Serum Of Multiple Myeloma Patients Research Articles

Gamma gap as a prognostic marker of treatment response in patients with multiple myeloma.

Multiple myeloma (MM) is defined by the excessive growth of cancerous plasma cells in the bone marrow, resulting in the production of an abnormal monoclonal paraprotein and signs of damage to vital organs. The diagnosis of multiple myeloma can be complex. This paper centers on the diagnosis of MM and examines the role of the gamma gap (GG) in prompting tests to assess treatment effectiveness. Sixty individuals newly diagnosed with MM were selected from medical facilities in Iraq, specifically Baghdad Hospital/Medical City and Hematological Center, between November 2022 and March 2023. Additionally, 30 healthy participants were included as a comparison group. The assessment of suspected MM patients involves confirming the presence of a monoclonal paraprotein using serum electrophoresis (SEPG) and immunofixation (SIF), as well as analyzing free light chains (FLC). Spectrophotometric methods were used to measure total serum protein and albumin levels and calculate the globulin-to-albumin ratio by subtracting serum albumin from total serum protein. In most MM cases the monoclonal immunoglobulin (as M-spike) was identified and quantified by SPEP. Based on the findings from IFE detection, the prevalence of M-spike, particularly type IgG, was observed in 45 cases (83.3%), of which 40% were type κ, 24.6% were type λ, and 19.3% were of an unknown type. Additionally, type IgA was the second most prevalent (10 cases, 16.7%), with 14.3% being type κ and 1.7% being type λ. Moreover, the results indicated a significant association between GG and M-spike, as well as a strong correlation between GG and pre- and post-treatment M-spike levels. The area under the curve was deemed acceptable (AUC = 0.735, p = 0.0001), demonstrating high diagnostic accuracy (86.2) in distinguishing MM patients before and after treatment. The evaluation of GG in the serum of MM patients demonstrated elevated levels, which were significantly associated with patients post-treatment. Therefore, our findings have significant implications for the use of GG as a prognostic indicator for monitoring treatment response in MM patients.

An automated workflow based on data independent acquisition for practical and high-throughput personalized assay development and minimal residual disease monitoring in multiple myeloma patients.

Minimal residual disease (MRD) status in multiple myeloma (MM) is an important prognostic biomarker. Personalized blood-based targeted mass spectrometry detecting M-proteins (MS-MRD) was shown to provide a sensitive and minimally invasive alternative to MRD-assessment in bone marrow. However, MS-MRD still comprises of manual steps that hamper upscaling of MS-MRD testing. Here, we introduce a proof-of-concept for a novel workflow using data independent acquisition-parallel accumulation and serial fragmentation (dia-PASEF) and automated data processing. Using automated data processing of dia-PASEF measurements, we developed a workflow that identified unique targets from MM patient sera and personalized protein sequence databases. We generated patient-specific libraries linked to dia-PASEF methods and subsequently quantitated and reported M-protein concentrations in MM patient follow-up samples. Assay performance of parallel reaction monitoring (prm)-PASEF and dia-PASEF workflows were compared and we tested mixing patient intake sera for multiplexed target selection. No significant differences were observed in lowest detectable concentration, linearity, and slope coefficient when comparing prm-PASEF and dia-PASEF measurements of serial dilutions of patient sera. To improve assay development times, we tested multiplexing patient intake sera for target selection which resulted in the selection of identical clonotypic peptides for both simplex and multiplex dia-PASEF. Furthermore, assay development times improved up to 25× when measuring multiplexed samples for peptide selection compared to simplex. Dia-PASEF technology combined with automated data processing and multiplexed target selection facilitated the development of a faster MS-MRD workflow which benefits upscaling and is an important step towards the clinical implementation of MS-MRD.

Expression and Clinical Significance of LINC00475 in Multiple Myeloma

To investigate the relative expression level and clinical significance of LINC00475 in serum of patients with multiple myeloma (MM). The expression of LINC00475 in serum of 108 MM patients and five MM cell lines including RPMI 8226, NCI-H929, U266, OPM2 and CAG were detected by real-time fluorescence quantitative PCR. The diagnostic value of LINC00475 in MM was evaluated by receiver operating characteristic (ROC) curve analysis. The correlation of LINC00475 with patients' characteristics was analyzed. Compared with control groups, the expression of LINC00475 was up-regulated in serum of MM patients and MM cell lines (all P < 0.05). ROC curve analysis showed that the optimal cut-off value of LINC00475 was 262.4, the area under curve (AUC) was 0.924(95%CI : 0.884-0.964), and sensitivity and specificity was 83.3% and 91.7%, respectively, which indicated that LINC00475 had good evaluation value in MM patients. Compared with low-LINC00475 expression group, patients in high-LINC00475 expression group had higher levels of β2microglobulin (β2-MG) and Cystatin C (Cys-C) but lower albumin (ALB) (all P < 0.05). Compared with MM patients with International Staging System (ISS) stage I, the expression level of LINC00475 was significantly higher in patients with stage II and III (both P < 0.05). LINC00475 is helpful to distinguish MM patients from healthy adults, which is correlated with the prognostic indicators such as β2-MG, ALB, and ISS stage.

The diagnostic value of serum lncRNA CATG00000112921.1 as a marker of multiple myeloma

Multiple myeloma (MM) is a malignant plasma cell disease. At present, numerous studies have shown that lncRNA plays a very important role in the occurrence, development and even drug resistance of multiple myeloma. It may become a potential diagnostic and prognostic marker of multiple myeloma and provide new ideas for targeted therapy. Based on the above research background, this study used gene chip technology to screen out the differentially expressed lncRNA in the serum of MM patients and healthy people, and verified more clinical serum samples to screen out the lncRNA with the largest difference as a biomarker for further research. In this research, the data of hospitalized patients diagnosed with MM and healthy people in the Affiliated Hospital of Guangdong Medical University were retrospectively collected. The lncRNA expression profile of serum samples from patients with multiple myeloma and healthy controls was analyzed by lncRNA chip technology. The serum samples were verified by real-time fluorescence quantitative PCR, and the candidate diagnostic markers were screened out. The ROC working curve was drawn to evaluate the diagnostic efficacy of the candidate markers and to determine their stability at different temperatures and time. A total of 44 MM patients and 37 healthy people were involved in this research. Among them, 4 patients with MM and 4 patients with HD were sent for microarray analysis. According to Fold Change ≥ 2 and P < 0.05, a total of 17 differentially expressed lncRNA molecules were screened, of which 9 were up-regulated RNA molecules and 8 were down-regulated RNA molecules. Through real-time fluorescence quantitative PCR verification, it was found that lncRNA CATG00000112921.1 was highly expressed in the healthy control group and diminished in patients with multiple myeloma, P < 0.001. The ROC curve demonstrated that the area under the curve (AUC) was 0.749, the sensitivity was 0.636, the specificity was 0.789, and the 95 % CI was 0.636-0.862 (P < 0.001). In addition, in order to verify the effects of temperature, time and repeated freezing and thawing on lncRNA, it was placed at 25°C, 4°C, -20°C, -80°C for 0h, 24h, 48h, 72h, and placed at-80°C repeated freezing and thawing 0 times, 2 times, 4 times, 8 times, and the expression level was not significantly changed. Serum lncRNA CATG00000112921.1 may be a potential candidate diagnostic marker for multiple myeloma. The ROC curve shows that it has good diagnostic value, and its high stability at different temperatures and different times is a required condition for becoming a diagnostic marker. As far as we know, this is the first study in the world to find differential expression of lncRNA CATG00000112921.1 in peripheral serum of healthy people and newly diagnosed multiple myeloma patients. This study also highlights the application of gene chip technology in screening differentially expressed genes.

Multiple Myeloma Induced IL-6 Reprograms Lipid Metabolism through a CD36 Dependent Mechanism

Multiple Myeloma (MM) requires a high metabolic turnover to allow for expansion and paraprotein production. MM metabolism is complex relying on various metabolic pathways to fuel its survival including fatty acid (FA) oxidation (1, 2). The liver is a master regulator of fatty acid metabolism and distribution. It also stores FA as lipid droplets during steady state or produces ketone bodies during a starvation state (3). This study aimed to determine whether MM alters FA uptake within the liver and by what mechanism this occurs. To investigate the role of FA metabolism we used a mouse model of MM (5TGM1 (GFP) cells into KaLwRij mice). Analysis of serum from 5TGM1 (GFP) engrafted KaLwRij mice showed increased FFA when compared to control animals. RT-qPCR of RNA isolated from liver samples showed a decrease in genes involved in FA uptake and metabolism. These genes included the long chain fatty acid transporter CD36, which belongs to the class B scavenger receptors and is essential for uptake of LCFA in hepatocytes. In vitro modelling showed that primary hepatocytes cultured in conditioned media from 5TGM1 cells had significantly reduced expression of CD36. Furthermore, serum isolated from MM patients (n=6) downregulated CD36 expression in the human hepatocarcinoma cell line, HepG2. These data support the hypothesis that MM induces a factor that alters lipid metabolism in the liver. To detemine the factor that MM secretes, and which regulates FA uptake in the liver, a cytokine array of the serum from 5TGM1 (GFP) engrafted KaLwRij mice was compared to control serum. Pathway analysis revealed interleukin-6 (IL-6) as possible key factor involved in regulating liver FA uptake. In vitro gene expressions analysis of mouse primary hepatocytes revealed that addition of IL-6 to the medium inhibited expression of CD36 and also showed that primary hepatocytes had decreased reliance on FA oxidation when treated with IL-6. Furthermore. 5TGM1 (GFP) engrafted KaLwRij mice showed increased serum IL-6 levels compared to controls. Finally, using the IL-6 neutralising antibody, we observed that this blocked CD36 downregulation in HepG2 cultured with MM patient serum. Here we demonstrate that MM changes FA metabolism in the liver through upregulation of IL-6. Gene expression of FA genes identifies CD36 is down regulated in hepatocytes in response to IL-6. These data suggest that MM is able to redirect FFA away from the liver by down regulating CD36, which mediates FFA uptake in the liver. References Panaroni C, Fulzele K, Mori T, Siu KT, Onyewadume C, Maebius A, et al. Multiple myeloma cells induce lipolysis in adipocytes and uptake fatty acids through fatty acid transporter proteins. Blood. 2022;139(6):876-88.Tirado-Vélez JM, Joumady I, Sáez-Benito A, Cózar-Castellano I, Perdomo G. Inhibition of fatty acid metabolism reduces human myeloma cells proliferation. PLoS One. 2012;7(9):e46484.Hodson L, Gunn PJ. The regulation of hepatic fatty acid synthesis and partitioning: the effect of nutritional state. Nat Rev Endocrinol. 2019;15(12):689-700.

EXOSOME MIRNAS PROFILING IN SERUM AND PROGNOSTIC EVALUATION IN PATIENTS WITH MULTIPLE MYELOMA

Introduction: Multiple Myeloma (MM) is an incurable hematological malignancy. Tightly crosstalk through microRNAs (miRNAs) carried by exosomes between the bone marrow microenvironment and MM cells plays pivotal roles in MM. Our study aimed to identify the expression profile of exosomal miRNAs (exo-miRNAs) in the serum of MM patients and investigate the regulation network and their potential functions. Methods: Exosomes in serum from 19 newly diagnosed multiple myeloma patients and 9 healthy donors were isolated and the miRNA profile was investigated by small RNA sequencing. Differential expression of exo-miRNAs was calculated and target genes of miRNAs were predicted. CytoHubba was applied to identify the hub miRNAs and core target genes. The LASSO Cox regression model was utilized to develop the prognostic model, and the ESTIMATE immune score was calculated to investigate the correlation between the model and immune status in MM patients. Results: Our study clarified 313 differentially expressed serum exo-miRNAs between MM patients and HD. GO analysis of target genes of differential miRNAs showed that these target genes are mainly involved in the critical biological processes related to MM pathogenesis such as proteasome-mediated ubiquitin-dependent protein catabolic process, indicating the indispensable roles of these miRNAs in MM pathogenesis. Top six hub differentially expressed serum exo-miRNAs (hsa-miR-4728-5p, hsa-miR-455-3p, hsa-miR-6779-5p, hsa-miR-124-3p, hsa-miR-615-3p, and hsa-miR-7106-5p) were identified. 513 target genes of six hub exo-miRNAs were confirmed to be differentially expressed in MM cells in Zhan Myeloma microarray dataset. Functional enrichment analysis indicated that these target genes mainly involved mRNA splicing, cellular response to stress, and deubiquitination. Thirteen core exo-miRNA target genes were applied to create a novel prognostic signature to facilitate the risk stratification of MM patients, and two groups of MM patients with diverse outcomes were recognized (Figure 1A). The high-score patients identified by thirteen core genes signature displayed a higher 70 high-risk gene set (UAMS-70) score and the 56 drug-resistance genes set score, which might partly explain their inferior outcomes (Figure 1B–C). Additionally, our results suggest that the thirteen core genes signature model was highly correlated with the status of the immune microenvironment, and patients in the low-score group had higher immune cell infiltration levels (Figure 1D). Besides, patients in the high-risk group displayed lower HLA family gene expression (Figure 1E). Encore Abstract - previously submitted to EHA 2023 Keywords: Bioinformatics, Computational and Systems Biology, Diagnostic and Prognostic Biomarkers, Risk Models No conflicts of interests pertinent to the abstract.

Lactate trafficking inhibition restores sensitivity to proteasome inhibitors and orchestrates immuno-microenvironment in multiple myeloma.

Metabolic changes of malignant plasma cells (PCs) and adaptation to tumour microenvironment represent one of the hallmarks of multiple myeloma (MM). We previously showed that MM mesenchymal stromal cells are more glycolytic and produce more lactate than healthy counterpart. Hence, we aimed to explore the impact of high lactate concentration on metabolism of tumour PCs and its impact on the efficacy of proteasome inhibitors (PIs). Lactate concentration was performed by colorimetric assay on MM patient's sera. The metabolism of MM cell treated with lactate was assessed by seahorse and real time Polymerase Chain Reaction (PCR). Cytometry was used to evaluate mitochondrial reactive oxygen species (mROS), apoptosis and mitochondrial depolarization. Lactate concentration resulted increased in MM patient's sera. Therefore, PCs were treated with lactate and we observed an increase of oxidative phosphorylation-related genes, mROS and oxygen consumption rate. Lactate supplementation exhibited a significant reduction in cell proliferation and less responsive to PIs. These data were confirmed by pharmacological inhibition of monocarboxylate transporter 1 (MCT1) by AZD3965 which was able to overcame metabolic protective effect of lactate against PIs. Consistently, high levels of circulating lactate caused expansion of Treg and monocytic myeloid derived suppressor cells and such effect was significantly reduced by AZD3965. Overall, these findings showed that targeting lactate trafficking in TME inhibits metabolic rewiring of tumour PCs, lactate-dependent immune evasion and thus improving therapy efficacy.

The Relationship between Serum miRNAs and Early Mortality in Multiple Myeloma Patients Treated with Bortezomib-Based Regimens.

Multiple myeloma (MM) is a hematological malignancy characterized by the clonal proliferation of plasma cells in the bone marrow (BM) microenvironment. Despite the progress made in treatment, some MM patients still die within the first year of diagnosis. Numerous studies investigating microRNA (miRNA) expression patterns suggest they may be good prognostic markers. The primary aim of this study was to analyze the expression of selected miRNAs in the serum of MM patients who were later treated with bortezomib-based regimens, and to determine their potential to predict early mortality. The study was conducted in 70 prospectively recruited patients with newly diagnosed MM admitted to the Department of Hematology of the Copernicus Memorial Hospital, Lodz (Poland) between 2017 and 2021. Among them, 17 patients experienced death within 12 months of diagnosis. The expression of 31 selected miRNAs was determined using a miRCURY LNA miRNA Custom PCR Panel. The obtained clinical data included patient characteristics on diagnosis, treatment regimen, response to treatment, and follow-up. Differential expression analysis found two miRNAs to be significantly downregulated in the early mortality group: hsa-miR-328-3p (fold change-FC: 0.72, p = 0.0342) and hsa-miR-409-3p (FC: 0.49, p = 0.0357). Univariate and multivariate logistic regression analyses were performed to assess the early mortality rate. The final model consisted of hsa-miR-409-3p, hsa-miR-328-3p, age, and R-ISS 3. It yielded an area under the curve (AUC) of 0.863 (95%CI: 0.761-0.965) with 88.2% sensitivity and 77.5% specificity. Further external validation of our model is needed to confirm its clinical value.

Serum soluble BCMA can be used to monitor relapse of multiple myeloma patients after chimeric antigen receptor T-cell immunotherapy

PurposeChimeric antigen receptor T-cell (CAR-T) therapy has been proven very effective in treating hematologic malignancies. Ciltacabtagene autoleucel (cilta-cel), a second-generation CAR-T cell with double B cell maturation antigen (BCMA) targeting binding domains, showed an 88% overall response rate (ORR) in patients with relapsed/refractory multiple myeloma (MM), which were carried out in our institute. This study aimed to assess the prognostic potential of soluble BCMA (sBCMA) in serum as a biomarker in MM after CAR-T therapy. Patients and methodsSerum samples (n = 44) from MM patients were collected before and after CAR-T therapy. The level of sBCMA was analyzed by enzyme-linked immunosorbent assay (ELISA). Additionally, three patients’ long-term longitudinal analysis were performed. ResultsSerum sBCMA level was correlated with the percentage of malignant plasma cells in bone marrow (r = 0.613). After CAR-T infusion, the sBCMA level in serum of MM patients decreased markedly (median: 508,513 pg/mL before CAR-T infusion, 89,198 pg/mL in the first month, 8448 pg/mL in the second months, and 6010 pg/mL in the third month after CAR-T infusion). In patients who obtained objective response (≥ PR), re-elevated sBCMA indicated the possibility of disease recurrence. At a cutoff 69,326.27 pg/mL, sBCMA shows high sensitivity (87.5%) and specificity (88.5%) for identifying relapse of MM after CAR-T therapy. ConclusionOur results suggested that serum sBCMA level changes in response to the clinical status of MM patients after anti-BCMA CAR-T therapy. Furthermore, sBCMA may be a auxiliary biomarker for disease monitoring in MM patients after CAR-T therapy.

Exosome Mirnas Profiling in Serum and Prognostic Evaluation in Patients with Multiple Myeloma

Objective: Multiple Myeloma (MM) is a hematological malignancy characterized by the clonal expansion of malignant proliferation of plasma cells in the bone marrow. Moreover, the proliferation of MM cells is highly dependent on the BM microenvironment. MicroRNA (MiRNAs) are short, noncoding RNAs that regulate gene expression at the posttranscriptional level. MiRNAs carried by exosomes play pivotal roles in the crosstalk and signal transferring between malignant cells and non-malignant cells, which are involved in the pathogenesis of MM. Here, we investigated the exosome miRNA profiling in peripheral blood serum of MM patients and compared it with healthy donors. Bioinformatics analysis was utilized to clarify the molecular pathways associated with exosome miRNAs via their target genes. Furthermore, a prognostic model based on the top 20 hub genes was constructed in MM. Methods and materials: The serum samples including 19 newly diagnosed MM patients and 9 healthy donors were analyzed. Exosome in serum was isolated and miRNA profiling was investigated. Differential expression of miRNAs in exosomes was calculated between MM patients and HDs using the edgeR package. STRING and CytoHubba were applied to identify the hub miRNAs and core mRNAs. The exosomal miRNA-mRNA regulatory network was constructed. Functional enrichment of the core target genes was implemented using Metascape. The ESTIMATE immune scores were used to analyze the infiltration levels of immune cells in MM patients with GSE136324. The proportion of 22 tumor-infiltration immune cells in each MM patient was calculated using the CIBERSORT score. Multivariate Cox analysis of hub mRNAs was utilized to construct a novel prognostic model based on the top 20 hub genes. Results: In the present study, we found 353 differentially expressed exo-miRNAs between MM patients and healthy donors, and 9564 target mRNAs by 353 exo-miRNAs were predicted. GO and KEGG analysis of target mRNAs showed that they were involved in biological processes including proteasome-mediated ubiquitin-dependent protein degradation process. The exosomal miRNA-mRNA regulatory network was constructed and six hub miRNAs were identified. Target genes of these six hub miRNAs and the differential expressed genes (DEG) in Zhan Myeloma were intersected, and 513 DEGs targeted by hub miRNAs were identified. The protein-protein interaction (PPI) network was established in the STRING database. According to module analysis, the top 3 three modules were identified in the PPI network including mRNA splicing, cellular response to stress, and deubiquitination. Moreover, a module related to cytokine-related signaling pathways was enriched as well. Further analysis suggests that Interleukin 6 Signal Transducer (IL6ST), Tyrosine Kinase 2 (TYK2), and Adenosine Deaminase (ADAR) exhibited high correlations with immune scores in MM and were significantly associated with infiltration of CD4 T cell subtypes including follicular helper T cells and CD4 naïve T cells. Taken together, these findings suggested that IL6ST, TYK2, and ADAR might play pivotal roles in tumor immune escape. In addition, the top 20 hub genes were selected from the PPI network. Multivariate Cox regression analysis showed that the 20 hub genes-based signature could be utilized to construct a prognostic model. Conclusions: In conclusion, our study successfully depicted the exo-miRNA expression profiles in serum exosomes of MM patients and constructed the miRNA-mRNA regulatory network. A novel prognostic model based on 20 hub genes is constructed that facilitates the risk stratification of MM patients with distinct outcomes.

P868: A CIRCULATING SERUM MIRNAS-BASED MODEL TO PREDICT EARLY MORTALITY IN MULTIPLE MYELOMA PATIENTS TREATED WITH BORTEZOMIB-BASED REGIMENS.

Background: Multiple myeloma (MM) is a hematological malignancy characterized by the clonal proliferation of plasma cells in the bone marrow. Despite the progress made in the treatment of MM, some patients die within the first year of diagnosis. Numerous studies underline the role of miRNAs in the pathogenesis of MM and their potential role in prognosis. Aims: The aim of this study was to analyze the expression of selected miRNAs in the serum of MM patients treated with bortezomib-based regimens and determine their potential to predict early mortality. Methods: The study was conducted in prospectively-recruited patients with newly-diagnosed MM who were qualified for bortezomib-based treatment regimens. All were admitted to the Department of Hematology, Medical University of Lodz between 2017 and 2021. Venous blood samples were collected from the patients before treatment initiation, processed, and stored for further use. Total RNA, including miRNA, was isolated from serum, and reverse-transcribed to cDNA. The expression of 31 selected miRNAs was determined using a miRCURY LNA miRNA Custom PCR Panel. The miRNA selection was based on the results of our previous study (Robak et al., Cancers Cancers (Basel) 2020; 12(9): 2569). Three miRNAs were used for expression normalization: hsa-miR-23b-3p, hsa-miR-151a-5p and hsa-miR-152-3p. Clinical data, including patient characteristics on diagnosis, treatment regimen, response to treatment and follow-up were obtained from hospital records. Differential expression analysis, univariate and multivariate logistic regression models were performed using R version 3.6.3. Results: A total of 69 MM patients were included in the study with a mean age at diagnosis of 64.9 ± 11.0 years. Among them, 17 patients experienced death within 12 months of diagnosis. Patients with early mortality were significantly older (72.6 vs. 62.3 years, p=0.0005), and more frequently men (70.6% vs. 42.3%, p=0.0429). No differences were observed in R-ISS distribution or CRAB symptoms between the groups, nor were any significant differences between percentage myeloma infiltration in the bone marrow and various laboratory findings, including LDH, serum M protein, albumin, CRP, and uric acid. In the differential expression analysis, two miRNAs were significantly downregulated in early mortality group- hsa-miR-328-3p (fold change- FC: 0.72, p=0.0342) and hsa-miR-409-3p (FC: 0.49, p=0.0357). Similarly, hsa-miR-328-3p (OR 0.44, 95%CI: 0.20-0.97, p=0.0415) and hsa-miR-409-3p (OR 0.69, 95%CI: 0.48-0.98, p=0.0400) were found to have a protective effect against early mortality in univariate analyses. The multivariate logistic regression analysis found that miRNAs retained their significance when established clinical prognostic factors were included. The final model consisted of hsa-miR-409-3p (OR 0.61, 95%CI: 0.37-0.99, p=0.0480), hsa-miR-328-3p (OR 0.33, 95%CI: 0.13-0.87, p=0.0254), age (OR 1.13, 95%CI: 1.03-1.23, p=0.0096) and R-ISS 3 (OR 2.91, 95%CI: 0.63-13.47, p=0.1723). A receiver operating characteristics (ROC) analysis for the model yielded an area under the curve (AUC) of 0.863 (95%CI: 0.761-0.965). At the optimal cut-off value of 0.28, the sensitivity and specificity reached 88.2% and 77.5%, respectively. Image:Summary/Conclusion: hsa-miR-409-3p and hsa-miR-328-3p are independent factors related to early mortality in MM patients. Our results were used to generate a multiple regression model that may have the potential to predict early mortality. Further external validation of our model is necessary to confirm its clinical value.

Combination therapy for treating drug-resistant multiple myeloma

Abstract Proteasome inhibitors (PIs) have remarkably improved the survival of multiple myeloma (MM) patients but dose-limiting toxicities and the development of drug resistance limit their long-term utility. Elevated TGF-β levels in MM patient sera correlate with chemoresistance, disease progression, metastasis and poor prognosis. Therefore, we evaluated the anti-MM therapeutic potential of a TGF-β type I receptor kinase inhibitor, Vactosertib. In vitro treatment of Vactosertib synergistically inhibited the growth of PI-resistant MM cells in combination with the ixazomib by suppressing TGF-β activation of Smad2/3 and expression of PSMB5, which encodes the proteasome 5 catalytic subunit targeted by PIs. Oral administration of Vactosertib as a single agent inhibited MM progression and induced a decrease in mortality and an increase in body weight of the mice bearing MM. Vactosertib alone also attenuated TGF-β activation of Smad2/3, reduced the expansion of CD11b+Gr-1+ myeloid derived suppressor cells (MDSCs) in bone marrow tumor microenvironment, and diminished the population of Foxp3+ regulatory T cells (Treg) in the spleen. Combination therapy of Vactosertib plus ixazomib was exhibited a synergistic anti-tumor effect when compared to either Vactosertib or ixazomib alone by greatly prolonged survival and significant a reduction in both MDSCs and Tregs. Taken together, our data provide the rationale for clinical evaluation of Vactosertib in MM and demonstrate proof-of-concept that combination of Vactosertib and PIs may overcome chemoresistance and enhance durable patient responses. Supported by 1R03CA259901-01A1

Serum hsa_circ_0087776 as a new oncologic marker for the joint diagnosis of multiple myeloma

ABSTRACT Multiple myeloma (MM) is a hematologic malignancy caused by abnormal proliferation of bone marrow plasma cells, which lacks diagnostic markers and has a general prognosis. At present, the understanding of its pathogenesis provides the basis for the combined diagnosis and new targeted therapy of the disease. In this study, quantitative real-time PCR was used to detect 136 MM patients and 74 healthy controls, and the clinical application value of hsa_circ_0087776 as a new tumor marker and combined diagnosis was evaluated. The results showed that the expression of hsa_circ_0087776 was significantly lower in serum of MM patients (P-value < 0.0001), and the expression was consistent in MM cells. In the analysis of clinicopathological parameters, it was found that there were significant statistical differences with MM stage and renal injury. In addition, it significantly increased the sensitivity with ALB, β₂-MG joint diagnosis, to provide a basis for diagnosis, improve the prognosis of the disease, improve the survival of patients and quality of life. These studies suggest that hsa_circ_0087776 can be used as a new oncology marker for the combined diagnosis of MM. Impact statement: Various evidences have shown that the role of circRNA in the occurrence and development of diseases is potentially unknown and untapped. Therefore, it has a broad prospect to find circRNA specifically expressed in MM patients for combined diagnosis and targeted therapy of MM. However, MM lacks such specific tumor markers. Therefore, the discovery of new specific tumor markers for combined diagnosis is an important milestone in the development of medical history. In the research, we founded hsa_circ_0087776 can be used as a new oncology marker for combined diagnosis of MM.

Non-invasive discrimination of multiple myeloma using label-free serum surface-enhanced Raman scattering spectroscopy in combination with multivariate analysis

We report non-invasive discrimination of multiple myeloma (MM) using label-free serum surface-enhanced Raman scattering (SERS) spectroscopy in combination with multivariate analysis. Colloidal silver nano-particles (AgNPs) were used as the SERS substrate. High quality serum SERS spectra were obtained from 53 MM patients and 44 healthy controls (HCs). The SERS spectral differences demonstrated variation of relative concentrations of biomolecules in the serum of MM patients in comparison to HCs. Multivariate analysis methods, including principal component analysis (PCA), linear discriminant analysis (LDA), and support vector machine (SVM), were used to build discrimination models for MM. Leave-one-out cross-validation (LOOCV) was used to evaluate the performances of the models, in terms of accuracy, sensitivity, specificity, and area under the receiver operating characteristic curves (AUC). Using the SVM model, the accuracy for discrimination of MM was achieved as 78.4%, and the corresponding sensitivity, specificity, and AUC values were 0.830, 0.727, and 0.840, respectively. The results show that the serum SERS in combination with multivariate analysis could be a fast, non-invasive, and cost-effective technique for discrimination of MM.

P-081: A novel circulating miRNA involved in T cell senescence of multiple myeloma patients

<h3>Background</h3> Many circulating miRNAs were reported as biomarkers of multiple myeloma (MM),but the functions of these circulating miRNAs are not clarified recently. The aim of this work is to identify novel biomarkers of circulating miRNAs in diagnosis and prognosis for MM and reveal their function in the pathogenesis of MM. <h3>Methods</h3> MiRNAs profiling in serum(n=15) and MM cells(CD138+, n=10) were analyzed by RNA-sequencing of MM patients. The common dysregulated miRNAs(P<0.05) were identified via Veen Diagram analysis in serum and MM cells between MM patients and healthy donors, then validated by RT-PCR. ROC analysis and Kaplan-Meier(K-M) analysis were performed to evaluate the differently expressed circulating miRNAs in diagnosis and prognosis in MM patients, respectively. Exosomes of serum were isolated and characterized by dynamic light scattering, transmission microscopy, and western blot analysis. <h3>Results</h3> There were stable and specific miRNA expression profiles in serum and tumor cells of MM patients. The miRNA profiling of MM serum showed that there were 74 differential circulating miRNAs(P<0.05), among which 56 miRNAs(75.7%) were down-regulated and 18 miRNAs were up-regulated(24.3%). MiRNA profiling of MM cells showed that there were 376 miRNAs differentially expressed(P<0.05), of which 87(23.1%) were down-regulated and 289(76.9%) were up-regulated. Veen analysis found 36 common dysregulated miRNAs both in serum and tumor cells of MM. There were 22.3% of differential miRNAs located on chromosome 1 and 17. Five circulating miRNAs(miR-27b-3p, miR-145-3p, miR-628-3p, miR-342-5p and miR-30e-3p) were further confirmed differentially expressed in MM patients(n=201) by RT-PCR. K-M analysis showed that MM patients with low level of miR-27b, miR-145, and miR-628 had shortened PFS and OS. Strikingly, comparing to the decreased level of miR-27b in MM patients serum, miR-27b was enriched in the exosome of serum. MM patients with low level of serum miR-27b displayed lower CD3+T cells and CD3+CD4+T cells in peripheral blood. Our further study found increased proportation of senescent T cells in MM patients with low level of serum miR-27b, especially in CD8+T cells. In order to confirm T cell senescent phenotype, PBMCs were co-cultured with MM patients serum exosomes which mimic the in vivo situation. Flow cytometry analysis showed the CD28 expression was notably decreased on the CD3+ T cells in the co-culture group. In addition, RT-PCR and flow cytometry analysis showed that overexpression of miR-27b significantly down-regulated the level of CD28 in jurkat cells, a T cell line. These findings suggested an immune suppressive microenvironment in MM patients with lower level of serum miR-27b. <h3>Conclusion</h3> Circulating miRNA works as a usful biomarker in diagnosis and prognosis of MM. Strigently, serum miR-27b is a novel circulating miRNA involved in T cell senescence of multiple myeloma patients which indicated the poor outcome of patients with MM.

Preclinical Evaluation of MEDI2228, a BCMA-Targeting Pyrrolobenzodiazepine-Linked Antibody Drug Conjugate for the Treatment of Multiple Myeloma

Despite remarkable progress in treatment over the past two decades, multiple myeloma (MM) remains an incurable disease. B-cell maturation antigen (BCMA, TNFRSF17) is a suitable therapeutic target for the treatment of MM due to its restricted expression on normal plasma cells and universal expression in myeloma cells. Targeting BCMA with an antibody-drug conjugate (ADC) is an attractive therapeutic option, combining the specificity of a monoclonal antibody with a potent cytotoxic drug to preferentially eliminate antigen-positive cells for the treatment of cancer.Here we describe MEDI2228, a novel ADC that targets BCMA. MEDI2228 is composed of a fully human antibody site-specifically conjugated to a DNA cross-linking pyrrolobenzodiazepine (PBD) dimer via a protease-cleavable linker. Flow cytometry studies show that MEDI2228 is rapidly internalized and trafficked to lysosomes. Upon release, the warhead binds to the minor-groove and cross-links DNA, leading to DNA damage and apoptotic cell death.The anti-tumor activity of MEDI2228 was assessed in vitro in 10 MM and plasma cell leukemia (PCL) cell lines encompassing several cytogenetic subtypes. MEDI2228 was highly active in 8 of 10 MM cell lines tested (IC50 range 6 to 210 ng/mL) including cell lines with high (~19,000 receptors/cell) or low (~930 receptors/cell) expression of BCMA. In addition, MEDI2228 is active in the presence of bone marrow stromal cells, which have been shown to play a role in chemotherapy resistance, and in cell models resistant to Lenalidomide. In vivo antitumor activity was evaluated in 4 separate MM and PCL subcutaneous xenograft models and 2 PCL disseminated xenograft models, where a single injection of MEDI2228 induced tumor regression at doses as low as 0.1 mg/kg.A soluble form of BCMA (sBCMA) has been detected in the serum of MM patients (Sanchez et. al. Br J Haematol 2012) and is comprised of the entire extracellular domain of the molecule (Laurent et. al. Nat Commun 2015). Therefore, sBCMA could diminish the effects of antibody-based therapies. We found that levels of sBCMA are elevated in the sera of MM patients compared with healthy donor controls, with levels ranging from 23 ng/mL to 728 ng/mL. The functional features of sBCMA and recombinant monomeric human BCMA are similar (Laurent et. al. Nat Commun 2015). Therefore, to mitigate the potential effects of sBCMA on the efficacy of our drug, the antibody component of MEDI2228 was selected because it possessed weak binding to recombinant monomeric human BCMA and strong binding to membrane-bound BCMA. The in vitro cytotoxicity of MEDI2228 in the presence of clinically-relevant levels of sBCMA was evaluated using NCI-H929 and MM.1S cells. MEDI2228 was cytotoxic to both MM.1S and NCI-H929 in vitro, killing an average of 95% of tumor cells in the presence of sBCMA at levels up to 720 ng/mL with little impact on IC50. ADCs developed from antibodies that possessed a similar affinity between monomeric BCMA and membrane-bound BCMA exhibited a sBCMA-dose dependent drop in potency, with a 20-fold shift in IC50 in the presence of 720 ng/mL sBCMA.MM is a clonally heterogeneous disease, and previously it has been reported that a small population of CD19+CD138- clonogenic cells can be identified in the bone marrow (BM) of MM patients (Matsui et. al. Blood 2004). We used flow cytometric sorting of CD19 and CD138 populations and outgrowth in methocult to confirm that CD19+CD138- cells are the clonogenic cells in MM BM samples. Furthermore, we found that this population contains the same light chain restriction, genetic translocation, and aberrant phenotype as the MM plasma cells. These cells express BCMA and can be killed by MEDI2228 in methocult outgrowth assays.These data demonstrate that MEDI2228 exhibits potent antitumor activity in preclinical models of MM. Importantly, in vitro experiments suggest that this activity is maintained in the presence of sBCMA. MEDI2228, bearing a potent PBD payload, may effectively target both the bulk myeloma plasma cells as well as the more quiescent, CD19+CD138- clonogenic cells, which may offer an opportunity for more durable clinical response in this genetically heterogeneous disease. DisclosuresKinneer:MedImmune: Employment. Meekin:MedImmune: Employment. Varkey:MedImmune: Employment. Xiao:MedImmune: Employment; Bristol-Myers Squibb: Employment. Zhong:MedImmune: Employment. Breen:MedImmune: Employment. Hurt:MedImmune: Employment. Thomas:MedImmune: Employment. Flynn:MedImmune: Employment. Hynes:MedImmune: Employment. Bezabeh:Salubris Biotherapeutics Inc: Employment; MedImmune: Employment. Chen:MedImmune: Employment. Wetzel:MedImmune: Employment. Chen:MedImmune: Employment. Anderson:Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; C4 Therapeutics: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Herbst:MedImmune: Employment. Tice:MedImmune: Employment.

A CK1δ/CK1ε-to-β-Catenin Circuit Is a Therapeutic Vulnerability for Primary and Drug Resistant Multiple Myeloma

Multiple Myeloma (MM) remains an incurable malignancy, despite the advent of several new therapeutic agents, including immunomodulatory drugs (IMiDs, e.g., Lenalidomide (Len)) and proteasome inhibitors (PIs, e.g ., Bortezomib (Btz)). Accordingly, there is an urgent need to identify new targetable vulnerabilities for MM patients. We developed an ex vivo 384-well assay that allows one to define drug sensitivities of primary patient CD138+ MM cells in the context of a reconstructed tumor microenvironment (TME), including allogeneic BMSC, extracellular matrix and MM patient serum. Using this platform and activity-based proteomic profiling (ABPP), we identified shared signaling pathways induced by the interactions of MM with the TME and integrated these data with screens performed using a bank of protein kinase inhibitors (PKI) and current anti-MM therapeutics. These analyses revealed that the serine/threonine kinases casein kinase-1δ (CK1δ) and CK1ε are high priority targets for MM. In support of this data, a highly selective and potent dual inhibitor of CK1δ/CK1ε coined SR-3029 is the most potent PKI versus MM. Further, our studies revealed SR-3029 has potent activity in 75/86 primary patient MM specimens tested thus far, including quad and penta-refractory MM.Previous studies have shown that SR-3029 targets CK1δ in triple negative breast cancer (TNBC) and that the therapeutic response relied on disabling the Wnt/β-catenin signaling. Aberrant activation of the Wnt/β-catenin pathway has well established roles in the development and progression of cancer. Indeed, gain-of-function somatic mutations in β-catenin and upstream receptors such as LRP5, and loss-of-function mutations in components of the β-catenin destruction complex, including the scaffold proteins APC and Axin, are hallmarks of several tumor types.Our analyses of whole exome sequencing (WES) of MM patients revealed that ~40% of tumors have functional protein-altering mutations in the Wnt/β-catenin pathway, and that this is manifest in both treatment-naïve and relapsed MM. Additionally, RNA-seq and gene expression profiling analysis of over 1,300 MCC MM patients specimens have show that components of the Wnt/β-catenin signaling pathway are elevated in some subsets of MM. Further, we have shown that SR-3029 treatment or inducible knockdown of CK1δ or CK1ε rapidly compromises the growth and survival of MM cell lines. Importantly, the anti-MM activity of SR-3029 is augmented in MM cell lines with selected resistance to Btz and Len relative to parental myeloma cells. Further, treatment of MM cells with this kinase inhibitor leads to marked reductions in total β-catenin protein, decreases in β-catenin target genes (e.g., MYC, CCND1 and WNT3), and to marked changes in the expression of components of the Wnt/β-catenin signaling pathway. Finally, using the established 5TGM1/Kal-Ridge (C57B6/KaLwRijHsd) syngeneic mouse model of multiple myeloma, we show that tumors derived from 5TGM1 MM cells, which rapidly die following exposure to SR-3029 ex vivo, are also sensitive to CK1δ/CK1ε inhibition in vivo, where the SR-3029 treated cohort of animals demonstrated decreased tumor burden as assessed by IgG2b levels and imaging, and by significantly improved survival relative tovehicle treated recipients. Collectively, these findings demonstrate that SR-3029 has potent activity in both naïve and therapy resistant multiple myeloma and establish CK1δ and/or CK1ε as attractive targets for anti-myeloma therapy. DisclosuresSiqueira Silva:AbbVie: Research Funding. Teer:Interpares Biomedicine: Consultancy. Shain:Celgene Corporation: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Janssen: Consultancy; Takeda Pharmaceuticals: Consultancy.

Promoting effects of MiR-135b on human multiple myeloma cells via regulation of the Wnt/β-catenin/Versican signaling pathway

MicroRNA (MiR)-135b and its mediated Wnt/β-catenin signaling pathway are involved in human malignancies. However, their roles in multiple myeloma (MM) remained poorly understood. Our study aimed to uncover their roles in MM. MiR-135b and Versican expressions were measured using quantitative real-time polymerase chain reaction (qRT-PCR). MM cell proliferation, apoptosis, migration and invasion were detected by cell counting kit-8 (CCK-8) assay, flow cytometry, wound healing assay and transwell assay, respectively. Relative expression of Wnt/β-catenin signaling pathway-related protein was quantified by Western blot. MiR-135b was upregulated in the serum of MM patients, and miR-135b upregulation promoted MM cell proliferation, migration and invasion but suppressed apoptosis. Also, miR-135b upregulation promoted activation of Wnt/β-catenin signaling pathway. However, downregulation of miR-135b caused an opposite effect. After incubating cells with miR-135b inhibitor and Wnt/β-catenin signaling pathway agonist Lithium chloride (LiCl), which reversed the effects of downregulating miR-135b. Versican is the downstream effector of the Wnt/β-catenin signaling pathway, and its silencing reversed the effects of LiCl on MM cells. In conclusion, miR-135b and its mediated Wnt/β-catenin signaling pathway promoted proliferation, migration and invasion but suppressed apoptosis of MM cells through regulating Versican, providing a possible treatment for MM.

The Expression of CD40/CD40L in MM Patients and Its Significance to the Prognosis of MM Patients

To explore the expression of CD40/CD40L in multiple myeloma(MM) patients and its influence on prognosis. Thirty patients with MM treated in Cangzhou People's Hospital from May 2016 to June 2017 were selected and divided into MM group, then 30 healthy people with a physical examination in our hospital at the same time were selected as the normal group. The serum CD40/CD40L levels of the patients in the two groups was detected by flow cytometry, and its correlation with the lymphocyte population, pathological grade and prognostic significance of MM patients was anaysis. The expression of CD40 in serum of the patients in MM group was significantly higher than those in normal group (P<0.05). The expression of CD40L in serum of the patients in MM group showed no significant difference as compared with those in normal group (P>0.05). The levels of CD40 and CD40L in the patients before and after chemotherapy showed no difference(P>0.05). The levels of Ts and NK cells in the patients of MM group were lower than those in normal group (P<0.05). The proportion of total B lymphocytes, Th and Th/Ts cells between the two groups showed no significant difference (P>0.05). The CD40 level was correlated with the serum total B lymphocyte level of the patients in MM group (r=0.877, P=0.005). There was a correlation with CD40L and Th cells in the serum of MM patients (r=-0.783, P=0.035). The expression of serum CD40 in the patients at phase III-IV was higher than those of the patients at phase I-II, the levels of serum CD40L in MM patients at different periods showed no significant difference(P>0.05). The survival rate of MM patients with high CD40 expression was lower than that of MM patients with low CD40 expression (χ2=1.639, P=0.201). The high level CD40 was the main factor affecting the prognosis of MM patients (95%CI: 1.156-4.125). The increasing of CD40 level in MM patients is related to the pathological grade of the patients. Chemotherapy can reduce the level of CD40. The increasing of CD40 is an important factor for the poor prognosis of MM patients. CD40L level is not meaningful for MM treatment and prognosis.

A High Throughput Quantitative Seroproteomics Analysis of Multiple Myeloma Patients on Tagraxofusp Therapy Identifies Novel Cytokine-Assisted Mechanism of Action

Background We showed a tumor-promoting and immunosuppressive role of plasmacytoid dendritic cells (pDCs; CD123/IL-3Rα+) in multiple myeloma (MM) pathogenesis (Chauhan et al. Cancer Cell, 2009;Ray et al, Leukemia, 2018). Importantly, tagraxofusp, an FDA-approved (for patients with blastic plasmacytoid dendritic cell neoplasm) novel targeted therapy directed against CD123, triggers anti-MM activity by reducing the viability of MM-promoting pDCs. These observations led to a recently completed phase 1 clinical trial of tagraxofusp and pomalidomide/dexamethasone in relapsed and refractory MM patients (NCT02661022). The treatment regimen demonstrated preliminary safety and efficacy, with 5 of 9 heavily pretreated patients achieving durable partial response (PR) (ASH 2019). Here, we report the initial results of our correlative science studies using bone marrow (BM), peripheral blood (PB), and serum from the study cohort. Materials and Methods Tagraxofusp is a bioengineered therapeutic protein developed by fusing human IL-3 to the catalytic translocation domain of truncated diphtheria toxin (DT) via a Met-His linker (Stemline Therapeutics, NY). pDCs and patient MM cells were purified from BM/PB samples after informed consent, and quantified using FACS, as described (Ray et al, Leukemia, 2018). A novel high-throughput seroproteomics platform SOMAscan was utilized to analyze 1,310 protein analytes in serum samples from MM patients (n = 9). SOMAscan data were subjected to meta-analysis to generate heatmaps, followed by hierarchical cluster analysis. SOMAscan results were validated with ELISA using supernatants from MM patient pDCs cultured with or without tagraxofusp. Results Analysis of BM/PB samples from MM patients receiving tagraxofusp therapy showed a marked reduction in the frequency of viable pDCs [average 2% at screening vs 0.75% post-tagraxofusp; n = 6; p = 0.036]. pDCs isolated from tagraxofusp-treated patients showed decreased ability to trigger MM cell growth. Seroproteomics analysis of MM patient serum before and after tagraxofusp therapy showed alterations in the levels of 100 proteins [Median Fold Change in expression: 0.39 to 4.5; n = 6; 3 each; p &amp;lt; 0.05]. Importantly, tagraxofusp treatment reduced pDC-related soluble proteins including IFN-α (fold change: 0.8, treated vs untreated; p &amp;lt; 0.05). Our earlier study showed that pDC-MM interactions triggered secretion of IL-3, which in turn promotes both pDC survival and MM cell growth. Importantly, tagraxofusp decreased serum IL-3 (fold change 0.75, treated vs untreated; p &amp;lt; 0.05), consistent with tagraxofusp decreasing survival of tumor-promoting pDCs. Conclusions Our current correlative science studies validate target specificity of tagraxofusp against MM pDCs in relapsed and refractory MM patients enrolled in a phase 1 clinical trial and support further evaluation for this novel therapeutic to improve the clinical outcome of patients with MM. Further combination studies are planned. Disclosures Mo: Celgene/BMS: Membership on an entity's Board of Directors or advisory committees. Olguin:Stemline Therapeutics: Current Employment. Chen:Stemline Therapeutics: Current Employment. Brooks:Stemline: Current Employment. Mughal:Stemline: Current Employment. Richardson:Celgene/BMS, Oncopeptides, Takeda, Karyopharm: Research Funding. Chauhan:consultant to Stemline Therapeutics, Inc., and Equity owner in C4 Therapeutics.: Consultancy, Other: Equity owner in C4 Therapeutics.; Oncopeptide AB: Consultancy. Anderson:Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees.