching themes: (1) beliefs about ASCT, (2) perceptions of information provided about MM and ASCT, (3) high levels of fear and anxiety due to COVID-19, (4) feelings about ASCT disruption or delay due to COVID-19, (5) perceptions of care, and (6) importance of social support. Example subthemes were beliefs that ASCT would provide a long-remission/best chance of normality including freedom from chemotherapy and associated side-effects, disappointment, and devastation at COVID-related treatment delays (despite high anxiety about infection) and exceptionally high levels of trust in the transplant team. Such insights will help us adjust our service and counselling approaches to be more in tune with patients’ priorities and expectations.

Autologous stem cell transplantation (ASCT) is standard of care in biologically fit, newly diagnosed multiple myeloma (MM) patients, offering better therapeutic outcomes and improved quality of life (QoL). However, with the UK’s 1st national lockdown on 23/03/2020, several guidelines recommended deferring ASCT due to risks of infection, with resource limitations forcing some units to suspend ASCT entirely. Such changes to patients’ treatment plans inevitably altered their lived experience during these uncertain times with expected impact on QoL. We conducted a qualitative study using semi-structured interviews to gain insight into MM patients’ understanding of their disease, initial therapy and ASCT, and their response to therapy changes. A clinical snapshot of how COVID-19 affected the MM ASCT service in a single UK institution is also provided, including changes to chemotherapy treatment plans, timing, and prioritisation of ASCT. Framework analysis identified 6 overarching themes: 1) beliefs about ASCT, 2) perceptions of information provided about MM and ASCT, 3) high levels of fear and anxiety due to COVID-19, 4) feelings about ASCT disruption or delay due to COVID-19, 5) perceptions of care and 6) importance of social support. Example subthemes were beliefs that ASCT would provide a long-remission/best chance of normality including freedom from chemotherapy and associated side-effects, disappointment, and devastation at COVID-related treatment delays (despite high anxiety about infection) and exceptionally high levels of trust in the transplant team. Such insights will help us adjust our service and counselling approaches to be more in tune with patients’ priorities and expectations.

BackgroundThe 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.MethodsThis 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.Results198 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.ConclusionThese data demonstrate the poor outcomes of patients failing lenalidomide-based treatments in the real world, the highlight need for more effective treatments.

Purpose Multiple myeloma (MM) is a clonal bone marrow disease characterized by the neoplastic transformation of differentiated postgerminal B cells. It is a heterogeneous disease both at the genetic level and in terms of clinical outcome. Immunoglobulin M (IgM) MM is a rare subtype of myeloma. Similar to Waldenström macroglobulinemia (WM), patients with MM experience IgM monoclonal gammopathy; however, both diseases are distinct in terms of treatment and clinical behavior. Materials and Methods To shed light on the presentation of IgM MM, its prognosis, and its gene expression profiling, we identified and characterized 21 patients with IgM MM from our database. Results One of these patients presented with a rare IgM monoclonal gammopathy of undetermined significance that progressed to smoldering myeloma. The median survival of the 21 patients was 4.9 years, which was comparable to a matched group of patients with non-IgM MM with similar myeloma prognostic factors (age, gender, albumin, creatinine, anemia, lactate dehydrogenase, β2-microglobulin, cytogenetics abnormalities), but much less than the median survival reported for patients with WM (9 years). We identified a cluster of genes that differ in their expression profile between MM and WM and found that the patients with IgM MM displayed a gene expression profile most similar to patients with non-IgM MM, confirming that IgM MM is a subtype of MM that should be differentiated from WM. Conclusion Because the prognosis of IgM MM and WM differ significantly, an accurate diagnosis is essential. Our gene expression model can assist with the differential diagnosis in controversial cases.

Background Achieving minimal residual disease (MRD) negativity during treatment for multiple myeloma (MM) is prognostic for disease free and overall survival. There is limited testing of MRD outside clinical trials, and the need to process fresh samples for multicolour flow cytometry (MCF) assays presents a challenge to laboratories. We set up an 8-colour MCF assay, in collaboration with a reference centre (Leeds HMDS), to identify and quantify malignant plasma cells in bone marrow (to limit of detection 10-5), using a BD LSRFortessaTM Flow Cytometer. We developed a fixation protocol, removing the need for immediate flow cytometer access and carried out a pilot study of MRD status in patients treated with standard non-trial protocols. Methods Initially, 43 fresh samples were processed in parallel with duplicate samples sent to Leeds, with 100% concordance in MRD result. We optimised a novel fixation technique, processing and staining samples within 24 hours, before using 0.4% paraformaldehyde (PFA) to fix, and storing at 4°C, permitting data acquisition up to 6 days later. We tested this on 24 samples and confirmed concordance between fresh and fixed samples, with good correlation in MRD level (Pearson’s correlation co-efficient; r=0.95 (95% CI 0.89-09.8); r2= 0.91; p= Results Between July 2020 and June 2021, MRD was performed on 76 samples, with 24% (18/76) fixed to facilitate processing. All samples were from transplant eligible MM patients, with 58 post-induction therapy, on aspirates performed for restaging prior to autologous stem cell transplantation (ASCT). This cohort represents a UK real-world snapshot, with complete response in 28% (16/58) post-induction with NICE-approved bortezomib-based triplet regimens. 17% (10/58) of samples were MRD negative, with no difference in post-induction MRD level between patients with or without R-ISS-defined high-risk cytogenetics (p=0.72). 18 post-ASCT samples were analysed, and 8 (44.4%) were MRD negative; in 8 of these with matched pre-ASCT samples (all MRD positive), 4 were MRD negative post-ASCT. The number of MRD negative samples was significantly higher post-ASCT (p=0.02). Conclusions In summary, we describe an adaptation of the Leeds HMDS 8-colour MRD assay to include a novel fixation step, permitting batching for flow cytometric analysis, without affecting sample validity. This real-world snapshot suggests that less than 20% of patients are MRD negative post standard UK induction therapy, but this increases post-ASCT. Further work on larger cohorts and longer follow up will confirm the prognostic value of MRD assessments outside the trial setting and establish its utility in real world practice.

A number of new treatments have been approved for patients with multiple myeloma (MM) and the survival of patients continues to improve. However, discrepancies in outcomes of clinical trials and th...

Introduction: Daratumumab in combination with bortezomib and dexamethasone (DVd) demonstrated a superior overall response rates (ORR) and progression free survival (PFS) compared to Vd in the CASTOR phase 3 trial for patients with RRMM. On this basis, DVd was recommended in March 2019 for UK patients with RRMM that had 1 prior line (PL). Discrepancies in outcomes between patients treated in clinical trials compared to routine practice is well recognised due to a combination of patient, disease and treatment-related factors. In addition, bortezomib is often administered once-weekly in routine practice to minimise neuropathy, while CASTOR used bi-weekly bortezomib dosing. As a result, the real-world outcomes of patients treated with DVd are yet to be determined. The primary aims of this analysis was to assess the ORR and PFS for patients with RRMM with 1PL treated with DVd in routine practice. Secondary aims were to assess OS, time to next treatment (TTNT), and efficacy in different sub-groups (high risk cytogenetics, previous proteasome inhibitor (PI) exposure, refractoriness of prior therapies, bi-weekly vs weekly bortezomib schedule, and previous treatment free interval (TFI)). Methods: This was a retrospective analysis from 14 centres (academic and community hospitals; 7 within the West Midlands Research Consortium (WMRC)) treated with DVd between March 2019 and June 2021. Patients received daratumumab (IV and then SC from June 2020) weekly in cycles 1-3, on day 1 of a 3-week cycle during cycles 4-8, and then monthly from cycle 9 to progression. SC Bortezomib was predominantly given weekly for cycles 1-8 although 5 centres used bi-weekly dosing for selected patients with aggressive disease. Adverse events were graded as per CTCAE criteria. Results: 288 patients were included, with a median age of 69 years (range 20-88) (Table 1). Patients received a median of 1 PL (range 1-2) with 93% (269) 1PL, 7% (18) 2 PL (due to COVID-19 measures). The majority had an ECOG performance status of 0-2 (98%) and most received weekly bortezomib (n=201). This population differed from those with 1PL treated on CASTOR in being older, more were ISS 3 (31% vs 19%, p=0.0145), and more had prior bortezomib exposure (71% vs 51%, p=0.0003), 4% were PI refractory, 9% had a GFR of The ORR was 76%, with >VGPR in 54% (Table 2), with no significant difference in response between patients receiving biweekly vs weekly bortezomib (85% vs 83%; p=0.71). The median time to response was 1.6m. With a median follow up of 15m, the median PFS was 14m (95% CI 11.6-16). High cytogenetic risk patients had inferior outcomes: median PFS 10m (95% CI 6-14) for high risk vs not reached for standard risk (p=0.043); as did those with advanced ISS: median PFS was not reached, 15 and 12m for stage I, II and III respectively (p=0.05). For 15 patients with extramedullary disease (EMD), the median PFS was 3m (95% CI 1-5). Median PFS for patients who were PI refractory was shorter (10m vs 15m for PI sensitive patients (p=0.006)). There was no difference in median PFS for patients with prior PI exposure vs no prior PI (15 vs 13m; p=0.75), or according to weekly or bi-weekly bortezomib schedule (11 vs 15m; p=0.14). The median TTNT was 21m (95% CI 17-25). Overall, the median duration of treatment was 8m and 25 patients (9%) stopped treatment to receive a second autologous stem cell transplant. Those that had a prior TFI of >12m had a longer median PFS of 21m vs 10m (p=0.0004). The median OS has not been reached, with an estimated 2-year OS of 74%. For patients with high risk cytogenetics the median OS was 16m (95% CI 9-23; vs not reached for standard risk; p=0.0006), with estimated 2-year OS in the high risk group of 36%. There was no difference in OS for patients treated with biweekly vs weekly bortezomib (not reached for either; p=0.38). DVd was generally well tolerated with 6% stopping due to adverse events (CASTOR 9.5%). Grade 3 or 4 toxicity occurred in 62 (22%) most commonly neutropenia and thrombocytopenia, with any grade infusion reactions reported in 27 (9%). Conclusions: These real-world data of DVd at 1 st relapse demonstrated good tolerability and high response rates with a weekly bortezomib schedule despite a more heterogenous population. However, high risk patients by cytogenetics, ISS or EMD had inferior outcomes as did those treated within 12 months from first line treatment. Figure 1 Figure 1. Disclosures Cook: Karyopharm: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Oncopeptides: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding. Pratt: Binding Site: Consultancy; BMS/Celgene: Consultancy; Gilead: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Amgen: Consultancy. Kishore: Celgene: Other: Attending fees; Jannsen: Other: Attending fees; Sanofi: Other: Attending fees; Takeda: Other: Attending fees. Yong: Amgen: Honoraria; Autolus: Research Funding; BMS: Research Funding; Janssen: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; GSK: Honoraria; Takeda: Honoraria. Popat: Abbvie, Takeda, Janssen, and Celgene: Consultancy; Takeda: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES; GlaxoSmithKline: Consultancy, Honoraria, Research Funding; AbbVie, BMS, Janssen, Oncopeptides, and Amgen: Honoraria; Janssen and BMS: Other: travel expenses.

Background Concerns have been raised about the ability of patients with plasma cell disorders (PCD) to mount adequate immune responses to vaccination, particularly considering the initial extension to vaccination intervals in the United Kingdom to up to 12 weeks in December 2020, the start of the vaccination roll-out. Protecting this vulnerable patient group with anti-SARS-CoV-2 vaccination is critical. Methods We measured the humoral responses in PCD patients after the first dose of the BNT162b2 and ChAdOx-1 nCOV-19 vaccines. Antibody levels were measured using Elecsys Anti-SARS-CoV-2S assay for quantitative detection of IgG Abs, specific for the SARS-CoV-2 spike-protein receptor binding domain. Positive cut-off of ≥0.80 U/mL defined serologic response. Testing was performed at (or closest to) 4 and 8 weeks post-dose. Baseline nucleocapsid Ab results were available from previous screening in a subset of patients. Clinical information was retrieved from medical records. Results 198 PCD patients (178 multiple myeloma, 15 amyloid, 3 SMM/MGUS, other 2 PCD), median age 63 (range 35-84), had serologic assessment after one vaccine dose against SARS-CoV-2. 69% (138) were on chemo-immunotherapy treatment (CIT) within 4 weeks of first dose. Previous COVID-19 infection and exposure was defined as a positive COVID-19 PCR swab or positive N-antibody at baseline and were excluded from analysis. Patients were tested at median 42 days (range 11-90) after their first dose. After one dose, 68% (135/198) were seropositive, with median Ab titres 21 U/mL (IQR 3.92-133.5). In those with negative baseline Ab test, seroconversion to one dose was 67% (64/96). Forty-one patients were tested more than once after one dose, at median 33 days (12-62) and again at 67 days (38-91). 22 were seronegative at first testing, of these, 6 seroconverted on second testing, prior to second dose. Age ≥70, light chain (LC) disease, disease status less than VGPR, CIT within 4 weeks, low IgM and ASCT more than 12m were statistically significant seronegative predictors on univariate analyses. LC disease, less than VGPR and IgM less than 0.4g/L, retained statistical significance on multivariate analyses. On linear regression analysis in seropositive patients, more lines of treatment and CIT within 4 weeks were significantly associated with lower antibody titres. Conclusion Serologic response to SARS-CoV-2 vaccination is lower in PCD patients than reported healthy controls at 68% after one dose. Further work in PCD is needed to understand how Ab levels correlate to neutralisation capability, cellular responses, protection from infection and how long seroconversion lasts to better define correlates of protection. Nearly a third do not respond to a single vaccine dose causing concerns that PCD patients are left vulnerable with a delayed vaccination strategy and should be prioritised to receiving the second dose at the recommended manufacturers time scale of 3 or 4 weeks.

Purpose: To determine whether a reduction in the intensity of Total Therapy (TT) reduces toxicity and maintains efficacy. Experimental Design: A total of 289 patients with gene expression profiling (GEP70)-defined low-risk multiple myeloma were randomized between a standard arm (TT4-S) and a light arm (TT4-L). TT4-L employed one instead of two inductions and consolidations. To compensate for potential loss of efficacy of TT4-L, bortezomib and thalidomide were added to fractionated melphalan 50 mg/m2/d for 4 days. Results: Grade ≥3 toxicities and treatment-related mortalities were not reduced in TT4-L. Complete response (CR) rates were virtually identical (P = 0.2; TT4-S, 59%; TT4-L, 61% at 2 years), although CR duration was superior with TT4-S (P = 0.05; TT4-S, 87%; TT4-L, 81% at 2 years). With a median follow-up of 4.5 years, there was no difference in overall survival (OS) and progression-free survival (PFS). Whereas metaphase cytogenetic abnormalities (CAs) tended to be an adverse feature in TT4-S, as with predecessor TT trials, the reverse applied to TT4-L. Employing historical TT3a as training and TT3b as test set, 51 gene probes (GEP51) significantly differentiated the presence and absence of CA (q < 0.0001), seven of which function in DNA replication, recombination, and repair. Applying the GEP51 model to clinical outcomes, OS and PFS were significantly inferior with GEP51/CA in TT4-S; such a difference was not observed in TT4-L. Conclusions: We identified a prognostic CA-linked GEP51 signature, the adversity of which could be overcome by potentially synergizing anti–multiple myeloma effects of melphalan and bortezomib. These exploratory findings require confirmation in a prospective randomized trial. Clin Cancer Res; 23(11); 2665–72. ©2016 AACR.

// Xenofon Papanikolaou 1 , Sarah Johnson 1 , Tarun Garg 1 , Erming Tian 1 , Ruslana Tytarenko 1 , Qing Zhang 1 , Caleb Stein 1 , Bart Barlogie 1 , Joshua Epstein 1 and Christoph Heuck 1 1 Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, Little Rock, AR Correspondence: Christoph Heuck, email: // Keywords : artesunate, apoptosis, caspases, myeloma, ROS Received : February 18, 2014 Accepted : March 22, 2014 Published : March 24, 2014 Abstract Although novel drugs have contributed immensely to improving outcomes of patients with multiple myeloma (MM), many patients develop drug resistance and ultimately succumb to MM. Here, we show that artesunate, an anti-malarial drug, reliably induces cell death in vitro in naive as well as drug-resistant MM cells at concentrations shown to be safe in humans. Artesunate induced apoptosis predominantly through the non-caspase mediated pathway by primarily targeting mitochondria and causing outer mitochondrial membrane permeabilization that led to cytosolic and subsequent nuclear translocation of mitochondrial proteins apoptosis inducing factor (AIF) and endonuclease G (EndoG). Nuclear translocation of AIF and EndoG was accompanied by low levels of reactive oxygen species (ROS) and increased mitochondrial production of superoxide. These effects were present before apoptosis was evident and were related to intracellular levels of bivalent iron (Fe +2 ). Artesunate’s unique mechanism probably was at least partially responsible for, its ability to act synergistically with multiple anti-myeloma agents. Our findings suggest that artesunate acts through iron to affect the mitochondria and induce low ROS and non-caspase–mediated apoptosis. Its potency, toxicity profile, and synergism with other drugs make it an intriguing new candidate for MM treatment.

Poster: "EANM 19 / EP-0316 / Impact of PET Reconstruction Algorithms on Assessment and Quantification of FDG-PETCT Findings in Patients with Multiple Myeloma" by: "S. Riaz, S. Arulogun, A. L. Smith, N. Rabin, S. Wan, J. Bomanji; NHS, London, "

Relapsed/refractory multiple myeloma represents a major challenge in multiple myeloma therapy. For patients with relapsed/refractory multiple myeloma, we developed a treatment schema of metronomically scheduled drug therapy. We identified 186 patients who had been treated with metronomic therapy between March 2004 and January 2012 with a median follow up of 24.2 months. Median age was 61 years (range 36–83). Median number of prior therapies was 14 (range 1–51). Median number of completed metronomic therapy cycles was 1 (range 1–5), while 45 of 186 (25%) received 2 or more cycles. Responses included complete remission in 11 of 186 patients (6%), very good partial remission in 12 of 186 (7%), partial remission in 65 of 179 (36%), and minimal response in 29 of 186 (16%), for an overall response rate of 63% (117 of 186). Median overall survival and progression-free survival were 11.2 and 3.6 months, respectively. Hematologic toxicity grading was problematic as 146 of 186 (78%) of patients presented with at least grade 2 thrombocytopenia within 90 days prior to starting metronomic therapy. Grade 4 leukopenia, anemia, and/or thrombocytopenia following metronomic therapy occurred in 108 of 186 (58%), 12 of 186 (6%), and 147 of 186 (79%) patients, respectively. Incidence of grade 3–4 neutropenic fever was 4 of 186 (2%). Most patients (177 of 186, 95%) were treated in an outpatient unit and secondary admissions due to regimen-related toxicity occurred in 37 of 186 (20%). Treatment-related mortality was evident in 2 of 186 (1%). In conclusion, metronomic therapy is an effective late salvage treatment in relapsed/refractory multiple myeloma, with a high overall response rate and a favorable toxicity profile.

Objectives Patients with multiple myeloma (MM) are at increased risk of arterial thrombosis. Our aim was to determine the risk factors, mechanisms and outcome of strokes in these patients. Methods We conducted a retrospective matched case-control study from our database of MM patients enrolled in Total Therapy (TT) 2, TT3a and TT3b protocols who developed a vascular event (transient ischemic attack, ischemic stroke, or intracerebral hemorrhage) from October 1998 to January 2014. Cases were matched for age-matched selected controls. Baseline demographics, risk factors, MM characteristics, laboratory values, and mortality of cases were compared to those of controls. Multivariate logistic regression analysis identified risk factors associated with stroke. Ischemic strokes (IS) were classified with modified Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria. Results Of 1,148 patients, 46 developed a vascular event (ischemic stroke, 33; transient ischemic attack, 11; hypertensive intracerebral hemorrhage, 2). Multivariate logistic regression analysis determined renal insufficiency (odds Ratio, 3.528; 95% CI, 1.36-9.14; P = 0.0094) and MM Stages I and II (odds Ratio, 2.770, 95% CI, 1.31-5.81; p = 0.0073) were independent predictors of stroke. In our study, strokes attributable to hypercoagulability, atrial fibrillation and small-vessel occlusion were common mechanisms. After a stroke, 78% of patients were discharged to home or a rehabilitation facility and 4% to a long-term nursing facility; in-hospital mortality was 15%. Despite suffering a stroke no significant differences in survival were observed. Conclusion In our cohort of multiple myeloma patients, renal failure and MM Stages I and II had increased risk of stroke.

Multiple myeloma is preceded by an asymptomatic phase, comprising monoclonal gammopathy of uncertain significance and smoldering myeloma. Compared to the former, smoldering myeloma has a higher and non-uniform rate of progression to clinical myeloma, reflecting a subset of patients with higher risk. We evaluated the gene expression profile of smoldering myeloma plasma cells among 105 patients enrolled in a prospective observational trial at our institution, with a view to identifying a high-risk signature. Baseline clinical, bone marrow, cytogenetic and radiologic data were evaluated for their potential to predict time to therapy for symptomatic myeloma. A gene signature derived from four genes, at an optimal binary cut-point of 9.28, identified 14 patients (13%) with a 2-year therapy risk of 85.7%. Conversely, a low four-gene score (

As part of Total Therapy (TT) 3b, baseline marrow aspirates were subjected to two-color flow cytometry of nuclear DNA content and cytoplasmic immunoglobulin (DNA/CIG) as well as plasma cell gene expression profiling (GEP). DNA/CIG-derived parameters, GEP and standard clinical variables were examined for their effects on overall survival (OS) and progression-free survival (PFS). Among DNA/CIG parameters, the percentage of the light chain-restricted (LCR) cells and their cytoplasmic immunoglobulin index (CIg) were linked to poor outcome. In the absence of GEP data, low CIg 5.5 mg/l, a percentage of LCR cells exceeding 50%, C-reactive protein ⩾8 mg/l and GEP-derived high centrosome index. Further analysis revealed an association of low CIg with 12 gene probes implicated in cell cycle regulation, differentiation and drug transportation from which a risk score was developed in TT3b that held prognostic significance also in TT3a, TT2 and HOVON trials, thus validating its general applicability. Low CIg is a powerful new prognostic variable and has identified potentially drug-able targets.

All cases of clinical myeloma (CMM) are preceded by an asymptomatic monoclonal gammopathy (AMG), classified as either monoclonal gammopathy of undetermined significance (MGUS) or asymptomatic multiple myeloma (AMM). We analyzed data from AMG patients (n = 331) enrolled in a prospective, observational clinical trial (S0120). Baseline data from clinical variables, gene expression profiles (GEP) of purified tumor cells, and findings of magnetic resonance imaging (MRI) were correlated with the risk of progression to CMM requiring therapy. GEP of purified tumor cells revealed that all molecular subtypes of CMM are also represented in the AMG phase. An increased risk score (>-0.26) (based on a 70-gene signature, GEP70) was an independent predictor of the risk of progression to CMM. Combination of elevated serum free light chain, M-spike, and GEP70 risk score identified a subset with high risk (67% at 2 years) of progression to CMM requiring therapy. Importantly, absence of these factors in AMM patients predicted low risk similar to MGUS. Detection of multiple (>1) focal lesions by MRI also conferred an increased risk of progression. These data demonstrate that signatures associated with high-risk CMM impact disease risk and support inclusion of genomic analysis in the clinical management of AMGs.

Collection of hematopoietic progenitor cells (HPC) after previous autologous hematopoietic progenitor cell transplant (aHCT) was studied in 221 patients with multiple myeloma (MM). With a total of 333 collections, the median number of CD34+ cells collected was 4.7 × 10(6) CD34+ cells/kg, and 74% of the patients collected ≥ 2.5 × 10(6) CD34+ cells/kg. Among 26 variables examined, the strongest predictor for poor collection was a platelet count

Multiple myeloma (MM) is a clonal plasma cell (PC) disorder characterized by end organ damage that is in turn characterized by CRAB criteria (calcium and creatinine elevation, anemia and bone lesions).1 It is commonly accepted that nearly all cases of MM are preceded by a clinically benign phase of monoclonal gammopathy of undetermined significance (MGUS) that evolves through a stage of smoldering multiple myeloma (SMM) without end organ damage,2 collectively referred to as asymptomatic monoclonal gammopathies (AMG).3 Although traditionally SMM is considered more prone to MM progression than MGUS, additional variables, such as involved-to-uninvolved free light-chain ratio4 and magnetic resonance imaging-defined focal lesion number and size,5 have been linked to progression to MM and form the basis for the newest International Myeloma Working Group criteria for MM.6 As the treatment of MM has been greatly advanced, emphasis has been placed on identifying patients with AMG at high risk of progression to MM so that, with earlier treatment, end organ damage can be minimized.7 Many new high-risk variables have indeed been identified such as level of circulating plasma cells8 and gene expression profiling (GEP).9, 10 We have previously reported that two-parameter flow cytometry of DNA and cytoplasmic light-chain immunoglobulin (DNA/CIG) is highly predictive of progression-free and overall survival in newly diagnosed MM treated with Total Therapy.11 In the current subset analysis of S0120, we have investigated whether the DNA/CIG assay can also identify patients with AMG at high risk for progression to MM requiring therapy (time to therapy, TTT).12 Of 254 patients enrolled at the University of Arkansas in the observational SWOG S0120 protocol with AMG, 110 had evaluable DNA/CIG information and retained AMG status according to the revised International Myeloma Working Group criteria for MM.6 All patients underwent detailed clinical staging as previously reported.9, 10 DNA/CIG assay was performed on whole bone marrow aspirates along with metaphase cytogenetics and GEP of CD138+ purified PC.13 Imaging studies involved metastatic bone surveys and, in the majority of the cases, magnetic resonance imaging examination of the axial and appendicular skeleton. Details of the DNA/CIG method have been published elsewhere.14, 15 A technical modification of the assay was applied uniformly since August 2006. The assay is based on the two-parameter flow cytometry of cytoplasmic immunoglobulin and DNA of whole bone marrow aspirates. Single-cell suspensions were exposed to anti-light-chain reagents (Dako Kappa and Lambda light chain F(AB)2/FITC conjugated) and then counterstained for DNA with propidium iodide with the addition of RNase. To quantitate the cellular DNA content, DNA index (DI)16 was determined and calculated as the ratio of the mean for each light-chain-positive G0/1 DNA peak divided by the mean of the light-chain-negative diploid G0/1 peak on the X axis. A DI between 0.99 and 1.01 was referred to as diploid, while hyperdiploid implied DI>1.01 and hypodiploid DI17% and CIg 17% and serum-M⩾3 g/dl; albumin

Extramedullary plasmacytomas constitute a rare and not well studied subset of multiple myeloma (MM) relapses. We report the incidence, clinical-laboratory features and outcome of patients with MM and extramedullary relapse (ExMeR). A total of 303 patients with symptomatic MM were recorded in a 13-year period in two institutions. Twenty-eight cases of ExMeR (9%) were recorded. There was an increased frequency of elevated lactate dehydrogenase (LDH) (p = 0.026), bone plasmacytomas (p = 0.001) and fractures (p = 0.002) at diagnosis, in patients with ExMeR compared to the others. ExMeR was associated with an ominous outcome, high LDH, constitutional symptoms and a statistically significant decrease of monoclonal paraprotein compared to levels at diagnosis (p = 0.009). Prior treatment with bortezomib was associated with a decreased hazard of ExMeR (p = 0.041). Overall survival (OS) was decreased in patients with ExMeR compared to the others (38 vs. 59 months, p = 0.006). Patients with MM with ExMeR have a lower OS and their clinical and laboratory features differ from those without.

Introduction: Total Therapy 4 (TT4) comprises a randomized phase III trial enrolling 289 patients with gene expression profiling defined low-risk MM in which patients were allocated to a standard arm (TT4-S) or a light arm (TT4-L) with as principal goal to reduce toxicity yet maintain efficacy in TT4-L. Methods: The TT4-S regimen was similar to TT3b and utilized 2 cycles of VDTPACE induction, tandem transplantation with melphalan 200mg/m2, 2 cycles of dose reduced VDTPACE consolidation and 3 years maintenance with VRD. In TT4-L the number of induction and consolidation cycles was reduced to one each and melphalan was given in a fractionated fashion (50mg/m2/d x 4days) to avoid peak levels of melphalan and reduce mucosal toxicity. Bortezomib and thalidomide were added to the fractionated melphalan conditioning regimen to explore synergistic effects and compensate for potential loss of efficacy. Results: Grade ≥3 toxicities in TT4-S and L occurred with similar frequencies. With a median follow-up of 4.5 years, the OS and PFS were similar in TT4-S and TT4-L at 90 and 87% respectively. The same applied to PFS (TT4-S 84% versus TT4-L 79%). The presence of metaphase defined cytogenetic abnormalities (CA) affected clinical outcomes. In TT4-S, patient with CA had a strong trend toward inferior OS compared to patients with no CA (2 year estimate 83 versus 94%, p=0.08), while the reverse applied to TT4-L (95 versus 81%, p=0.07). Non-significant trends in similar directions were noted for PFS. Complete remission duration tended to be inferior in patients with CA-type MM in TT4-S (2 year estimate, 79 vs. 92%, p=013) with no significant differences in TT4-L. Time to relapse was significantly shorter for CA patients on the TT4-S arm (2 year estimate, 15.4 versus 3.9%), but was not affected by CA in the TT4-L (15.8 vs 7.9%, p=0.79. The observation of CA's favorable OS impact in TT4-L was not anticipated. We next analyzed whether the presence or absence of metaphase CA was linked to specific gene probes which could help to explain better outcomes in TT4-L. Among a training set of 266 untreated patients enrolled in TT3a with available baseline GEP studies, 90 (34%) exhibited CA. Among a test set of 164 patients with baseline GEP accrued to TT3b, 67 (41%) qualified as having CA. Fifty-one probes were different in patients with and without CA (q Conclusions: A prognostic CA-linked GEP signature can identify patients who benefit from conditioning with fractionated melphalan dosing together bortezomib, thalidomide and dexamethasone which negates the adverse impact of CA. Patients who lacked a CA-type gene signature were best served with single high dose melphalan. These exploratory findings need to be confirmed in a prospective randomized trial. Figure 1. PFS according to 51-gene model predicting CA versus no-CA according to arm (TT4-S, 1A; TT4-L, 1B) Figure 1. PFS according to 51-gene model predicting CA versus no-CA according to arm (TT4-S, 1A; TT4-L, 1B) Disclosures van Rhee: University of Arkansa for Medical Sciences: Employment. Mitchell:Cancer Research and Biostatistics: Employment. Zangari:Millennium: Research Funding; Novartis: Research Funding; University of Arkansas for Medical Sciences: Employment; Onyx: Research Funding. Sawyer:University of Arkansas for Medical Sciences: Employment. Waheed:University of Arkansas for Medical Sciences: Employment. Heuck:Millenium: Other: Advisory Board; Janssen: Other: Advisory Board; Celgene: Consultancy; Foundation Medicine: Honoraria; University of Arkansas for Medical Sciences: Employment. Thanendrarajan:University of Arkansas for Medical Sciences: Employment. Schinke:University of Arkansas for Medical Sciences: Employment. Jethava:University of Arkansas for Medical Sciences: Employment. Grazziutti:University of Arkansas for Medical Sciences: Employment. Petty:University of Arkansas for Medical Sciences: Employment. Steward:University of Arkansas for Medical Sciences: Employment. Panozzo:University of Arkansas for Medical Sciences: Employment. Bailey:University of Arkansas for Medical Sciences: Employment. Hoering:Cancer Research and Biostatistics: Employment. Crowley:Cancer Research and Biostatistics: Employment. Davies:University of Arkansas for Medical Sciences: Employment; Celgene: Consultancy; Janssen: Consultancy; Onyx: Consultancy; Millenium: Consultancy. Barlogie:University of Arkansas for Medical Sciences: Employment. Morgan:MMRF: Honoraria; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Weismann Institute: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; CancerNet: Honoraria; University of Arkansas for Medical Sciences: Employment; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees.

The proteasome inhibitor Bortezomib (Bz), the first agent of a new class of drugs in Multiple Myeloma (MM), has shown remarkable activity and forms an integral part of modern MM treatment. Nevertheless, resistance to Bz eventually develops in a significant proportion of patients, with adverse effects on survival. Numerous publications have addressed this issue through in vitro developed models of acquired Bz resistance (BzR). However the results were quite different in each publication, none of the produced Bz myeloma cell lines was provably stable, no common mechanism of resistance could be demonstrated, and hence were of minimal relevance to the clinical setting. In order to address these issues an effort was made for the development of an in vitro model of acquired BzR that would resemble the clinical reality in the most accurate way. Two myeloma cell lines were used, one resembling a multisensitive (JJN3) and the other a multiresistant (U266) drug behavior, that were both sensitive to Bz. An at least 20 fold increase in the 48h Bz IC50 was noted for both cell lines. The increase in the IC50 was able to be verified a year after culturing the cell lines in normal medium thus ensuring a stable resistance phenotype. To delineate the molecular mechanisms that underlie the development of BzR a combined genetic/Gene Expression Profile (GEP) and functional/Proteomics approach was used with emphasis in the common elements of both cell lines. The hypothesis was that if certain pathways are activated in the cells that actually produce the phenotype of BzR they must fulfil two important criteria: 1) They must be present in all the levels of the BzR, 2) The gene changes have to be verified in the level of the gene encoded proteins thus securing their functional importance. GEP of the naïve cell lines along with the GEP of the Bz resistant cells at different levels of BzR (5-fold, 10-fold, 20-fold) were used. The statistical analysis revealed 100 gene probes common in both cell lines that achieved their highest change as soon as BzR was established and remained stable at that level for all later versions (P The proteomics analysis of the Bz resistant cell lines at their latest level of resistance (20-fold) revealed 262 proteins common in both cell lines that were up-regulated and 263 common in both cell lines that were down-regulated (change >10% to be considered significant). The intersection of the list of the common genes with the list of the common proteins revealed 47 gene-proteins all but one novel in MM. They can be grouped in distinct biological categories with the most prominent ones being the ROS/Mitochondrial Factor category comprising of 10 gene-proteins, the E3 Ubiquitin Pathway 6 genes-proteins and Translation Regulation 5 genes-proteins. Even more importantly 30 of them have profound survival implications in MM -all of them novel in MM- both for Overall Survival (OS) and Progression Free Survival (PFS) in both Bz (TT3) and non Bz (TT2) containing protocols implying that myeloma cells apply both Bz specific and non-specific mechanisms to acquire BzR. Based on these 30 genes-proteins a GEP risk score (GEP-30) was constructed that was able to achieve remarkable statistical power in both Bz containing and non containing trials of both newly diagnosed (TT2 with and without thalidomide i.e. TT2+ and TT2-, TT3a, TT3b, HOVON, MRC IX, Figure 1A,B,C) and relapsed MM (TT6 , OS: NR vs 1.52 yr P Figure 1. KM plots for OS and PFS of GEP-30 for newly diagnosed MM Figure 1. KM plots for OS and PFS of GEP-30 for newly diagnosed MM Figure 1B. Figure 1B. Figure 1C. Figure 1C. Disclosures Stein: University of Arkansas for Medical Sciences: Employment. Barlogie:University of Arkansas for Medical Sciences: Employment. Epstein:University of Arkansas for Medical Sciences: Employment. Heuck:Janssen: Other: Advisory Board; Celgene: Consultancy; Millenium: Other: Advisory Board; Foundation Medicine: Honoraria; University of Arkansas for Medical Sciences: Employment.

Introduction: Despite the enormous progress in MM therapy brought about by the rapid development of many novel agents, many patients end up with limited treatment options. We have previously reported on the efficacy and safety of metro16 in RRMM (Papanikolaou, Haematology 2013). Here we are reporting on an extension of such treatment to 28 d (metro28). Patients and Methods: The treatment consisted of a cycle of 28d continuous iv infusions of ADR and DDP each at 1mg/m2/d, along with thalidomide 50 to 100mg/d x 28; bortezomib 0.8 to 1.0mg/m2 on days 1, 4, 7, 10, 13, 16, 19, 22, 25, 28; DEX 8 to 12mg on days 1-4, 7-10, 13-16, 19-22, 25-28; some patients also received vincristine 0.07mg flat dose by CI for 28 days. This was off-protocol therapy that patients provided written informed consent for. The IRB permitted data retrieval and analysis. Results: 150 patients were identified, virtually all had received prior tandem transplants, bortezomib, lenalidomide, carfilzomib and pomalidomide. The median age was 64yr; B2M was elevated >=3.5mg/L in 48%, abnormal cytogenetics (CA) were present in 86%, and 44% had GEP70-defined high risk MM. Figure 1 portrays clinical outcomes. As of April 2015, 60 patients had died, and the 2-yr OS estimate was 45% (Figure 1A); the 6-mo PFS estimate was 31% although 15% had no progression at 18mo (Figure 1B). Analysis by GEP70 and GEP5 risk revealed 18-mo OS estimates of 80% among the 53 patients with low risk in both models, whereas the presence of high risk (HRMM) in either model conferred a significantly reduced 18-mo OS estimate of 25% (p=ULN (25%, HR=3.46, p=3.5mg/L (51%, HR+2.63, p=0.014) and thrombocytopenia Conclusion: Metro28 represents a well-tolerated additional tool in the treatment armamentarium for RRMM. Figure 1. Clinical outcomes A: Overall survival B: Progression-free survival C: Overall survival according to GEP70 and GEP5 risk designations Figure 1. Clinical outcomes. / A: Overall survival. / B: Progression-free survival. / C: Overall survival according to GEP70 and GEP5 risk designations Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Schinke: University of Arkansas for Medical Sciences: Employment. Thanendrarajan:University of Arkansas for Medical Sciences: Employment. Rosenthal:Cancer Research and Biostatistics: Employment. Petty:University of Arkansas for Medical Sciences: Employment. Zangari:University of Arkansas for Medical Sciences: Employment; Onyx: Research Funding; Millennium: Research Funding; Novartis: Research Funding. Heuck:Celgene: Consultancy; Foundation Medicine: Honoraria; Janssen: Other: Advisory Board; University of Arkansas for Medical Sciences: Employment; Millenium: Other: Advisory Board. van Rhee:University of Arkansa for Medical Sciences: Employment. Epstein:University of Arkansas for Medical Sciences: Employment. Yaccoby:University of Arkansas for Medical Sciences: Employment. Morgan:Weismann Institute: Honoraria; CancerNet: Honoraria; MMRF: Honoraria; University of Arkansas for Medical Sciences: Employment; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Davies:University of Arkansas for Medical Sciences: Employment; Celgene: Consultancy; Janssen: Consultancy; Millenium: Consultancy; Onyx: Consultancy. Barlogie:University of Arkansas for Medical Sciences: Employment.

Introduction: The objective of study was to determine the risk factors, stroke mechanisms and outcome following a stroke in Multiple myeloma (MM) patients. Materials and Methods: We conducted a matched cohort study from a prospective database of MM patients enrolled in TT2, TT3A, TT3B protocols who developed a vascular event (transient ischemic attack, ischemic stroke and intracerebral hemorrhage) from 1998 to 2014 with age, sex and treatment matched controls. Comparison of baseline demographics, risk factors, myeloma characteristics, laboratory values and mortality between both groups was performed using Pearson's Chi-square test for categorical variables and student T test for continuous variables. Multivariate logistic regression analysis was performed to identify risk factors associated with stroke. For statistical analysis SAS 9.4 software was used and p value of ≤ 0.05 was considered significant. Strokes were classified using the modified Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria. Results: Of 1148 patients, 46 developed a vascular event (Ischemic stroke (TIA)-33, Transient ischemic attack-11, Hypertensive intracerebral hemorrhage-2). On univariate analysis, predictors of stroke were a positive smoking history (26.1% vs 13% p=0.0381), renal insufficiency (23.9% vs 8.0% p=0.0039), hemodialysis (10.9 vs 0.7% p=0.004) and MM Stage I and II as opposed to Stage III (Stage I - 23.9% vs 9.4%, Stage II - 17.4% vs 12.3, Stage III - 58.7% vs 78.3% p=0.025). Despite the lack of significant difference in the baseline laboratory values between both groups, among the cases, there was a significant decrease in the platelet count (112.6 vs 255.2, p On multivariate analysis, independent predictors of stroke were renal insufficiency (Odds Ratio, 3.528, 95% CI, 1.36-9.14; p=0.0094) and MM Stage I and II (Odds Ratio, 2.770, 95% CI, 1.31-5.81; p=0.0073). The ischemic strokes subtypes were: Large vessel disease 6%, cardioembolic 18%, small vessel disease 21%, other known etiologies 49% (hypercoaguable state; watershed; others) and cryptogenic in 6%. Following ischemic event, antiplatelet agents were used in 16 patients, anticoagulation in 7 patients but 23 patients were ineligible for both due to thrombocytopenia. In our cohort, 78% were discharged home or rehabilitation facility, 4% were transferred to long-term nursing facility and in hospital mortality was 15%. Conclusion: In MMpatient'srenal insufficiency andMM Stage I and II were associated with increased stroke risk. Besides hypercoagulability other mechanisms like atrial fibrillation, watershed strokes and small vessel disease played major role. Table 1.Demographic and disease characteristics of multiple myeloma patients experiencing a stroke compared to controlsVariableStroke (N=46)No Stroke (N=138)p-valueAge [mean (sd)]60.6 (7.7)60.7 (7.8)0.8960Female50.0 (23)41.6 (57)0.3023Race Caucasian95.7 (44)90.6 (125)0.3635#Hypertension54.4 (25)43.5 (60)0.2003HPL33.3 (15/45)26.1 (36)0.3464Diabetes17.4 (8)9.4 (13)0.1409CAD10.9 (5)10.9 (15)>0.99CHF4.4 (2)8.7 (12)0.5237AFIB17.4 (8)9.4 (13)0.1409Smoking26.1 (12)13.0 (18)0.0381ETOH2.2 (1)1.5 (2)>0.99Malignancy8.7 (4)16.1 (22/137)0.2159Nephropathy23.9 (11)8.0 (11)0.0039Hemodialysis10.9 (5)0.7 (1)0.0040Protocol TT2 TT3A TT3B54.4 (25) 32.6 (15) 13.0 (6)57.2 (79) 21.0 (29) 21.7 (30)0.3036MM Stage I II III23.9 (11) 17.4 (8) 58.7 (27)9.4 (13) 12.3 (17) 78.3 (108)0.0182MM Isotype IgG IgA FLC-κ FLC-λ Other58.7 (27) 21.7 (10) 8.7 (4) 10.9 (5) 049.3 (68) 24.6 (34) 10.1 (14) 10.9 (15) 5.1 (7)0.6128MM Risk [mean (sd)] Death-0.13 (0.61; N=37) 65.2(30)0.09 (0.67; N=88) 51.5(71)0.0941 0.19OSA: Obstructive sleep apnea, CAD: coronary artery disease, CHF: Congestive heart failure, AFIB: Atrial fibrillation, HPL hyperlipidemia. Table 1. Results on multivariable logistic model Variable Odds Ratio 95% CI p-value Nephropathy 3.528 1.36 to 9.14 0.0094 MM Stage I or II 2.770 1.31 to 5.81 0.0073 Disclosures Hinduja: University of Arkansas for Medical Sciences: Employment. Limaye:University of Arkansas for Medical Sciences: Employment. Ravilla:University of Arkansas for Medical Sciences: Employment. Sasapu:University of Arkansas for Medical Sciences: Employment. Waheed:University of Arkansas for Medical Sciences: Employment.

Therapy related myeloid malignancies are an increasingly recognized treatment complication in patients undergoing therapy for multiple myeloma. The main predisposing factors are the alkylating agents, topoisomerase II inhibitors and radiotherapy, but recently questions have been raised regarding the immunomodulatory agent lenalidomide. Little is known about the new antimyeloma agents in the context of therapy related myeloid malignanices. The duration of treatment and the time from diagnosis are the main contributing factors in alkylating induced myeloid malignancies which occur 5-10 years after treatment, chromosome 5 and 7 abnormalities being the characteristic finding. High dose therapy (HDT) does not seem to be a major contributing factor per se in multiple myeloma. In a number of large published series, all the factors related with therapy-induced myelodysplasia were defined prior to HDT. Topoisomerase II inhibitors induce mainly acute leukemias which invariably correlate with dysregulation of the MLL gene. Radiotherapy causes therapy related myelodysplasia if applied in bone marrow producing areas, especially if combined with chemotherapy. Therapy related myeloid malignancies generally herald a poor prognosis. Karyotypic abnormalities seem to be the main prognostic factor. In all cases the risk for therapy related myeloid malignancies drops sharply by 10 years after the treatment.