Clonal Plasma Cells In Bone Marrow Research Articles

Biological and Clinical Significance of Undetectable Circulating Tumor Cells (CTCs) in Patients (Pts) with Multiple Myeloma (MM)

Background: There is evidence suggesting that CTCs are responsible for the spreading of clonal plasma cells (PC) inside and outside the bone marrow (BM). However, some pts unexpectedly have no CTCs at diagnosis. Here, we hypothesize that the absence of CTC defines a specific MM subtype with unique biological and clinical features. To our knowledge, this has never been investigated. Aim: Define the frequency and the characteristics of MM pts with undetectable CTCs. Methods: The presence of CTCs was assessed by NGF in 1,687 blood samples from pts with MGUS (n=467), smoldering (SMM, n=368), newly diagnosed (NDMM, n=652), and relapsed refractory MM (RRMM, n=200). All pts were enrolled in prospective PETHEMA/GEM multicenter studies. BM clonal PC from pts with (n=239) and without (n=17) detectable CTCs were isolated by FACS prior exome and RNA sequencing. Results: The frequency of pts with undetectable CTCs progressively decreased ( p <.001) from MGUS (54%) to SMM (21%) and NDMM (8%) and RRMM (11%). In those with detectable CTCs, the median percentage was 0.0003, 0.002, 0.01 and 0.008, respectively ( p <.001). The 1-log increment observed in the progression from MGUS to SMM and NDMM underpins the link between CTC levels and malignant transformation. The similarity between NDMM and RRMM is probably related with the fact that the latter were analyzed in between different lines of therapy. When compared to other SMM pts, those with undetectable CTCs were more frequently staged with low risk according to the 20/2/20 IMWG model (32% vs 49%; p <.001). Absence of CTCs (HR: 0.38; p =.042) and the 20/2/20 IMWG model (HR: 0.46; p =.011) had independent prognostic value in a multivariate analysis. SMM pts with undetectable vs detectable CTCs showed lower risk of transformation (progression rates at 2 years of 7% vs 24%, p =.004). When compared to other NDMM pts, those with undetectable CTCs had significantly less anemia and BM infiltration. There were no differences in the incidence of lytic lesions, renal insufficiency and hypercalcemia. Only one pt without CTCs was staged with R-ISS 3. These findings, together with a higher frequency of plasmacytomas, suggest that the MM subtype with undetectable CTCs may include pts with macrofocal disease. In transplant eligible NDMM, pts with undetectable vs detectable CTCs had PFS rates at 7 years of 90% and 44% (HR: 0.14; p <.001), and OS rates of 97% and 72% (HR: 0.10; p =.02). In transplant ineligible NDMM, pts with undetectable vs detectable CTCs had PFS rates at 2 years of 83% and 54% (HR: 0.31; p =.02), and OS rates of 94% and 76% (HR: 0.33; p =.13). In a multivariate analysis including the R-ISS and transplant eligibility, the absence of CTCs showed independent prognostic value for PFS (HR: 0.25; p <.001) and OS (HR: 0.24; p =.014). Among pts with undetectable CTCs, there were no differences in PFS and OS according to the presence of standard vs high risk cytogenetic abnormalities. In contrast with pts having detectable CTCs, the PFS and OS of those without CTCs was not significantly reduced if CR or MRD negativity were not achieved after treatment. The cytogenetic profile of BM clonal PC from pts with undetectable (n=112) vs detectable (n=846) CTCs was characterized by a lower frequency of +1q21 (16% vs 45%, p <.001) and del(1p32) (2% vs 8%, p =.02). Other alterations were not significant. There was no correlation between the number of mutations and the percentage of CTCs. There were 66 differentially expressed genes (DEG) between BM clonal PC from pts with detectable vs undetectable CTCs. Of note, 9 of the 66 DEG were in Chr1, with up- and downregulation of genes in 1p and 1q, respectively. There was a significant correlation between CTC levels and the expression of genes associated with MM dissemination ( FLNA, LGALS1and CXCR4). These findings, together with the modest correlation between the percentage of CTCs and BM clonal PC determined by morphology (r=.45, p <.001) and NGF (r=.49, p <.001), suggest that the selective egress of tumor cells from BM into the PB might depend on specific genetic alterations and transcriptional priming more than tumor burden. Conclusions:This study uncovered that ~8% of MM pts may progress due to mechanisms different from CTC dissemination. This subgroup showed unique features and achieved unprecedented outcomes, particularly if eligible to intensive therapy. Accordingly, our results endorse the recognition of pts with undetectable CTCs as a new biological and clinical subtype of MM.

Clonal Bone Marrow Plasma Cells Are Common in Patients with Vexas Syndrome

Introduction Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant plasma cell disorder. Its prevalence increases with age, reaching 5.3% at 70 years and older. MGUS progression rate to multiple myeloma (MM) or lymphoplasmacytic malignancies is about 1% per year. Patients with autoimmune diseases are at increased risk for developing MGUS and MM. VEXAS ( vacuoles, E1 enzyme, X-linked, autoinflammatory and somatic syndrome), an autoinflammatory disorder caused by somatic mutations in ubiquitin-activating enzyme, UBA1 gene, has been associated with plasma cell disorders, specifically MGUS, but the rate of progression is unknown. Cyclin D1, important in cell cycle progression, is regulated by ubiquitin-proteasome mediated degradation, and deregulated cyclin D1 degradation appears to be responsible for the increased levels of cyclin D1 in several cancers. We aim to assess marrow characteristics of plasma cell disorders (MGUS and MM) in VEXAS, and the impact of a defective UBA1 gene product on protein expression of cyclin D1. Methods We assessed 28 patients with VEXAS for the presence of plasma cell disorders, and studied the clinical and laboratory characteristics of those with clonal plasma cells (PC) in the marrow. Bone marrow biopsies and aspirates were evaluated by morphology, immunohistochemistry (IHC) dual staining CD138/cyclin D1, dual staining MUM-1/CD20), in situ hybridization (Kappa and Lambda light chains) and flow cytometry (FC) using antibodies for CD45, CD19, CD20, CD38, CD138, CD56, CD27, CD81, and cytoplasmic Kappa and Lambda immunoglobulins. Serum free kappa and lambda light chains, IgG, IgM, and IgA, and level of M paraprotein by serum protein electrophoresis with immunofixation were obtained. Fluorescent in situ hybridization (FISH) analysis for MM was performed in most patients with clonal PCs. Results Patients' characteristics are shown in Table 1. Eight out of 28 (28.6%) patients with VEXAS syndrome, all males, had clonal plasma cells in their bone marrows: 3 MGUS, and 5 MM (3 smoldering, SMM; 2 active MM). Median age at diagnosis of plasma cell disorder was 69.5 years (range 60-75). All patients had pathogenic UBA1 somatic mutations (5 M41T, 1 each with M41V, M41L, and splice). Clinically, monoclonal protein size was small, with no specific selection of the type of M protein. Strikingly, most (7/8) clonal plasma cells showed cyclin D1 expression by IHC (dual immunohistochemical stain cyclinD1/CD138 shows double-positive plasma cells; figure not shown), displaying nuclear immunoreactivity in 6 cases, and cytoplasmic immunoreactivity in 1 case. FC showed positive CD20 expression of clonal plasma cells in 4 cases, dim to negative CD19 expression in 4 cases, variable CD56 (positive in 3 cases, partial positive in 1 case, negative in 2 cases), dim to negative CD27 in 3 cases, dim to negative CD81 in 3 cases. FISH for MM revealed: t(11;14) in 4 cases, gain of 11q (CCND1) in 2 cases (in one case, CCND1 gain was present in addition to t(11;14); in the second case, CCND1 gain was associated with gains of 1q and 17p(TP53). The cyclin D1-negative MM case was negative for t(11;14) but had t(4;14). All cases with t(11;14) were positive for cyclin D1 by IHC (nuclear staining). The one MM case with negative nuclear staining for cyclin D1 but positive cytoplasmic staining, was negative for t(11;14) but had gain of 11q (CCND1).One patient evolved from SMM to active MM after 4 years. No evolution to MM was seen in patients with MGUS with median follow-up of 30 months. Conclusions Patients with VEXAS have a high prevalence of clonal plasma cell disorders. Clonal plasma cells in VEXAS patients are associated with t(11;14), and with nuclear cyclin D1 expression by IHC. Cytoplasmic cyclin D1 staining in the absence or weak nuclear staining was observed with CCND1 gain and absence of t(11;14). Cytoplasmic cyclin D1 expression may be related to lack of cyclin D1 protein degradation by a defective UBA1 gene product. Our results suggest that defective UBA1 and autoinflammation pose an increased risk for developing MGUS or multiple myeloma.

Relapsed and Refractory Multiple Myeloma with Pleural Involvement: A Case Series

Introduction. Development of novel treatments and immunotherapeutics continue to improve outcomes in patients with multiple myeloma (MM), especially in those patients with standard and intermediate risk disease; patients with high-risk disease continue to have dismal outcomes. Secondary extramedullary disease (sEMD) is a high-risk disease feature associated with an expected survival less than one year (Zanwar, AJH, 2023). Detailed description of patients presenting with sEMD involving the pleural has not been previously published. In the present project, we aimed to identify and characterize this patient population. Methods. This study was approved by the University of Utah (UU) IRB (#00156207). Inclusion was determined by search of the UU Department of Radiology database for all imaging including PET, CT, and MRI; search terms included pleura, pleural, lung, extramedullary, plasmacytoma, and multiple myeloma. Descriptive analysis was completed and included patient demographics, treatment, and outcomes. Survival was described with Kaplan-Meier analysis. Results. Fourteen patients treated at the UU between 2004 and 2023 were identified as having sEMD and pleural involvement. Demographics are highlighted in Table 1. The median age was 59 (range 34-79), 50% of patients were male, 86% were Caucasian, and 8% were Hispanic. Patients were treated with a median of 2.5 (range 1-9) lines of therapy prior to sEMD development and 6 (range 2-13) in total. 50% of the patients were penta-class refractory. The median follow-up after MM diagnosis was 34.5 months (range 13.0-162.0), median follow-up after pleural EMD diagnosis was 9.0 months (range 0.4-92.0), and the median time to development of pleural EMD following diagnosis was 18.2 months (range 3.8-142.8). At initial diagnosis, half of the patients had > 50% bone marrow clonal plasma cells, 29% had R-ISS stage 3 disease and 36% had R2-ISS stage III/IV. Of the 11 patients with available cytogenetic information at the time of sEMD diagnosis, 73% had high-risk cytogenetics (defined as presence of del(17p), t(4;14), t(14;16) and/or +1q). The most common cytogenetic abnormalities were +1q in 73%, including 36% with 3 copies and 36% with > 4 copies, amp(1q), and del(17p) in 36% . Of these patients, 18% had double hit and 18% had triple hit disease. The most frequent concomitant cytogenetic abnormalities were +1q and del(17p) seen in 36% of the patients. On imaging, 79% had additional sites of EMD other than the pleura. The most frequent other sites of sEMD were skin/soft tissue, intrabdominal extrahepatic, and hepatic. Pleural effusion was present in only one patient. Following diagnosis of sEMD with pleural involvement, the overall response rate (ORR) was 93% and the ORR was 88% in patients with 1q21 abnormalities. In this population, the median OS from diagnosis was 2.5 years (range 1.1-14) and mOS from diagnosis of pleural EMD was 0.64 years (range 0.03-7.7). Conclusion. To the best of our knowledge, this is the largest case series focused on the characterization of patients with sEMD and pleural involvement. The survival results align with those recently presented by Zanwar et al., suggesting that pleural involvement does not portend worse prognosis than EMD in general. We highlight that the majority of patients have 1q21 abnormalities and additional sites of EMD. Ultimately, patients with sEMD and pleural involvement have extremely high-risk disease, are responsive to treatments but have a drastically shortened survival.

Evaluation of Metabolic Profile in Monoclonal Gammopathy of Undetermined Significance and Asymptomatic Multiple Myeloma

Background: Multiple myeloma (MM) is an incurable, heterogeneous neoplasm of clonal plasma cells preceded by the precursor conditions monoclonal gammopathy of undetermined significance (MGUS) and asymptomatic multiple myeloma (AMM). Features of metabolic syndrome including obesity and diabetes mellitus (DM) have been linked to MM development and mortality. A study of serum metabolomics in patients with clinical MM revealed alterations in the citrate cycle, pyrimidine, purine, and amino acid metabolism (Chanukuppa V, et al, 2019). However, the role of dysregulated metabolism in progression of monoclonal gammopathy across the disease spectrum requires further investigation. We performed a pilot study to compare metabolic profiles in patients with MGUS and AMM. Methods: Clinical data and peripheral blood plasma samples were collected on 20 patients with non-immunoglobulin M MGUS and 20 patients with AMM. Global metabolomic studies by liquid chromatography (LC) tandem mass spectrometry (MS) were performed on plasma using a SCIEX TripleTOF 6600+ mass spectrometer. Each sample was run on Hypercarb and Reverse phase LC columns in both positive and negative MS modes. Standard targeted and untargeted metabolomic platforms were used with assessment of changing metabolites between the two groups in a one-to-one comparison. Correlation analysis and differential expression analysis were completed and top changing metabolites determined based on ranking of log fold change and p value cutoff (at least <0.05). Pathway enrichment data was generated with advanced data analysis. Results: Median age was 70 and 67 years in the MGUS and AMM groups, respectively. 65% of patients in the AMM group were male versus (vs) 40% in the MGUS group. Black individuals represented 15% of MGUS and 30% of AMM patients. Mean serum monoclonal (M) protein was higher in AMM patients (0.87 g/dL vs 0.28 g/dL, p=0.009); mean bone marrow clonal plasma cells in AMM was 17% (range 10-40%). Three AMM patients had high risk cytogenetics inclusive of chromosome 1 abnormalities. We observed that more individuals in the AMM group had a diagnosis of DM (35% vs 15%), were on hyperlipidemia (HLD) medications (80% vs 45%) and were on ≥3 hypertension (HTN) medications (35% vs 20%). Increasing weight and hemoglobin a1c values were seen with rise in serum M protein in the combined groups. For global metabolomics, 2,513 metabolites were annotated from targeted and untargeted peak curation and results underwent data normalization and standardization with exclusion of outliers from the data set. Using targeted data, Partial Least Squares-Discriminant Analysis showed separation of the MGUS and AMM groups (Figure 1). There were 8 significantly increased metabolites (dihydrouracil, quinate, melatonin, azelate, phosphonoacetate, malate, L-alanine, and lysine) and 9 significantly decreased metabolites (diethanolamine, xanthosine, nervonate, quinolinate, L-dopa, ethylmalonate, butanoate, carnosine, and thyroxine) in AMM when compared to MGUS. Pathway enrichment analysis revealed several pathways increased in AMM (Figure 2), including D-glutamine and D-glutamate metabolism and the citrate cycle. Pathways decreased in AMM included purine, pyrimidine, and glutathione metabolism. Conclusions: In our study, patients with AMM were more likely to demonstrate features of metabolic syndrome, including higher rate of DM and need for HTN and HLD medications. Additionally, a trend of higher weight and hemoglobin a1c values occurred as serum M protein increased. Global metabolomics identified unique metabolites that differed in AMM relative to MGUS patients. The unsaturated fatty acid nervonate, decreased in AMM in our study, was previously shown to have potential preventive effects on obesity-related metabolic disorders (Oda E, et al, 2005). Our findings in these precursor cohorts are concordant with prior serum metabolomics in MM showing alterations in amino acid metabolism including glutamine (Chanukuppa V, et al, 2019), with glutaminase inhibitors currently under study in relapsed/refractory MM (Bolzoni M, et al, 2016). Additional investigation of metabolic profile in larger groups across the spectrum of monoclonal gammopathy will help further elucidate the role of this dysregulated metabolism in myelomagenesis and could lead to preventative interventions targeting the associated metabolic syndrome earlier in disease course.

Hyperphosphatemia: A Clue to Diagnosis

Serum phosphate level reference range in adults is 2.5 mg/dL to 4.5 mg/dL and in children is 3 mg/dL to 6 mg/dL. The causes of hyperphosphatemia fall into four categories. These are decreased renal excretion of phosphorus, exogenous phosphorus administration, redistribution of phosphorus, and pseudohyperphosphatemia. We report a 69-year-old gentleman presented with the history of swelling of feet and facial puffiness of 1 month duration. He had renal failure with normal sized kidneys. Serum phosphorus was high. Advanced investigations revealed plasma cell proliferative disorder (clonal bone marrow plasma cells >10%) on bone marrow examination, presence of M band at the junction of beta-2 and gamma region, and elevated serum IgG and serum beta-2 microglobulin. Hyperphosphatemia in multiple myeloma may be true, or pseudohyperphosphatemia. Diligent history, examination, and investigations have yielded the possibility of pseudohyperphosphatemia owing to multiple myeloma in our patient. The interference with the phosphomolybdate ultraviolet assay for serum phosphorus estimation is one of the reasons of pseudohyperphosphatemia in multiple myeloma. The other mechanism of pseudohyperphosphatemia could be the direct binding of paraprotein to phosphorus.

Subcutaneous daratumumab (DARA SC) versus active monitoring in patients (pts) with high-risk smoldering multiple myeloma (SMM): Randomized, open-label, phase 3 AQUILA study.

TPS8075 Background: Standard of care for SMM includes active monitoring until progression to multiple myeloma (MM); however, recent evidence suggests pts with high-risk features may benefit from early treatment. DARA is a human IgGκ monoclonal antibody targeting CD38 that is approved as monotherapy for relapsed/refractory MM (RRMM) or in combination with standard of care for RRMM or newly diagnosed MM. Results from the phase 3 COLUMBA study showed that DARA SC demonstrated similar efficacy to intravenous (IV) DARA but with a lower rate of infusion-related reactions and shorter administration time. Based on the promising single-agent activity observed with IV DARA in intermediate- or high-risk SMM pts during the phase 2 CENTAURUS study, we hypothesized that DARA SC may delay progression to MM versus active monitoring in pts with high-risk SMM. Methods: AQUILA is an ongoing, randomized, open-label, multicenter phase 3 study of DARA SC versus active monitoring in pts with high-risk SMM. DARA SC (DARA 1,800 mg + recombinant human hyaluronidase PH20 [rHuPH20; 2,000 U/mL; Halozyme]) is administered by manual injection over approximately 5 minutes at alternating locations on the abdomen weekly in Cycles 1 and 2, every 2 weeks in Cycles 3-6, and every 4 weeks thereafter until 39 cycles (28 days/cycle), up to 36 months, or until disease progression. Eligibility criteria include confirmed diagnosis of SMM for ≤5 years, factors indicating high risk of progression to MM (clonal bone marrow plasma cells [BMPCs] ≥10% and ≥1 of the following: serum M protein ≥30 g/L, IgA SMM, immunoparesis with reduction of 2 uninvolved Ig isotypes, serum involved:uninvolved free light chain ratio ≥8 to < 100, or clonal BMPCs > 50% to < 60% with measurable disease), and ECOG performance status ≤1. The primary endpoint is progression-free survival (PFS), assessed by an independent review committee, with disease progression defined according to International Myeloma Working Group diagnostic criteria for MM. Secondary endpoints include time to biochemical or diagnostic (SLiM-CRAB) progression, overall response rate, complete response rate, duration of response, time to response, time to first-line treatment for MM, PFS on first-line treatment for MM (PFS2), overall survival, and incidence of MM with adverse prognostic features. The study completed enrollment on May 6, 2019; 390 pts have been randomly assigned to DARA SC or active monitoring. The primary efficacy analysis will be performed after approximately 165 PFS events have been observed. Clinical trial information: NCT03301220.

Identification of Immune-Related Genes for Risk Stratification in Multiple Myeloma Based on Whole Bone Marrow Gene Expression Profiling.

Background: Multiple myeloma (MM) is characterized by abnormal proliferation of bone marrow clonal plasma cells. Tumor immunotherapy, a new therapy that has emerged in recent years, offers hope to patients, and studying the expression characteristics of immune-related genes (IRGs) based on whole bone marrow gene expression profiling (GEP) in MM patients can help guide personalized immunotherapy. Methods: In this study, we explored the potential prognostic value of IRGs in MM by combining GEP and clinical data from the GEO database. We identified hub IRGs and transcription factors (TFs) associated with disease progression by Weighted Gene Co-expression Network Analysis (WGCNA), and modeled immune-related prognostic signature by univariate and multivariate Cox and least absolute shrinkage and selection operator (LASSO) regression analysis. Subsequently, the prognostic ability of signature was verified by multiple statistical methods. Moreover, ssGSEA and GSEA algorithm reveled different immunological characteristics and biological function variation in different risk groups. We mapped the hub IRGs by protein-protein interaction network (PPI) and extracted the top 10 ranked genes. Finally, we conducted vitro assays on two alternative IRGs. Results: Our study identified a total of 14 TFs and 88 IRGs associated with International Staging System (ISS). Ten IRGs were identified by Cox -LASSO regression analysis, and used to develop optimal prognostic signature for overall survival (OS) in MM patients. The 10-IRGs were BDNF, CETP, CD70, LMBR, LTBP1, NENF, NR1D1, NR1H2, PTK2B and SEMA4. In different groups, risk signatures showed excellent survival prediction ability, and MM patients also could be stratified at survival risk. In addition, IRF7 and SHC1 were hub IRGs in PPI network, and the vitro assays proved that they could promote tumor progression. Notably, ssGSEA and GSEA results confirmed that different risk groups could accurately indicate the status of tumor microenvironment (TME) and activation of biological pathways. Conclusion: Our study suggested that immune-related signature could be used as prognostic markers in MM patients.

The Significance of CD319 and CD269 in the Detection of Immunophenotyping and Minimal Residual Disease in Multiple Myeloma Patients

To investigate the expression of CD319 and CD269 in plasma cells of patients with multiple myeloma (MM) and the feasibility of using CD319 instead of CD38 as a gating antigen in immunophenotyping and minimal residual disease (MRD) monitoring. The expression levels of CD319 and CD269 antigens in clonal bone marrow plasma cells of 387 patients were detected by CD38/CD138 gating strategy with 8-color flow cytometry, and the stability of antigens was also analyzed, and the sensitivity and correlation of two different gating strategies employing CD319/CD138 and CD38/CD138 were compared as well. The control group consisted of 53 cases with non-malignant blood disease matched by age and sex. Monoclonal plasma cells were detected in 303 of 387 MM patients, among which 277 cases (91.42%) were positive for CD269, and all cases were positive for CD319 (100%). In newly diagnosed MM (NDMM) and recurrent refractory MM (RRMM) patients, the expression levels of CD269 were 97.53% (0-99.92%) and 94.96% (0.22%-99.99%), respectively, while levels of CD319 were 99.90% (87.77%-100%) and 99.78% (63.12%-100%), respectively. The expression levels of CD269 and CD319 in the control group were 97.00% (77.00%-100%) and 100% (89.00%-100%), respectively. There were no significant differences in the expression levels of CD269 and CD319 among NDMM, RRMM and the control group. Patients acquiring therapeutic effects were divided into complete remission (CR) group, very good partial response (VGPR) group and partial response (PR) group. Gating with CD38/CD138, median MRD values were 0.76% (0-1.88%), 0.77% (0-4.96%) and 1.75% (0.09%-10.90%) in the three groups, respectively, while gating with CD319/CD138, median MRD values were 0.57% (0.18%-1.96%), 1.07% (0.12%-4.85%) and 1.77% (0.08%-8.22%), respectively. There was no significant difference in MRD level by the two gating strategies, but a good correlation between the two (r=0.808, P<0.05). In addition, in 4 patients treated by CD38 monoclonal antibody (DARA), the expression level of CD38 was observed to be down-regulated or even negative after treatment. When the MRD level was very low, CD38/CD138 gating resulted in false MRD-, while CD319/CD138 gating resulted in MRD+. CD319 and CD269 express stably and continuously in plasma cells of MM patients at different disease stages. CD319 can be used as an alternative of CD38 for immunophenotyping and MRD detection, especially for MRD detection after DARA treatment, while CD269 is suitable for detection before BCMA-CAR-T treatment.

Genomics of Smoldering Multiple Myeloma: Time for Clinical Translation of Findings?

Simple SummaryIn this review we summarized the most relevant biological features concerning smoldering multiple myeloma (SMM). We outlined the genetic architecture of the disease and how it is entering in the SMM risk stratification. In particular, we pointed out how the identification of a high-risk setting, meaning the population with the risk of faster progression to the symptomatic phase, is crucial and, despite huge improvements in recent years, still represents an unmet clinical need. Indeed, the correct identification of these patients will drive to an early therapeutical intervention. Moreover, we also discussed the role of the microenvironment, which is highly relevant in the symptomatic disease but still understudied in the SMM setting. Finally, we debated the state-of-the-art current available therapies and ongoing clinical trials, and envisioned possible strategies to introduce a biological-based stratification approach within the daily clinical practice.Smoldering multiple myeloma (SMM) is an asymptomatic disorder of clonal bone marrow (BM) plasma cells (PCs) in between the premalignant condition known as monoclonal gammopathy of undetermined significance and overt multiple myeloma (MM). It is characterized by a deep biological heterogeneity that is reflected in a markedly variable progression risk among patients. Recently proposed risk stratification models mainly rely on indirect markers of disease burden and are unable to identify cases in whom clonal PCs have already undergone the “malignant switch” but major clonal expansion has not occurred yet. In the last years, the application of next-generation sequencing (NGS) techniques has led to profound advances in the understanding of the molecular bases of SMM progression, and in all likelihood, it will contribute to the needed improvement of SMM prognostication. In this Review, we describe the recent advances in characterizing the genomic landscape of SMM and intrinsic determinants of its progression, highlighting their implications in terms of understanding of tumor evolution and prognostication. We also review the main studies investigating the role of the microenvironment in this early disease stage. Finally, we mention the results of the first randomized clinical trials and discuss the potential clinical translability of the genomic insights.

Safety and Tolerability of Daratumumab in Patients with Relapsed Light Chain (AL) Amyloidosis: Preliminary Results of a Phase II Study

Daratumumab is a human IgG1k monoclonal antibody targeting the CD38 surface antigen on plasma cells, and has proven efficacy in multiple myeloma. AL amyloidosis is a plasma cell disorder in which clonal bone marrow plasma cells produce immunoglobulin light chains that misfold into fibrils that are deposited in organs, leading to organ dysfunction and failure. While the biology of the clonal plasma cell in AL amyloidosis is distinct from that of myeloma, clonal plasma cells in AL amyloidosis do express surface CD38, providing a rationale for the use of daratumumab in AL amyloidosis. Infusion reactions (IR) of 48% were reported in patients who received daratumumab as monotherapy for relapsed multiple myeloma. Therefore, we designed a clinical trial to study tolerability of daratumumab in patients with relapsed AL amyloidosis (clinicaltrials.gov identifier: NCT02841033). The primary objective was to determine the safety and tolerability of infusion of daratumumab, with respect to IR. The secondary objectives were to assess hematologic response, clinical response and time to next treatment.Accrual on this clinical trial started in April 2017. Patients with relapsed AL amyloidosis after ≥1 prior therapy, and involvement of at least one major vital organ , eGFR of >20 mL/min, AST/ALT < 3xULN, NTproBNP <8500 pg/mL, LVEF >30%, FEV1 >50% in patients with known COPD or chronic smokers, and ECOG performance status of <3, received daratumumab at 16 mg/kg IV infusion weekly for weeks 1-8, followed by every 2 weeks for weeks 9-24 and every 4 weeks thereafter until progression or unacceptable toxicities, for up to 24 months. The first infusion of daratumumab was administered in 1000 mL, the second infusion in 500 mL if no grade 1 or greater reactions occurred throughout the first 3 hours of the first infusion, and subsequent doses were administered in 500 mL of saline. All patients received acetaminophen, diphenhydramine, loratidine, famotidine, monteleukast and methyprednisolone (100 mg for the first 2 infusions and 60 mg thereafter) 30-60 minutes prior to infusion. Ondansetron was added after developement of grade 1 nausea/vomiting in the first 2 patients. Diphenhydramine, famotidine and methyprednisolone (40 mg) were also administered 2 hours after the start of infusion during the first 2 infusions even if there was no reaction. Methylprednisolone 20 mg (or its equivalent) and monteleukast were adminsitered 24 and 48 hours after start of infusion for the first 2 infusions and then monteleukast was optional. All patients received prophylaxis with acyclovir.At data cut-off (July 23, 2017) 8 patients have been enrolled. The median age was 63 years (range, 60-83), and median number of prior therapies was 3 (range, 1-6): 6 (75%) had received HDM/SCT, 5 (62.5%) had received immunomodulatory agents, and 7 (87.5%) proteosome inhibitors. Three patients were refractory to prior therapy and the median time from last therapy was 15 months. The median time from diagnosis to enrollment on this trial was 68 months. The median number of organ systems involved was 2 (range, 1-4). Five patients (62.5%) had involvement of >2 organ systems. All 8 (100%) patients had cardiac biomarker stage II or III disease. Median NT-proBNP level was 1650.5 pg/mL (range, 656-3962) and urine protein excretion was 0.41 g/24 h (range, 0-10.1), while median dFLC was 196.8 mg/L (range, 20.4-854). At the time of this report, 7 patients continue on study and a total of 65 infusions have been completed. The median number of infusions received per patient is 9 (range, 2-12). One patient was removed from the protocol after developing progression of plasma cell dyscrasia markers after 2 cycles. No patient experienced a grade 3-4 IR. Only 2 patients experienced grade 1 nausea and vomiting during first infusion, which resolved after an antiemetic, without experiencing IR. There was no interruption or delay of infusion. No hospitalization for IR was required. The median time of first infusion was 6.5 hours and 2nd infusion was 4.2 hours. Hematologic responses were rapid and are shown in Figure 1.Daratumumab infusion is well tolerated in patients with relapsed AL amyloidosis, when administered with appropriate pre and post-medications. Infusion related reactions were minimal and not comparable to reported incidence in myeloma. Preliminary data suggest a rapid hematologic response after 1 dose of daratumumab in patients with AL amyloidosis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Autologous Stem Cell Transplant in the Era of Bortezomib-Based Induction for AL Amyloidosis Results in Improved Hematologic Response Rates: a Single Institution 11 Year Experience

Introduction: The current standard of practice for patients with systemic immunoglobulin light chain amyloidosis (AL) if transplantation-eligible is to proceed directly to high-dose melphalan (HDM) and autologous stem cell transplant (ASCT) without induction therapy. This approach has been associated with higher response rates and improved overall survival compared to chemotherapy alone. However multiple recent studies have shown the merits of bortezomib induction in AL patients who are transplantation-eligible and ineligible, and it is also increasingly used when significant bone marrow clonal plasma cells are present. These therapies have shown to be well-tolerated with favorable hematologic and organ responses, often enabling patients who were originally transplantation-ineligible to become eligible. The purpose of this study is to update our previously performed retrospective analysis of a single-institution population of patients with AL treated with bortezomib induction followed by HDM + ASCT.Methods: All patients with biopsy-confirmed AL who underwent HDM + ASCT at Oregon Health and Science University from 2006 to 2017 were included (n=53). Organ involvement and responses were assessed using the consensus criteria reported by the Roundtable on Clinical Research in Immunoglobulin Light-chain Amyloidosis. Institutional eligibility criteria for performing HDM + ASCT for AL patients included: Eastern Cooperative Oncology Group performance status ≤2, left ventricular ejection fraction >45%, New York Heart Association class <3, cardiac biomarker stage 1 or 2, carbon monoxide diffusing capacity >50%, and serum creatinine ≤3.0 mg/dL unless receiving dialysis. Patients received melphalan 200 mg/m2 or dose-reduced melphalan (100-140 mg/m2) for those at higher risk.Results: Of the 53 total patients, 13 proceeded directly to ASCT after diagnosis, whereas 34 received a bortezomib-containing induction regimen (increased from 12 in our previous study), and 6 received some other form of induction treatment (Table 1). Twenty-three patients received dose-reduced melphalan prior to ASCT. Twenty-five patients had cardiac involvement. Two patients (3.7%) died within 100 days after ASCT (one due to exsanguination from AL-involved splenic rupture, the other due to congestive heart failure). The overall hematologic (ORR) and organ response rates (OR) in the entire cohort after ASCT were 85% and 67%, respectively. ORR was 94% in the group that received bortezomib induction compared to 69% in the group that received no induction (p=0.04); OR in these groups were 77% and 54%, respectively (p=0.17). The mean time to maximum hematologic response was significantly shorter in those who received bortezomib induction compared to no induction (3.4 months vs. 16.8 months, p<0.0001). The overall cardiac response rate was 65.0%, with a rate of 71.4% in those pre-treated with bortezomib versus 43% in those who did not receive induction therapy (p=0.35). With a median follow-up of 2.9 years, 3-year progression-free (PFS) and overall survival (OS) in the entire cohort were 62% and 82%, respectively (Figure 1); in those with cardiac involvement, these rates were 59% and 83%, respectively. 3-year OS (Figure 2) in the bortezomib-induction group was 87% compared to 77% in the group that received no induction (p=0.22), 3-year PFS (Figure 3) in these groups were 61% vs. 69%, respectively (p=0.52)Conclusion: With the update of our institutional analysis, we have continued to observe that bortezomib-based induction is well-tolerated in patients with and without cardiac involvement and is associated with higher ORR and shorter time to hematologic response compared to those who receive no induction. In our analysis, bortezomib-based induction did not significantly impact OR, cardiac response rate, PFS, or OS, but these are likely limited by sample size and short follow-up time. Our results otherwise appear comparable to those seen at other major transplant centers. These findings therefore continue to support the emerging roles for bortezomib-based induction regimens for the purposes of improving response and even increasing eligibility for patients to safely undergo HDM + ASCT. Larger scale analyses in the form of prospective multi-center trials would be beneficial to further study these roles. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Multiple myeloma – 2020 update on diagnosis and management

There has been remarkable progress made in the diagnosis and treatment of multiple myeloma (MM). The median survival of the disease has doubled as a result of several new active drugs. These advances have necessitated a revision of the disease definition and staging of MM. Until recently, MM was defined by the presence of end-organ damage, specifically hypercalcemia, renal failure, anaemia, and bone lesions (CRAB features) that can be attributed to the clonal process. In 2014, the International Myeloma Working Group (IMWG) updated the diagnostic criteria for MM to add three specific biomarkers that can be used to diagnose the disease in patients who did not have CRAB features: clonal bone marrow plasma cells greater than or equal to 60%, serum free light chain (FLC) ratio greater than or equal to 100 provided involved FLC level is 100 mg/l or higher, or more than one focal lesion on MRI. In addition, the definition was revised to allow CT and PET-CT to diagnose MM bone disease. With the introduction of immunomodulatory agents (IMiDs) and proteasome inhibitors (PIs), major improvements have been achieved in the treatment and outcome of MM. Different treatment combinations are now in use and newer the­rapies are being developed. However, nearly all MM patients ultimately relapse, even those who experience a complete response to initial therapy. Management of the relapsed disease remains a critical aspect of MM care and an important area of ongoing research. The aim of this review is to summarise the current methods of diagnosis and treatment of MM.

Daratumumab + bortezomib, thalidomide, and dexamethasone (D-VTd) in transplant-eligible newly diagnosed multiple myeloma (TE NDMM): Baseline SLiM-CRAB based subgroup analysis of CASSIOPEIA.

8538 Background: In the phase 3 CASSIOPEIA study, D-VTd significantly improved outcomes vs VTd in TE NDMM pts at an 18.8-mo median follow-up. To allow earlier diagnosis and treatment of MM, the IMWG added 3 validated biomarkers (≥60% clonal bone marrow plasma cells, serum free light chain ratio ≥100, and &gt; 1 focal bone lesion by MRI; “slim”) to the conventional “CRAB” diagnostic criteria. We present a subgroup analysis of CASSIOPEIA based on baseline slimCRAB criteria. Methods: TE NDMM pts were randomized 1:1 to 4 pre-ASCT induction and 2 post-ASCT consolidation cycles of D-VTd or VTd. The “slim-only” subgroup excludes pts with ≥1 conventional CRAB criterion based on data collected at baseline; the remaining pts were included in the “CRAB” subgroup. Results: Of 1085 randomized pts (543 D-VTd; 542 VTd), 81 were included in the slim-only subgroup (36 D-VTd; 45 VTd) and 1004 were included in the CRAB subgroup. In slim-only vs CRAB pts, 22% vs 54% had an ECOG score of ≥1, 4% vs 16% had ISS Stage III disease, and 11% vs 16% had high-risk cytogenetics. For D-VTd vs VTd pts in the slim-only group, these rates were 22% vs 22%, 8% vs 0%, and 6% vs 16%, respectively. Overall response rates (ORR) and rates of sCR, ≥CR, and MRD negativity were similar between slim-only and CRAB pts; for slim-only pts, rates were significantly higher for D-VTd vs VTd (Table). After an 18.8-mo median follow-up, progression-free survival (PFS) was not significantly different in slim-only vs CRAB pts, or in D-VTd vs VTd slim-only pts (Table). For D-VTd vs VTd CRAB pts, 18-mo PFS rates were 92% vs 84%, and 24-mo PFS rates were 89% vs 76%. Conclusions: Baseline characteristics indicate that slim-only pts were slightly fitter and of lower risk status vs CRAB pts; however, response rates, MRD-negativity rates, and PFS did not differ significantly between these groups. Among slim-only pts, significantly higher response and MRD-negativity rates were achieved with D-VTd vs VTd. Among CRAB pts, PFS rates were higher with D-VTd vs VTd. Clinical trial information: NCT02541383 . [Table: see text]

Multiple myeloma: 2020 update on diagnosis, risk-stratification and management.

Multiple myeloma accounts for approximately 10% of hematologic malignancies. The diagnosis requires ≥10% clonal bone marrow plasma cells or a biopsy proven plasmacytoma plus evidence of one or more multiple myeloma defining events (MDE) namely CRAB (hypercalcemia, renal failure, anemia, or lytic bone lesions) features felt related to the plasma cell disorder, bone marrow clonal plasmacytosis ≥60%, serum involved/uninvolved free light chain (FLC) ratio ≥100 (provided involved FLC is ≥100 mg/L), or >1 focal lesion on magnetic resonance imaging (MRI). The presence of del(17p), t(4;14), t(14;16), t(14;20), gain 1q, or p53 mutation is considered high-risk multiple myeloma. Presence of any two high risk factors is considered double-hit myeloma; three or more high risk factors is triple-hit myeloma. In transplant eligible patients, induction therapy consists of bortezomib, lenalidomide, dexamethasone (VRd) given for approximately 3-4 cycles followed by autologous stem cell transplantation (ASCT). In high-risk patients, daratumumab, bortezomib, lenalidomide, dexamethasone (Dara-VRd) is an alternative to VRd. Selected standard risk patients can get additional cycles of induction, and delay transplant until first relapse. Patients not candidates for transplant are typically treated with VRd for approximately 8-12 cycles followed by lenalidomide; alternatively these patients can be treated with daratumumab, lenalidomide, dexamethasone (DRd). After ASCT, standard risk patients need lenalidomide maintenance, while bortezomib-based maintenance is needed for patients with high-risk myeloma. Most patients require a triplet regimen at relapse, with the choice of regimen varying with each successive relapse.

Circulating Tumor Cells (CTCs) for Comprehensive and Multiregional Non-Invasive Genetic Characterization of Multiple Myeloma (MM)

Background: Genetic characterization is becoming relevant to predict risk of progression in smoldering MM and is fundamental to estimate survival in active MM. Thus, patients undergo multiple bone marrow (BM) aspirates for genetic screening that beyond painful, may not be fully representative due to patchy BM involvement, spatial genomic heterogeneity, or extramedullary disease. Accordingly, cell-free DNA has been investigated and showed high concordance with BM aspirates, but information is typically restricted to a few recurrent mutations since comprehensive genetic characterization (eg. whole-exome sequencing, WES) is applicable to &lt;25% of MM patients (those with ≥10% tumor DNA). By contrast, CTCs are detectable in virtually all smoldering and active MM patients and their numbers are prognostically relevant, but their applicability for non-invasive genetic characterization of MM has been poorly investigated. Aim: To compare the genetic landscape of CTCs vs matched BM clonal plasma cells (PCs) and extramedullary (EM) plasmacytomas, and validate standardized assays for CTCs' detection, isolation and genetic characterization. Methods: We used EuroFlow next-generation flow (NGF) cytometry to detect and isolate peripheral blood (PB) CTCs and matched BM clonal PCs from 38 MM patients (25 at diagnosis and 13 at relapse). In 8 cases, clonal PCs from EM plasmacytomas were also FACSorted. PB T cells were always used as matched germline control. In the training set, we performed custom WES (preceded by triplicates of whole-genome amplification) in matched CTCs, BM and EM clonal PCs from the 8 patients with all three spatially distributed clones. Only those mutations present in 2/3 libraries analyzed per sample were considered positive. In the validation set, we compared mutations, copy number alterations (CNA) and translocations present in CTCs and BM clonal PCs using the Chromium Exome Solution for low DNA-input (n=8), and solely CNA using the Affymetrix CytoScan HD platform (n=22). Read mapping, variant and structural calling were performed with the Multisample Exome (Dreamgenics) and LongRanger (10XGenomics) pipelines. The Chromosome Analysis Suite software (Affymetrix) was used to analyze CNA. Only those mutations with ≥10% variant allele frequency (VAF) and CNA larger than 1Mb were considered. Results: In the training set, 193/226 (85%) and 231/269 (86%) of total mutations present in BM and EM clonal PCs, respectively, were detectable on CTCs. All MM recurrent mutations (eg. BRAF) found in BM or EM clonal PCs were present in CTCs. Of note, there were 39 mutations in EM plasmacytomas that were detectable in CTCs but absent in BM clonal PCs. Furthermore, up to 50 mutations were present in CTCs while undetectable in BM clonal PCs (n=44) or EM plasmacytomas (n=6). After showing that CTCs harbor most mutations present in both medullary and extramedullary disease and even unveil mutations undetectable in single BM aspirates or individual EM plasmacytomas, we sought to evaluate the performance of standardized assays suitable to screen mutations and/or CNA from low cell numbers (ie. CTCs). Using 10XGenomics, 250/266 (94%) of total mutations and 17/17 (100%) of MM recurrent mutations present in BM clonal PCs were detectable on CTCs (eg. KRAS, BRAF, TP53 or FAM46C). The VAF of private mutations ranged between 0.1 and 0.3, suggesting these were subclonal in their respective spatial regions. Using 10XGenomics, 101/119 (85%) CNA and 2/2 (100%) IgH Tx present in BM clonal PCs were detectable in CTCs. Using the Cytoscan HD, there was 100% concordance between CNA in CTCs and BM clonal PCs, both at the chromosomal arm and interstitial levels. All mutations in TP53 were detectable in CTCs. Furthermore, +1q, del(1p), del(17p) or t(4;14) were always detected in CTCs whenever present in BM clonal PCs, and confirmed by FISH. Conversely, such comprehensive genetic characterization unveiled innumerous CNA and translocations not tested by routine FISH panels [eg. MYC amplification or t(6;14)]. Conclusions: Using two different standardized methods, we showed in the largest series in which CTCs were genetically characterized, that these are a reliable surrogate of MM patients' genetic landscape inside and outside the BM. Because NGF is broadly used, quantification, isolation and genetic characterization of CTCs may emerge as an optimal and standardized approach for non-invasive risk-stratification of MM patients. Disclosures Rios: Janssen: 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. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria. Paiva:Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche and Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene, Janssen, Sanofi and Takeda: Consultancy.

Impact of sFLC Ratio on Outcome in Patients with MM: Validating the Utility of sFLC in Response Definition

Background: The treatment of Multiple Myeloma (MM) has evolved significantly in the past decade with the introduction of novel agents and drug combinations, thus enhancing treatment efficacy and allowing more patients to achieve complete response (CR). This has created a need to identify surrogates for depth of treatment response. Serum free light chain (sFLC) ratio normalization has been shown to be prognostic for progression free survival as well as overall survival in patients achieving a complete response to therapy. Consequently, it has been incorporated as a defining feature for stringent CR, along with lack of clonal plasma cells by immunohistochemistry (IHC) or low sensitivity flow cytometry. The routine use of multiparametric flow cytometry with higher sensitivity to detect residual disease than IHC or the older 4-color flow cytometry, has raised the question as to whether sFLC ratio is still a valid indicator of response depth. Moreover, in nearly half of the patients with an abnormal sFLC ratio after treatment, the abnormality is secondary to suppression of one or both serum light chains. Therefore, we designed a retrospective study to address these issues. Patients and Methods: This is a retrospective study using the Multiple Myeloma Database at Mayo Clinic, Rochester. We included patients who, after any line of therapy, had negative serum and urine immunofixation and absence of clonal bone marrow plasma cells by flow cytometry (PC-PRO), which has a sensitivity of &gt;10-4. Simultaneous sFLC data was also extracted. Patients were grouped into three categories based on their sFLC ratios: 1) normal ratio (normal), 2) abnormal ratio due to suppression of the uninvolved light chain (LC), involved LC, or both (Abn-suppressed) and 3) abnormal ratio due to elevation of the involved LC (Abn-inv elevated). The primary endpoint was the median time to next treatment (TTNT), defined as the time from sample collection to the time of initiation of the subsequent therapy or time of last follow up if a subsequent line of treatment was not initiated. Results: The cohort consisted of 510 patients. 285 (56%) were males and 225 (44%) females. Median age was 61 years (IQR: 55-67). Median Follow-up was 41 months. The last treatments administered prior to data collection included stem cell transplant (SCT) (with or without maintenance) in 290 (57%) patients, and non-SCT regimens in the others. The sFLC ratio was normal in 337 (66%) and abnormal in 173 (34%) patients. Among the patients with abnormal sFLC ratios, 81 had elevated involved LC, 25 had suppression of the involved LC, 45 had suppression of the uninvolved LC and 22 had suppression of both LCs. We first examined the TTNT for the three groups and found that the TTNT was identical for those with a normal ratio and those with an abnormal ratio due to suppression of one or both light chains (Figure 1). So, we combined these two groups (Normal-Abn suppressed) and compared their outcomes to the patients with abnormal sFLC ratio due to elevated involved LC. The Abn-inv elevated group had a shorter TTNT as shown in Figure 2 (log-rank 0.06, Wilcoxon &lt;0.01). The Abn-inv elevated group also had decreased overall survival compared to the other group (log-rank: 0.05, Wilcoxon: 0.01) (Figure 3). Conclusion: This study provides 2 important observations. First, patients with an abnormal ratio due to suppression of one or both LCs have outcomes similar to those with a normal ratio, suggesting a need to clarify the current definition of stringent CR. Second, the study suggests an important prognostic value for an abnormal sFLC ratio due to elevated involved LC, suggesting this as an important surrogate for depth of response. Disclosures Kapoor: Janssen: Research Funding; Takeda: Honoraria, Research Funding; Cellectar: Consultancy; Celgene: Honoraria; Sanofi: Consultancy, Research Funding; Amgen: Research Funding; Glaxo Smith Kline: Research Funding. Dispenzieri:Akcea: Consultancy; Intellia: Consultancy; Janssen: Consultancy; Pfizer: Research Funding; Takeda: Research Funding; Celgene: Research Funding; Alnylam: Research Funding. Gertz:Ionis: Honoraria; Alnylam: Honoraria; Prothena: Honoraria; Celgene: Honoraria; Janssen: Honoraria; Spectrum: Honoraria, Research Funding. Lacy:Celgene: Research Funding. Dingli:Karyopharm: Research Funding; Rigel: Consultancy; Millenium: Consultancy; Janssen: Consultancy; alexion: Consultancy. Leung:Takeda: Research Funding; Aduro: Membership on an entity's Board of Directors or advisory committees; Prothena: Membership on an entity's Board of Directors or advisory committees; Omeros: Research Funding. Kumar:Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Takeda: Research Funding.

BCMA expression in AL Amyloidosis

AL Amyloidosis is a multisystem disorder of clonal plasma cells (PCs) that produce an abnormal light chain which misfolds and deposits in organs causing cellular stress, organ dysfunction and eventually death. Available therapies target PCs to stop the production of amyloidogenic light chains. The burden of clonal PCs in bone marrow (BM) is typically less in AL than in Multiple Myeloma (MM) which is potentially advantageous for immunotherapeutic strategies. B-Cell Maturation Antigen (BCMA) is a transmembrane protein that is involved in the regulation of B cell proliferation and survival as well as maturation/differentiation into PCs.

Prognostic Nomogram for the Overall Survival of Patients with Newly Diagnosed Multiple Myeloma.

To establish a nomogram for predicting the overall survival (OS) of patients with newly diagnosed multiple myeloma (MM), 304 patients with newly diagnosed MM were recruited between June 1, 2010, and June 30, 2015, from the Beijing Chaoyang Hospital, Capital Medical University, and randomly divided into training (n=214) and validation (n=90) cohorts. The Kaplan-Meier method and the Cox proportional hazards regression model were used to evaluate the prognostic effects of multiple clinical and laboratory parameters on survival. Significant prognostic factors were combined to build a nomogram. The discriminative ability and predictive accuracy of the nomogram were evaluated using the index of concordance (C-index) and calibration curves and compared with the five staging systems currently used for MM. Multivariate analysis of the training cohort revealed that the age at diagnosis, clonal bone marrow plasma cells, serum lactate dehydrogenase, serum β2-microglobulin, and del (17p) were independent risk factors for OS and were used to establish the nomogram. The C-index value of the nomogram for predicting OS was 0.749, which was significantly higher than the C-indices of the five most common staging systems currently used for MM. In the validation cohort, the C-index for nomogram-based predictions was 0.711 for OS, and the nomogram discrimination was better than the above mentioned five staging systems (P<0.001). All calibration curves revealed good consistency between predicted and actual survivals. The proposed nomogram is more accurate in predicting the prognoses of patients with newly diagnosed MM.

Transcriptomic Profiling of Circulating Tumor Cells (CTCs) in Multiple Myeloma (MM): A New Model to Understand Disease Dissemination

Background. The number of CTC predicts risk of transformation in smoldering MM and survival in active MM. Growing evidence suggests that as the tumor progresses and the microenvironment becomes hypoxic, clonal plasma cells (PC) constantly invade new regions of the bone marrow (BM) through induced systemic recirculation. Of note, the frequency of CTCs is typically low and thus, it is conceivable that the dissemination of MM depends on few tumor cells with unique features that induce them to egress the BM and spread the disease through peripheral blood (PB). This hypothesis has not been yet demonstrated because the transcriptional profile of CTCs in MM has not been investigated.Aim. To identify gene regulatory networks related to MM dissemination by comparing the transcriptional profile of CTCs with patient-matched BM clonal PCs.Methods. We used FACS to isolate CTCs and BM clonal PCs of paired PB and BM samples from 34 patients: 24 newly diagnosed MM, 9 relapsed MM and 1 MGUS. Transcriptomes were analyzed using Affymetrix arrays (n =31) and the BD WTA Precise assay was used for single-cell RNA sequencing (scRNAseq, n =3). Data was analyzed using Gene Set Enrichment Analysis (GSEA) and Limma for bulk and Seurat for scRNAseq data. The prognostic value of deregulated genes (FDR <0.1 & logFC >0.5) was investigated using a Cox-regression model in the CoMMpass dataset (n =553, IA11 release). The role of specific deregulated genes was evaluated by shRNA knockdown and blocking using a monoclonal antibody (mAb).Results. Transcriptomic profiling of patient matched CTCs and BM clonal PCs revealed a high correlation in gene expression (r =0.93; p =10-16). Only 45 genes emerged as significantly deregulated in CTCs, and GSEA unveiled biological functions related to inflammatory and interferon response (e.g. CCL5), signaling by IL-6/JAK/STAT3, IL-2/STAT5 and TNF via NFKB (CD44), the epithelial mesenchymal transition (EMP3), mitotic spindle and G2M checkpoints (TOP2A), or E2F targets (BIRC5). A high correlation in gene expression was also observed by scRNAseq (r =0.9; p =10-16), with only 31 genes (e.g. MALAT1, B2M, RHOH, ENAM or DUSP5) differentially expressed (adj.p <0.01). Under the hypothesis that genes deregulated in CTCs could be markers of dissemination and therefore of a more aggressive disease, we evaluated their prognostic significance in CoMMpass, assuming that BM clonal PCs with higher expression of these genes would be enriched in tumor cells with CTC-features. Accordingly, the expression levels of 12/33 upregulated genes in CTCs from bulk RNAseq data were associated with significantly different progression free survival (PFS). In the multivariate analysis including the above mentioned 33 genes and the R-ISS, only FLNA retained independent prognostic value; patients showing higher FLNA expression (n =185/553) had a significantly inferior PFS vs the remaining cases (medians of 22 and 47 months, respectively; p <.001). Of note, patients with higher FLNA expression also displayed significantly higher mutational burden (p =.003). After confirming that genes deregulated in CTCs were clinically meaningful, we selected two genes upregulated in CTCs (FLNA and CD44) to investigate their functional role. Interestingly, knockdown of both genes in the U266 MM cell line significantly altered actin polymerization (p ≤.003) leading to reduce migration (2-fold, p ≤0.01). Furthermore, we evaluated the effect of an anti-CD44 mAb in CB17/Icr-PrKdcscid/Crl immunodeficient mice treated with clodronate to avoid potential phagocytosis. After inoculation of MM1S-GFP intravenously and MM1S-tomato subcutaneously in Matrigel, mice were left untreated (n =11) or treated with a weekly intravenous injection of anti-CD44 (n =11) for one month and monitored weekly by bioluminescence. Overall, there was a significant reduction in the plasmacytoma volume (5-fold, p <.001) and cell engraftment of circulating MM1S cells (42-fold, p =.003) in treated mice.Conclusions. This is the first study analyzing the transcriptional profile of CTCs in MM. Our results reveal that gene expression of CTCs is almost identical to that of patient-matched BM clonal PCs, except for a few genes that are involved in interferon and inflammatory response, hypoxia, cell cycle and migration. Importantly, some of these genes are related to more aggressive disease and therefore, may represent novel therapeutic targets to overcome disease dissemination. [Display omitted] DisclosuresRios:Amgen, Celgene, Janssen, and Takeda: Consultancy. Martinez-Lopez:Pfizer: Research Funding; Vivia: Honoraria; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; BMS: Research Funding; Novartis: Research Funding. Hajek:Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding. San-Miguel:Sanofi: Honoraria; Celgene: Honoraria; Janssen: Honoraria; Amgen: Honoraria; Novartis: Honoraria; BMS: Honoraria; Roche: Honoraria.

59-Year-Old Man With Fatigue, Weight Loss, and Hepatomegaly

59-Year-Old Man With Fatigue, Weight Loss, and Hepatomegaly