Clonal Plasma Cell Malignancy Research Articles

Global Access to Multiple Myeloma Medications (GLAMM-2 Study): Access and Barriers to Chemoimmunotherapies and Transplant

Introduction Multiple myeloma (MM) is a clonal plasma cell malignancy and is the second most common type of hematologic cancer worldwide. Treatment options for MM have evolved in the last two decades with the introduction of novel therapeutic agents such as immunomodulatory drugs (IMiDs), proteasome inhibitors (PI), anti-CD-38 monoclonal antibodies (anti-CD38 MoAb), and recently BCMA-directed therapies (BDT). The variable access globally, especially for novel agents, has been a concern. We aim to study the global availability of 14 chemoimmunotherapy options in addition to autologous stem cell transplant (ASCT) which is approved in the US (excluding BDT due to limited availability), to identify regions with restricted access and analyze the main barriers to access. Methods A global cohort of oncologists outside the USA who treat MM participated in an online survey to evaluate the patterns of accessibility of MM therapies worldwide. The survey, conducted in collaboration with the US Myeloma Innovations Research Collaborative (USMIRC), was sent electronically to 176 oncologists worldwide (outside the US) from June 18th, 2023, to July 17th, 2023. It included questions on demographics, access, and perceived barriers. The respondents chose a response for each therapy as being “easily accessible”, “moderately accessible,” “not readily accessible” or “no access”. Countries were then classified as having “adequate access” if 80-100% of respondents affirmed easy/moderately easy access, while <30% were considered “limited access”, 60-79% were “high-intermediate access” and 30-59% were “low-intermediate access”. We classified countries into low- and high-income (LIC and HIC) based on the United Nations (UN) World Economic Classifications based on per capita gross national income (GNI). Means were calculated for regions of interest. An interim analysis is reported. Results Demographics: Ninety-Five (54%) oncologists from 33 countries completed the survey. Most were from Asia (56%). Others were Europe (19%), South America (12%), Africa (7%), Oceania (3%), and North America (Canada and Mexico, excluding the US) (3%). Only 17% specialized in plasma cell disorders; 50.5% were affiliated with university academic centers; 18% with private or community practices; 12% with community academic centers; and 17% were hybrid. Public-funded centers represented 61.1%, while 76% were transplant centers. Global Patterns: Most countries had adequate access to chemotherapies, IMiDs, PIs, Anti-CD38, MoAbs, and ASCT (Figure 1). Notably, ixazomib, isatuximab, and selinexor had low intermediate access, while elotuzumab had limited access. Access by Income: Most MM therapies were available in LIC and HIC. Of the therapies listed, 6 were adequately available in LIC and HIC: cyclophosphamide, lenalidomide, pomalidomide, daratumumab, bortezomib, and ASCT (Table 1). Elotuzumab and selinexor were limited in both LIC and HIC. Ixazomib and isatuximab were also limited in LIC but were low-intermediate in HIC. Access by Continent: Out of the 14 therapies, Europe had adequate or high-intermediate access to most modalities (12), followed by Oceania and Asia (10), and South America (9). North America and Africa had the lowest number of adequate or high intermediate access therapies (7) (Table 1). Barriers: The most common reason cited for limited access to MM therapies globally was the financial burden for the healthcare system, followed by limitations in local agency approval, inadequate chemotherapy suite resources, and inadequate staffing. Conclusion While most therapies were available, novel therapies such as isatuximab, ixazomib, selinexor, and elotuzumab were less readily accessible. Moreover, North America and Africa had the least access to therapies. This is likely because LIC and HIC have prioritized access to treatments with the best evidence of cost-effectiveness, while those with lower cost-effectiveness are generally limited. The financial burden on healthcare is a major limiting factor. USMIRC plans to investigate potential solutions and pursue global collaborative efforts to reduce disparities in therapies.

Utilizing a Machine Learning Approach to Define Racial Difference in Multi-Systemic Impact of Monoclonal Protein in Patient with Multiple Myeloma

Multiple myeloma (MM) is the second common hematologic malignancy characterized by the clonal proliferation of plasma cells. While the clinical outcomes of MM have been improved significantly, there remains an intriguing observation regarding the potential influence of race on disease biology and treatment response. In MM, the malignant plasma cell clone not only disrupts the local bone marrow microenvironment but also exerts a systemic impact on various physiological processes result in significant derangements in immune function, hematopoiesis, renal function, bone metabolism, and electrolyte homeostasis. Understanding the extent of this systemic impact and its potential variation between racial groups is crucial for optimizing patient care and treatment strategies. Here, we hypothesized that the systemic impact of the plasma cell clone does not differ significantly between African American and Caucasian patients. We employed a machine learning approach to capture the full spectrum of systemic effects comprehensively. Our novel model was trained to predict the magnitude of the plasma cell clone, as represented by serum monoclonal protein levels. By systematically comparing model performance with and without the inclusion of race as a variable, we sought to evaluate the potential influence of race on the systemic impact of the plasma cell clone. Method: A total of 171 patients with plasma cell dyscrasias including 1,472 observations were analyzed. where the upper limit of the observed M-spike was 3.5 gr/dL. Forty three clinical and lab variables as predictors of M-spike were fed into the machine learning model. Two lagged variables as the last two preceding M-spike values by the same subject were included. The random forest model was used, where regression forests are an ensemble of different regression trees and are used for nonlinear multiple regression. The goal of using a large number of trees was to train enough that each feature had a chance to appear in several models. The data was randomly split into a training set (80%) and a test set (20%), and a regression tree was built with the training set and then validated using the test set. Bootstrapping was used to generate a collection of data sets (n=500). Importance was measured by leaving a covariate out of models and comparing performance with its inclusion. Result: The training set used to develop the ML algorithm comprised 749 patient-based observations, which represented ~50% of the patient-based observations. The number of observations at each M-Spike value and the distribution of M-Spike values measured by SPEP are shown (Fig. 1). The residual distribution of the RF model indicated that nearly all M-spike values determined using the 43 variables distributed equally on either side of zero. The addition of race as variables did not significantly change the residual plots. The weighted value of each of the 43 independent variables was determined by individually removing a variable from the ML algorithm and measuring its effect on the mean squared error (MSE). As shown, removal of the first lagged M-spike, serum total protein, second-lagged M-spike, serum IgG, serum IgM, and serum IgA, had the greatest effects on the ML algorithm. The effect of race was then added to the ML algorithm. However, patient race had a low weighted value compared to the other variables included in the ML algorithm (Fig-2). M-spike values determined using the ML algorithm correlated highly with M-spike values determined using the laboratory measured SPEP values as indicated by the proximity of the Pearson and Spearman correlation coefficients to +1 . Using the 43 independent variables, the Pearson coefficient was 0.96 and the Spearman coefficient was 0.91, when M-spike values determined using the ML algorithm were compared to laboratory determined M-spike values. When race was added as a variable in the ML algorithm, the Pearson coefficient was 0.95 and the Spearman coefficient was 0.94 which is not significantly different. Conclusion: Here, we present the results of our machine learning analysis, which further supports our hypothesis and sheds light on the potential racial difference in the overall systemic impact of MM. Understanding the factors underlying racial disparities in MM will pave the way for more personalized and equitable treatment strategies, ultimately improving patient outcomes and overall disease management.

Cellular immunity in the era of modern multiple myeloma therapy.

Multiple myeloma (MM) is a relapsing clonal plasma cell malignancy and incurable thus far. With the increasing understanding of myeloma, highlighting the critical importance of the immune system in the pathogenesis of MM is essential. The immune changes in MM patients after treatment are associated with prognosis. In this review, we summarize currently available MM therapies and discuss how they affect cellular immunity. We find that the modern anti-MM treatments enhance antitumour immune responses. A deeper understanding of the therapeutic activity of individual drugs offers more effective treatment approaches that enhance the beneficial immunomodulatory effects. Furthermore, we show that the immune changes after treatment in MM patients can provide useful prognostic marker. Analysing cellular immune responses offers new perspectives for evaluating clinical data and making comprehensive predictions for applying novel therapies in MM patients.

Identifying associations between race and gender in the incidence and mortality of patients with multiple myeloma.

e20052 Background: Multiple Myeloma (MM) is a plasma cell malignancy due to proliferation of malignant clonal plasma cells. Prior studies have shown that overall incidence of MM was 8.47 per 100,000 individuals. It has also been seen that the incidence of MM has trended upwards in non-Hispanic white (NHW) males and non-Hispanic blacks (NHB) across all age groups. This retrospective, database-driven population study sought to further assess racial and gender-based differences in the trends of the incidence of MM and mortality from MM. Methods: Data obtained from the National Cancer Institute Surveillance, Epidemiology, and End Results (SEER) registry 17 were available to assess trends of multiple myeloma incidence and mortality from 2000 to 2019. Trends were described as average annual percentage change (APC) rates. Results: A total of 102,467 MM patients were identified in this data set; 56,508 (55.1%) were males and 45,949 (44.8%) were female. The distribution of MM by race was as follows (in order from highest to lowest): Non-Hispanic White [NHW] patients was 63.2% (N = 64,776), Non-Hispanic Black [NHB] patients was 18.6% (n = 19,099), Hispanic patients was 11.6 % (11,841), Non-Hispanic Asian [NHA] patients was 5.6% (5,769), and Non-Hispanic American Indian [NHAI]/Alaska Native patients was 0.5% (523). Incidence of MM (cases per 100,000 persons) was highest in males (7.5; 95% CI: 7.5, 7.6) and blacks (13; 95% CI: 12.8, 13.2). Overall trend in the incidence of MM has increased significantly for all gender and ethnicity groups over the period from 2000-2019, except for NHA patients. The incidence-based mortality of MM was highest in male (5.2; 95% CI: 5.2, 5.3) and black (8.7; 95% CI: 8.5, 8.8) patients. The overall incidence-based mortality increased significantly in males APC 4.1% (95% CI: 2.2,5.9; p < 0.001), females APC 3.6% (95% CI, 1.8,5.5; p < 0.001) and all ethnicities - NHW patients APC 4.1%, (95% CI, 2.2,6.0; p < 0.001), NHB (APC 4.2%, 95% CI, 2.1,6.3; P < 0.001), NHAI (APC 4.7%, 95% CI, -2.2,7.2; p < 0.001), NHA (APC 3.0%, 95% CI: 0.8,5.2; P = 0.009) and Hispanic (APC 2.9, 95% CI:1.1,4.7; P = 0.003). Conclusions: Population-based trends in MM incidence and mortality, as reported in this study, were increased commensurate with prior reports. Of note, mortality was noted to be highest amongst the NHB and male populations, indicating a need for further research focused efforts in this potentially high-risk population.

Unraveling the Heterogeneity of Multiple Myeloma Cells By Single-Cell RNA Sequencing Analysis

Multiple myeloma (MM) is a malignancy of clonal plasma cells with identical variable-diversity-joining (VDJ) region recombination of immunoglobulin loci (called repertoire) and extensive genome or transcriptome heterogeneity. Even though many new treatment modalities have been developed and the prognosis of patients has improved significantly, most cases of MM remain incurable. Recent evidence suggests that non-genetic cell plasticity and changes in cell state underlie the therapy resistance and tumor relapse, but the mechanisms remain largely unknown. Here, we have attempted to understand comprehensive architecture of the entire tumor cell populations using repertoire clonality as fingerprint. First, we subdivided bone marrow samples of primary MM at different stages (n=8) into 11 fractions based on the known surface antigens of MM and performed bulk RNA sequencing (bulk RNA-seq). Analysis of the repertoire revealed that tumor cells were also present in some minor fractions other than the main fraction (Lin-/CD19-/CD38++/CD138+) as the stage progressed. Importantly, cells with clonal repertoire were detected in CD138 negative fraction (Lin-/CD19-/CD38++/CD138-) in most samples (Monoclonal gammopathy of undetermined significance (MGUS) / Smoldering multiple myeloma (SMM) = 4.15±2.28%, Primary MM = 6.78±3.59%, Relapse MM = 7.8±5.82% [mean±95%Cl], n=24). The CD138 positive and negative cells were also morphologically different, suggesting that they were composed by cells of distinct characteristics. Indeed, comparison of the transcriptome data revealed 296 differentially expressed genes (DEGs) between these fractions (250 up-regulated and 46 down-regulated in CD138 negative fractions). Of interest, several pathways related to H3K4 methylation were positively enriched in CD138 negative fraction (p <0.001), which included KMT2A, KMT2B, KMT2C, KMT2D, and ASH1L, suggesting that differential methylation of histone H4 accounts for the heterogeneity of MM. In order to achieve a high-resolution and comprehensive evaluation of the entire tumor cell populations, we performed single-cell RNA sequencing (scRNA-seq) and single-cell VDJ targeted sequencing (scVDJ-seq) concurrently on Lin-/CD38++ sorted cells of 8 bone marrow samples from 7 patients including pre- and post-treatment status samples. This method allowed us to define normal cells and MM cells at the single-cell level by clonal repertoire sequence independent of transcriptome characteristics. We divided 18,031 MM cells into 23 clusters after integration. We first defined CD138 positive and negative MM gene signature using bulk RNA-seq data and then evaluated the scRNA-seq data. Notably, the CD138 axis appeared to clearly subdivide main MM populations into those with CD138 positive and negative MM gene signature. We also found that a part of clusters, which showed CD138 negative MM gene signature, tended to remain at high proportions after treatment. These populations were characterized by low expression levels of MHC class-I components and TNFRSF17/BCMA as well as high expression of MCL1, MALAT1 and NEAT1, which have been previously implicated in treatment resistance, and were present in all samples. H3K4 methylation related genes were also up-regulated in these populations. Furthermore, we found several clusters with unique and interesting characteristics independent of the CD138 axis. Trajectory and velocity analysis suggested that all cell populations transit to each other. Taken together, our results establish the heterogeneity of MM cells by the CD138 axis, which may be characterized by epigenetic plasticity. They also highlight CD138 negative population as a potential cause of treatment resistance and relapse. By using information of repertoire, we have achieved a comprehensive and accurate single-cell analysis of MM cell diversity that was not limited by cell surface antigens or transcriptome characteristics. We are currently analyzing near minimal residual disease (MRD) level samples using the same scRNA-seq methods and the functional and epigenetic properties of CD138 positive and negative MM cells.

Novel 17-Plex Flow Cytometry Assay for Comprehensive Immunophenotyping and Characterization of Plasma Cells (PC) in Relapsed/Refractory (RR) Multiple Myeloma (MM) Patients

Background: Multiple Myeloma (MM) is a hematological malignancy also known as plasma cell myeloma or myeloma, a type of bone marrow cancer characterized by the proliferation of clonal malignant plasma cells (PCs). Characterization of multiple myeloma plasma cells (MMPCs) remains challenging due to their plasticity, especially in response to emerging targeted therapies such as anti-BCMA and anti-CD38. The need for novel options for comprehensive phenotyping remains high due to the evolving target expression profiles and dynamics across MM disease evolution and treatment lines. Recent progress in flow cytometry offers the opportunity to analyze more than 30 surface markers in one panel. This technology advance will help to address the current challenges, answer clinically relevant questions and generate data to enable translational research and identification of new drug targets or combination partners. Methods: We present a novel 17-plex flow cytometry assay for immunophenotyping of PCs in RRMM patients enrolled in a global Phase I multi-center clinical study (NCT04557150). The panel relies on 5 markers for the identification of malignant plasma cells: CD38, CD138, CD45, CD319 and CD229. The inclusion of less commonly used markers (CD229, CD319) allows to overcome some of the challenges in MMPCs identification, such as CD138 instability and CD38 downregulation due to prior therapies. In addition, the assay includes markers commonly used to differentiate normal from malignant PCs (CD19, CD56, CD27, CD81, CD117, CD28). The panel also includes 2 immunotherapy target ligands (PD-L1 and 4-1BBL) and offers the possibility to measure another 3 clinically relevant targets on MMPCs, including BCMA. With this configuration, the assay allows relevant target antigen density quantification via the use of molecules of equivalent soluble fluorochrome (MESF) beads in 3 channels. Results: We present examples of how to apply this strategy for a reliable identification of MMPCs in RRMM patient samples after up to 14 previous lines of therapies. The assay design permits a deep phenotypical characterization of MMPCs and pharmacodynamic changes in markers targeted by immunotherapy drugs, like anti-CD38 or anti-BCMA. As an example, we characterize BCMA and CD38 expression on the surface of MMPCs at the time of enrolment into a Phase I study. Finally, with the goal of discovering new biomarkers that could be overlooked in the traditional manual gating hierarchy applied in flow cytometry, we have visualized the results using dimensionality reduction analysis (Uniform Manifold Approximation and Projection, UMAP). Using this approach, we have observed high inter-patient heterogeneity of MMPC phenotypes. Furthermore, we could compare intra-patient differences in MMPC phenotype in two compartments, whole blood and bone marrow aspirate. Updated data will be presented. Conclusion: The multi parameter flow cytometry assay described here represents a novel tool to reliably identify MMPCs in RRMM patients independently of commonly used unstable surface markers. Furthermore, it represents an important tool in the era of surface antigen targeted therapies (e.g., anti-BCMA) helping to understand drug resistance due to target loss. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genomic Landscape of t(11;14) in Multiple Myeloma

Background Multiple myeloma (MM) is a clonal plasma cell malignancy characterized by multistep genetic alterations, including translocations, copy number aberrations (CNA), and mutations. Translocations involving the immunoglobulin heavy chain (IGH) locus contribute to plasma cell immortalization and consequent commitment to the MM disease pathway, however other genetic events largely underlie the complex genomic landscape of MM with molecular and clinical heterogeneity. The t(11;14) translocation is the most common IGH translocation in MM and has been identified as a predictive biomarker of response to venetoclax in relapsed/refractory MM. The objective of this study was to confirm that t(11;14) is stable across the course of disease and across lines of therapy, and to assess the unique genomic landscape of t(11;14) MM. Methods This was a retrospective, single-center, non-interventional study. To confirm t(11;14) stability, longitudinal assessment of 272 patients for t(11;14) status by plasma cell-enriched FISH using the Vysis IGH/CCND1 XT DF FISH probe was performed on bone marrow aspirates (BMAs) collected at diagnosis and relapse. To assess the genomic landscape, 139 patient BMAs were collected from an approved biobank with informed consent and were analyzed by a targeted NGS panel. NGS was performed as described in Bolli et al. (Bolli et al. 2016) using an Agilent capture panel for sequencing on the Illumina NextSeq platform. Raw sequencing data was processed by an in-house bioinformatics pipeline to detect mutations, copy number aberrations and structural variants (SVs). Protein coding mutations with variant allele frequency >10% and minimum sequencing depth >=20X were selected. CNAs were defined as log ratio above 0.3 (copy number gain) or below -0.4 (copy number loss). SVs with 5 or more supporting reads were examined to detect translocations within the IGH locus. Results Among the 272 patients assessed by longitudinal t(11;14) FISH, 118 were t(11;14)+ and 154 were t(11;14)-. All t(11;14)+ patients remained positive from their initial assessment to first and subsequent longitudinal assessments (Figure 1). The t(11;14) FISH fusion (F) patterns were also consistent across longitudinal assessments with 90% of patients possessing and maintaining either a 1F (18%), 2F (69%), or ≥3F (3%) translocation pattern. No t(11;14)- patients acquired the translocation at relapse. Among the 139 samples analyzed for t(11;14), 130 samples had results from both FISH and NGS with a 100% concordance rate; 66 were t(11;14)+ and 64 were t(11;14)-. The mutational landscape in highly recurrent genes was heterogeneous (Figure 2). MAPK pathway mutations were the most common (47/130 pts, 36.2%), with recurrent mutations in BRAF gene more prevalent in t(11;14)- vs t(11;14)+ pts (18.8% vs 6.1%, p<0.05). Conversely, mutations in DIS3 gene were more common in t(11;14)+ vs t(11;14)- pts (21.2% vs 4.7%, p<0.05). DIS3 and BRAF mutations have been associated with poor prognosis in MM. High CNAs are associated with increased genomic instability, resistance to therapy, and decreased survival in MM. Increased CNAs were observed in t(11;14)- vs t(11;14)+ MM (p<0.001). Conclusion This study confirms in a large, longitudinal cohort of patients with MM that t(11;14) is stable from diagnosis to relapse. These analyses also begin to delineate the differentiated genetic architecture of t(11;14) MM, including fewer CNAs associated with increased genomic stability, but increased rates of DIS3 mutations that confer poor prognosis in MM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Tool to Longitudinally Assess the Secretory Capacity of Myeloma Cells throughout the Course of Disease

Multiple myeloma (MM) remains an incurable clonal plasma cell malignancy characterized by the production of monoclonal protein (M-protein). However, MM presents with variable M-protein secretory status. Plasma cells are professional antibody factories that secrete vast amounts of immunoglobulins (Igs) and myeloma cells are exquisitely sensitive to disruption of protein homeostasis. Proteasome inhibitors (PI) target the unfolded protein response by inhibiting degradation of ubiquitinated proteins (and paraproteins), leading to protein accumulation within the endoplasmic reticulum that culminates in cell death. International Myeloma Working Group (IMWG) criteria to assess tumor volume rely on the secretory function of MM cells determined by the concentration of the circulating monoclonal protein to infer tumor burden. Hence, changes in M-protein levels may be indicative of therapeutic response or disease progression. PIs can selectively target the most highly secretory MM, while residual cells, the source of relapse, are less secretory. Circulating M-protein levels are a product of the number of MM cells and the amount of M-protein secreted by each individual cell. IMWG criteria assumes that the secretory power of an individual myeloma cell remains constant throughout the course of treatment. However, this assumption has not been tested, due to the inability to accurately measure MM cell numbers. Since Next Generation Sequencing (NGS) allows for direct measurement of tumor cell number, we developed a method to define a "Secretory Power Index” (SPI) as a longitudinal tool to assess tumor burden. Methods: NGS on patient BM samples was checked while disease level was below VGPR throughout the disease course as standard-of-care. Patients with IgA and light chain MM were excluded due to different half-life of circulating M-protein. The ratio between IgG monoclonal proteins in mg/L to number of tumor cells measured by NGS was defined as SPI. The SPEP value 4 weeks before and after NGS was utilized to calculate SPI. If NGS could not count any myeloma cells (negative test) but there was circulating M-protein, the SPI was replaced with highest value was detected among the patient population. If NGS test was positive but there was no circulating protein the SPI was counted as zero. Mann-Kendall's non-parametric statistical test was used to assess the significance of SPI trend over the time. The comparison was performed intra-patient and not inter-patient. Results: 47 patients with >2 MRD assessment were included and 5 patients were excluded due to SPEP unavailability. Median age was 70 years (range 53-78), 25 patients were male (60%), 15 of patients were African-American (36%), and 27 patients were Caucasian (64%). 30 patients were IgG Kappa (71%) and the rest were IgG lambda (Table 1). Median total nucleated cells evaluated was 1.3x106 (range 0.5-3.2x106). Median number of dominant sequence identified was 2 sequences. All patients had first SPI after first line of therapy, 26 (62%) second SPIs were measured after second line of therapy and the rest in later lines. 8 (19%) patients had extramedullary disease with attachment to the bony compartment and none had known visceral disease at diagnosis. Median first SPI was 75 (range 0-1040). Mann-Kendall trend tests showed a statistically significant decrease in SPI over time (S = 2460.0, p < 0.001) which was significant after entering factors, e.g., age, race, light chain type, triple vs. quadruple induction. Conclusions and Limitations: Here, we developed a method to assess secretory power of myeloma cells. The results suggest that the SPI decreases over the time as patients go through different lines of therapy. The spatial heterogeneity of disease in BM may interfere with NGS sampling, however high predictability of long remission based on NGS negative BM highlights the high validity of this method. We will validate this tool in a larger prospective cohorts to demonstrates that IMWG criteria could under-estimate the response measured by global secretory function. This phenomenon is observed in light chain escape when MM progression presents as slight or no increase in monoclonal MM antibody with rapid rise in light chains. Also, non-secretory escape is not uncommon at very late stages of MM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Artificial Intelligence to Assess the Systemic Impact of Malignant Plasma Cell Clone in Patients with Plasma Cell Dyscrasia

Multiple Myeloma (MM) presents as a multi-system disease with significant derangements in the immune system, hematopoiesis, renal function, bone metabolism and electrolyte homeostasis. End-organ damage is the key factor that differentiates plasma cell (PC) dyscrasia requiring therapy, i.e., active MM, from disease precursor stages (Monoclonal Gammopathy of Undetermined Significance, MGUS and Smoldering MM, SMM) that do not require therapy. The prevailing assumption is that MGUS and SMM patients do not harbor significant or measureable systemic impact of clonal PCs. Therefore, a watch-and-wait approach is employed in MGUS and SMM patients until end-organ damage is evident. Here, we hypothesized that the systemic impact of clonal PCs is a continuum throughout disease progression, rather than a binary process, and that the impact on organ systems correlates directly with the volume of the malignant clone as measured by monoclonal protein levels. Therefore, to capture the systemic impact of PC clones, we utilized a Machine Learning (ML) approach that incorporates all available clinical and lab variables. Methods: A total of 171 patients with PC dyscrasias including 1,472 observations were analyzed. Forty-three clinical and lab variables were identified as predictors of M-spike protein levels and then fed into the ML model. Two lagged variables as the last two preceding M-spike values by the same subject were included. When needed, imputation for missing values was applied through interpolation from subject-level linear trend analysis. The random forest model was used, where regression forests are an ensemble of different regression trees and are used for nonlinear multiple regression. The default number of trees was set at n = 500, and the number of variables considered at each split after random selection was 13. The goal of using a large number of trees was to train enough that each feature had a chance to appear in several models. The data was randomly split into a training set, N=136 (80%) and a test set, N=35 (20%), and a regression tree was built with the training set and then validated using the test set. Bootstrapping was used to generate a collection of data sets (n=500), leading to a random forest of regression trees. Results and estimates were combined across trees. Importance was measured by leaving a covariate out of models, and comparing performance with its inclusion. All analyses were performed using R v3.6.2 and its libraries. Results: Median age of patients included in the study cohort was 73 years (range: 42-96), and 44% were male. Median M-spike value was (0.7 gr/dL, range: 0.1-3.5). Fig. 1 shows the number of observations and magnitude distribution for M-spike levels among the patients included in our study. As shown in Fig. 2, M-spike levels predicted by the ML algorithm, calculated M-spike in the test set, correlated highly with the observed M-spike levels in the test set (R-square: 94% and RMSE of 0.21). The Pearson and Spearman coefficients were 0.97 and 0.95, respectively. Fig. 3b. Indicates the residual distribution for the RF model with most of values are close to and on both side zero value. Conclusions: Here, we assessed the multi-system impact of plasma cell clone using a ML algorithm. Our results show that the degree of systemic involvement induced by the clone correlates directly with magnitude of the ML-calculated M-spike value. Applying this algorithm in longitudinal studies of patients with MGUS and SMM is warranted to detect the optimal point of multi-systemic impact of clonal PCs to start therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MULTIPLE MYELOMA: NARRATIVE REVIEW

Multiple myeloma (MM) is a disease of unknown etiology, characterized by the accumulation of malignant clonal plasma cells in the bone marrow. Among its possible causes, exposure to toxins or viruses and in some cases, radiation has been considered, representing around 10% of malignant hematological neoplasms. Chromosomal abnormalities related to the immunoglobulin heavy chain change region located on the long arm of chromosome 14 have been seen. Among its clinical manifestations, bone pain, pathological fractures, anemia, frequent infections, hypercalcemia, kidney failure and episodes of abnormal bleeding will be highlighted. Its diagnosis is based on the demonstration of an increase in plasma cells &gt;10% in the bone marrow. There is no known cure for MM. However, in recent years a stable remission of the disease has been achieved in patients that can last up to several years through a combination of chemotherapy and autologous stem cell transplantation.

Molecular Impact of the Tumor Microenvironment on Multiple Myeloma Dissemination and Extramedullary Disease.

Multiple myeloma (MM) is the most common malignant monoclonal disease of plasma cells. Aside from classical chemotherapy and glucocorticoids, proteasome inhibitors, immunomodulatory agents and monoclonal antibodies are used in the current treatment scheme of MM. The tumor microenvironment (TME) plays a fundamental role in the development and progression of numerous solid and non-solid cancer entities. In MM, the survival and expansion of malignant plasma cell clones heavily depends on various direct and indirect signaling pathways provided by the surrounding bone marrow (BM) niche. In a number of MM patients, single plasma cell clones lose their BM dependency and are capable to engraft at distant body sites or organs. The resulting condition is defined as an extramedullary myeloma (EMM). EMMs are highly aggressive disease stages linked to a dismal prognosis. Emerging literature demonstrates that the dynamic interactions between the TME and malignant plasma cells affect myeloma dissemination. In this review, we aim to summarize how the cellular and non-cellular BM compartments can promote plasma cells to exit their BM niche and metastasize to distant intra-or extramedullary locations. In addition, we list selected therapy concepts that directly target the TME with the potential to prevent myeloma spread.

Inhibition of the Protein Arginine Methyltransferase PRMT5 in High-Risk Multiple Myeloma as a Novel Treatment Approach.

Multiple myeloma (MM) is an incurable clonal plasma cell malignancy. Subsets of patients have high-risk features linked with dismal outcome. Therefore, the need for effective therapeutic options remains high. Here, we used bio-informatic tools to identify novel targets involved in DNA repair and epigenetics and which are associated with high-risk myeloma. The prognostic significance of the target genes was analyzed using publicly available gene expression data of MM patients (TT2/3 and HM cohorts). Hence, protein arginine methyltransferase 5 (PRMT5) was identified as a promising target. Druggability was assessed in OPM2, JJN3, AMO1 and XG7 human myeloma cell lines using the PRMT5-inhibitor EPZ015938. EPZ015938 strongly reduced the total symmetric-dimethyl arginine levels in all cell lines and lead to decreased cellular growth, supported by cell line dependent changes in cell cycle distribution. At later time points, apoptosis occurred, as evidenced by increased AnnexinV-positivity and cleavage of PARP and caspases. Transcriptome analysis revealed a role for PRMT5 in regulating alternative splicing, nonsense-mediated decay, DNA repair and PI3K/mTOR-signaling, irrespective of the cell line type. PRMT5 inhibition reduced the expression of upstream DNA repair kinases ATM and ATR, which may in part explain our observation that EPZ015938 and the DNA-alkylating agent, melphalan, have combinatory effects. Of interest, using a low-dose of mTOR-inhibitor, we observed that cell viability was partially rescued from the effects of EPZ015938, indicating a role for mTOR-related pathways in the anti-myeloma activity of EPZ015938. Moreover, PRMT5 was shown to be involved in splicing regulation of MMSET and SLAMF7, known genes of importance in MM disease. As such, we broaden the understanding of the exact role of PRMT5 in MM disease and further underline its use as a possible therapeutic target.

Involvement of the NF-κB and PI3K/Akt/mTOR pathways in cell death triggered by stypoldione, an o-quinone isolated from the brown algae Stypopodium zonale.

Multiple myeloma (MM) is a clonal plasma cell malignancy that remains incurable to date. Thus, the aims of this study were to evaluate the involvement of the NF-κB and PI3K/Akt/mTOR pathways in the cytotoxicity of stypoldione, an o-quinone isolated from the brown algae Stypopodium zonale, in MM cells (MM1.S). The cytotoxic effect was evaluated in MM1.S cells and peripheral blood mononuclear cells (PBMCs) by MTT assay. The stypoldione reduced the cell viability of MM1.S cells in a concentration and time-dependent manner (IC50 in MM.1S from 2.55 to 5.38 μM). However, it was also cytotoxic to PBMCs, but at a lower range. Additionally, no significant hemolysis was observed even at concentration up to 10 times the IC50 . Apoptotic cell death was confirmed by cell morphology and Annexin V-FITC assay. Stypoldione induced intrinsic and extrinsic apoptosis by increasing FasR expression and reactive oxygen species (ROS) production, inverting the Bax/Bcl-2 ratio, and inducing ΔΨm loss, which resulted in AIF release and caspase-3 activation. It also increased Ki-67 and survivin expression and inhibited the NF-κB and PI3K/Akt/mTOR pathways. These results suggest that stypoldione is a good candidate for the development of new drugs for MM treatment.

Monitoring multiple myeloma in the peripheral blood based on cell-free DNA and circulating plasma cells

With the advent of novel, highly effective therapies for multiple myeloma (MM), classical serologic monitoring appears insufficient for response assessment and prediction of relapse. Moreover, serologic studies in MM are hampered by interference of therapeutic antibodies. The detection of malignant plasma cell clones by next generation sequencing (NGS) or multiparameter flow cytometry (MFC) circumvents these difficulties and can be performed in the peripheral blood (pB) by targeting circulating cell-free DNA (cfDNA) or circulating plasma cells (CPCs), thus also avoiding an invasive sampling procedure. Here, we applied NGS of VJ light chain (LC) rearrangements in cfDNA and MFC of magnetically-enriched CD138-positive CPCs (me-MFC) to investigate disease burden in unselected MM patients. Sequencing was successful for 114/130 (87.7%) cfDNA samples and me-MFC results were analyzable for 196/205 (95.6%) samples. MM clones were detectable in 38.9% of samples taken at initial diagnosis or relapse (ID/RD), but only in 11.8% of samples taken during complete remission (CR). Circulating MM plasma cells were present in 83.3% of ID/RD samples and 9.9% of CR samples. Residual disease assessment by NGS or me-MFC in samples taken during very good partial remission or CR was 80% concordant. Notably, 4/4 (NGS) and 5/8 (me-MFC) positive CR samples were from patients with oligo- or non-secretory myeloma. The time to progression was shorter if there was evidence of residual myeloma in the pB. Together, our findings indicate that our two novel analytical approaches accurately indicate the course of MM and may be particularly valuable for monitoring patients with serologically non-trackable disease.

Talking about Complimentary and Alternative Medicine: A Conversation Analysis Study

The FlourishCare Model (FCM) transforms primary care sites by addressing all determinants of health and focusing on helping patients to flourish. The FlourishCare Index (FCI) is a clinical measure to assess the effectiveness of the FCM to address all determinants of health. We will present data on the effectiveness of the FCM serving 159 older adults with MCCs. The sample was mostly female (77%), White (64%), retired (54%), married (30%) or widowed (20%) and living in urban areas (64%). The mean age was 69 (SD=15), with 13 years education (SD=3). Patients changed significantly over time on total FCI scores (57%-72%;□=3.80,SE=0.63). Results show significant growth over time for individual health behaviors (58%-67%;□=2.14,SE=0.84), health care access (71%-89%;□=4.43,SE=1.00) and social determinants (62%-85%;□=5.54,SE=1.02) with psychological determinants (54%-61%;□=1.74,SE=0.95) and environmental determinants (70%-81%;□=2.81,SE=1.62) showing a trend to significance. Interaction effects with time show that the FCM supported patients with lower education attainment to improve at a higher rate than those with higher education attainment, for the total FCI score (□=-0.59,SE=0.24) and health care access (□=-0.94,SE=0.38). Receiving mental health counseling resulted in more improvement in psychological determinants than those who did not receive counseling (□=3.43,SE=2.04). The FCM was able to support rural patients at a higher rate than urban patients to gain access to health care (□=4.13,SE=2.02). The FCM supported Hispanic patients the most in improving social determinants of health (□=8.40,SE=3.93). This study showed the importance of a systems approach to care using measures that focus on what matters most to older adults who value quality-of-life outcomes.

Talking about Complimentary and Alternative Medicine: A Conversation Analysis Study

Multiple Myeloma (MM) is a clonal plasma cell malignancy characterized by low blood counts and increased risk of infection, and primarily afflicts older adults. Although MM is incurable, advances in treatment, including autologous stem cell transplant (ASCT) has improved the lifespan of patients. MM patients commonly use over-the-counter complementary and alternative medicines (CAM) alongside conventional cancer therapies which, often without recognition by health care practitioners, may impact their treatment. Using data from an 18-month ethnographic study, we applied conversation analysis to examine 1180 minutes of audio-recordings to describe how patients and nurses interacted about CAM during ASCT education visits. Patients (n=12) had a median age of 62 years (IQR= 54-73), were mostly white (n=12, 75%), male (n=9, 56%), and had a moderate score on the FACT-G7 of 15 (IQR= 10-20). All patients had a caregiver present during their visit. Nurses (n=3) were aged 39 (IQR= 29-49) all with at least five years providing care to patients with blood cancers. Results suggested that nurses rarely provided direct feedback about CAM modalities, instead providing brief responses, and moving on to other topics. Excerpts were categorized into three groups, (1) demonstration of implicit epistemic authority, (2) demonstration of deferred epistemic authority in patient-initiated conversations, and (3) demonstration of deferred epistemic authority in nurse-initiated conversations. Understanding how conversations surrounding CAM are navigated can provide insights into patient-communication in general, and methods for improving ASCT education.

Identification of a Novel Epigenetic Mechanism of MYC Deregulation in Smoldering and Newly Diagnosed Multiple Myeloma Patients

Enhanced expression of the MYC oncogene is associated with the initiation and maintenance of many human cancers, including multiple myeloma (MM). MM is a malignancy of clonal plasma cells, in which MYC deregulation is a key event in the progression from the precursor stages of monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) to symptomatic MM. Translocation and amplification of the 8q24.21 MYC locus are known mediators of MYC deregulation at premalignant stages for some patients. However, DNA and RNA sequencing of MM patients show that cases with an intact MYC locus also exhibit MYC deregulation, indicating that additional mechanisms are involved in the deregulation of MYC in MM. Here we describe a new epigenetic mechanism of transcriptional deregulation of MYC in malignant plasma cells. We show that activation of a novel non-coding regulatory region through the binding of MM-specific transcription factors (TFs) is associated with MYC dysregulation in MM.To define the MM-specific MYC epigenetic regulation mechanisms, we performed a high-throughput CRISPR interference (CRISPRi) screen in ANBL6 MM cells that harbor no MYC genetic aberrations. We infected ANBL6 cells with a library of >111,000 sgRNAs, tiling across ~1.2 Mb of sequence around MYC and induced expression of KRAB-dCas9 to epigenetically repress putative regulatory elements. We then sequenced the distribution of sgRNAs in the population before and after 14 passages of growth. Because the expression of MYC quantitatively tunes cellular growth, sgRNAs that reduce MYC expression are less abundant at passage 14. This screen identified a ~13 kb region that significantly reduced cellular proliferation when targeted with sgRNAs. We assessed the function of each enhancer region with individual sgRNAs in different MM cell lines and detected an 89% reduction in MYC mRNA levels on average 48 hours after activating KRAB-dCas9.To further characterize the new enhancer region, we performed chromatin immunoprecipitation (ChIP)- and assay for transposase-accessible chromatin (ATAC)- sequencing on MM cell lines and malignant cells obtained from the bone marrow of 13 SMM and 8 MM patients and normal plasma cells from 3 healthy donors. We found that enhancer elements were enriched for H3K27ac and showed greater chromatin accessibility in tumor cells than normal plasma cells. Motif analysis of the enhancer region recovered putative binding sites for multiple TFs, such as IRF4 and MAF, that play key roles in MM pathogenesis. ChIP-sequencing for these enhancer-associated TFs and luciferase reporter assays targeting their binding sites confirmed the binding and involvement of IRF4 and MAF in activating enhancer elements in MM cells with intact MYC loci.MYC abnormalities are well-known secondary genetic events that trigger the progression of precursor diseases to MM. To define the genetic status of the identified enhancer elements in malignant cells, we examined whole-genome sequencing (WGS) data of 906 MM patients from the MMRF CoMMpass cohort. We found focal amplification of the enhancer region in 2.8% (n = 26) of patients. Transcriptional analysis on the same patient cohort revealed a significant increase in MYC mRNA levels in enhancer-amplified patients compared to MM cases with no MYC aberrations. These results indicate that enhancer activity is required to induce MYC expression and progression of patients with intact MYC loci.Collectively, our findings reveal a novel mechanism of MYC deregulation in malignant plasma cells: selective gain of chromatin accessibility at the enhancer elements and amplification of the enhancer region allow for binding of regulatory factors IRF4 and MAF, which increase the transcription of MYC in the absence of the known MYC chromosomal abnormalities. Our results point to the importance of non-coding regulatory elements and their associated TF networks as drivers of MM progression and suggest a new approach to identify predictive biomarkers and therapeutic targets that could improve patient outcomes in MM and other cancers. DisclosuresFulco: Bristol Myers Squibb: Current Employment. Ghobrial: AbbVie, Adaptive, Aptitude Health, BMS, Cellectar, Curio Science, Genetch, Janssen, Janssen Central American and Caribbean, Karyopharm, Medscape, Oncopeptides, Sanofi, Takeda, The Binding Site, GNS, GSK: Consultancy.

INVESTIGATION OF THE QUALIFICATION OF RADIOLOGICAL TECHNIQUES TO DETECT OSTEOLYTIC LESIONS, FRACTURES, AND OSTEOPOROSIS IN MULTIPLE MYELOMA PATIENTS

Multiple myeloma(MM) is a malignancy of clonal plasma cells. Osteolytic lesions represent a criterion for symptomatic myeloma and are associated with bone loss, pathological fractures, and osteoporosis. Skeletal surveys with other sophisticated techniques and dual-energy x-ray absorptiometry (DEXA) are used to screen lytic lesions, and bone mineral loss, respectively. Here, we aimed to investigate the detection rate of osteolytic lesions and bone mineral loss by several imaging techniques in MM. Three-hundred and ten symptomatic MM patients were screened retrospectively. The results of radiological techniques were recorded. The detection rate of osteolytic lesions, fractures, and plasmacytomas by imaging techniques, as well as bone mineral loss with DEXA was recorded. Also, associations with gender, MM type, lytic lesions, and osteoporosis were investigated. Skeletal survey and PET-CT detected lytic lesions in 71.3% and 81.2% of patients, respectively. PET-CT had a sensitivity of 96.1% and specificity of 90.6% to detect lytic lesions. MRI was only used for patients with suspicious fractures and detected them for all patients who underwent MRI. The osteoporosis rate was 83% for 113 patients who underwent DEXA. Any association between lytic lesions and gender or MM type was not detected. Our study demonstrated that osteolytic lesions are not correlated with gender or MM type. PET-CT is a sensitive and specific method for detecting osteolytic lesions. Although DEXA is sensitive, its specificity is limited to detect osteoporosis in patients with lytic lesions.

The importance of cytogenetic and molecular aberrations in multiple myeloma

Multiple myeloma (MM) is a heterogeneous clonal malignancy of plasma cells characterized by cytogenetic and molecular abnormalities. Chromosomal abnormalities are present at diagnosis and can evolve during the progression of MM. Metaphase karyotyping and fluorescence in situ hybridization are considered the standard diagnostic procedures performed in clinical practice. These test results are required to determine the Revised International Staging System classification, treatment algorithms, and short- and long-term prognoses. Given the dynamic development of cytogenetic and molecular research, we should expect further progress in better understanding the biology of MM and changes to patient care in the coming years.

Histone Methyltransferases G9a and GLP; Identification and Validation of Novel Therapeutic Targets for Multiple Myeloma Treatment

Multiple myeloma (MM) is a virtually incurable clonal plasma cell malignancy which mainly resides in the bone marrow. Like in other cancers, numerous epigenetic aberrations have been described in MM, resulting in deregulated gene expression, disease progression and drug resistance. Targeting epigenetic modifiers involved in this deregulated epigenetic landscape therefore represents an interesting therapeutic approach. G9a (EHMT2) and GLP (EHMT1) are 2 histone methyltransferases which catalyze mono- and dimethylation of histone 3 lysine 9 (H3K9). Importantly, G9a is overexpressed in several cancers, correlating with a poor prognosis. Currently, data about the expression and role of G9a/GLP in MM is lacking. The aim of this study is therefore to investigate the functional role of G9a and GLP in MM pathogenesis.Here we report that high expression levels of both G9a and GLP are associated with a worse disease outcome in the UAMS-TT2 newly diagnosed MM patients cohort (n=345, GSE4581). Gene set enrichment analysis of high G9a/GLP expressers displays a significant enrichment of genes involved in pathways associated with MM disease progression, including the RAS pathway, NF-ĸB canonical pathway, IRF4 multiple myeloma program and mRNA splicing. Using a panel of 10 human cell lines, 3 murine cell lines and 5 primary samples we further evaluated the effect of the specific small molecule G9a/GLP inhibitors UNC0638 and BIX01294 on MM cell viability, cell cycle progression and apoptosis. We found BIX01294 and UNC0638 to significantly and potently reduce MM cell viability. Moreover, both inhibitors also induce cell cycle arrest and apoptosis. When comparing between both inhibitors, BIX01294 was found to be the most potent to induce apoptosis. Mechanistic studies for BIX01294 furthermore indicate that BIX01294 treatment results in autophagic programmed cell death as evidenced by a strong increase in the formation of LC3B puncta and an increase in LC3II and beclin-1 protein levels. In addition, we found that BIX01294 sensitizes MM cells to the clinically relevant proteasome inhibitor bortezomib and the Bcl-2 inhibitor ABT199. The in vivo anti-MM activity of therapeutic BIX01294 treatment was tested using the murine 5TGM1 model. Difference in overall survival between the vehicle and BIX01294 treated groups was assayed with a log-rank test and survival curves plotted using the Kaplan-Meier method. Therapeutic treatment of 5TGM1 inoculated mice with BIX01294 resulted in a clear delay in tumor progression, as evidenced by a clear decrease in tumor burden and a significant increase in the overall survival of BIX01294 treated mice compared to vehicle treated mice. Lastly, 5TGM1 inoculated mice were treated with a suboptimal dose of bortezomib or BIX01294, and the combination of both in a therapeutic setting. We observed a significant reduction in BM plasmacytosis in mice treated with bortezomib (p<0.05) and with the combination of both bortezomib and BIX01294 (p<0.001) compared to vehicle mice. Although there was no significant difference between BM plasmacytosis levels in mice treated with bortezomib and in the combo treated mice, we could observe a clear trend of an additional reduction in tumor burden in combo treated mice versus bortezomib treated mice.In conclusion, our data indicates for the first time the importance of the histone methyltransferases G9a/GLP in MM pathogenesis. Furthermore, we demonstrate that specific targeting of G9a/GLP induces MM cell apoptosis, enhances MM sensitivity to ABT- 199 and bortezomib and significantly delays tumor progression in the murine 5TGM1 model. Thus, G9a/GLP targeting represents a promising strategy to improve treatment of MM. DisclosuresNo relevant conflicts of interest to declare.