Expression In Multiple Myeloma Research Articles

T Cell Ig and ITIM Domains (TIGIT) Expression in Multiple Myeloma

Background Neoplastic myeloma cells show a wide variety of mechanisms that induce the microenvironment, allowing immune evasion and promoting their proliferation, survival, migration and drug resistance. In this context, immunocheckpoints are particularly important, representing the modulators of signal pathways responsible for immune tolerance, a mechanism that prevents the destruction of cancer cells by the immune system. T cell Ig and ITIM domains (TIGIT), a member of the poliovirus (PVR) /nectin receptor family, is a new immune checkpoint that negatively regulates T cell functions. In Multiple Myeloma (MM), TIGIT could play a relevant role leading to immunological escape. TIGIT performs its task mainly through binding ligands: CD155 and CD112, through which it inhibits the cytotoxicity of CD8+ T cells, as well as that of NK. In This Study, we evaluate the presence of TIGIT in bone marrow using instant digital pathology. AIMS Aims of this study are to develop a valid and reproducible workflow for instant digital pathology, to demonstrate the presence of TIGIT-positive T lymphocytes in bone marrow of patients with MM and to evaluate its correlation with clinical features PATIENTS and Methods 18 patients with newly diagnosed MM not previously treated, were evaluated in this study. The sample was enrolled from November 2022 to May 2023. Bone marrow blood was loaded on a porous matrix able to retain cells (Cytomatrix, UCS Diagnostics srl, Rome) for real time assessment of sample adequacy, morphological details preservation, and composition of hematopoietic subpopulations, using an ex-vivo fluorescent confocal microscope (FCM). Expert pathologists compared the digital FCM images with permanent Formalin Fixed Paraffin Embedded (FFPE) sections of the matrix and trephine bone marrow biopsy. Immunohistochemistry for TIGIT (TG-1 Clone, OncoDianova) was performed on matrix FFPE sections. RESULTS Our results showed that 15/17 (88%) patients resulted positive for TIGIT and 2/17 (12%) were negative. TIGIT expression was found mainly on CD8+ T lymphocytes, TIGIT positivity was correlated with parameters that define the aggressiveness of myeloma disease such as PC > 60% or FLC > 100, ISS score, R-ISS score, cytogenetic risk and LDH > 220mU/ml. Among the patients defined as “ultra-High Risk”, 9/10 (90%) resulted positive for TIGIT. Comparing TIGIT with the MM International Staging System (ISS), we observed that all patients with scores II and III were positive for TIGIT (100%) while 78% of them with ISS I results positive. Conclusions Our results suggest that FCM can be applied immediate evaluation of bone marrow aspirates, preserving the cellular sample for final histological analysis. “Furthermore, our data shows the positivity of TIGIT in 88% of patients, demonstrating the presence of this immune checkpoint in the bone marrow microenvironment. These data will need to be confirmed in a larger group of patients, but they open up the possibility of new target treatments in multiple myeloma

Preclinical In Vivo Imaging of Multiple Myeloma with [ 18f]Fluorohomoleucine (FHL) PET/CT

Purpose: Imaging of patients with multiple myeloma (MM) remains challenging due to its multi-focal nature affecting bone marrow, bone structures and soft tissue. As a result, current diagnostic methods like X-rays, CT and MRI scans only provide a partial understanding of disease activity and treatment response, lacking a comprehensive overview. Although 2-deoxy-2-[ 18F]fluoro-D-glucose (FDG) positron emission tomography (PET) has become the standard for detecting multifocal cancer spread, its use in MM patients is limited due to metabolic heterogeneity of MM and limited sensitivity in detecting extramedullary disease leading to false negative and false positive scans. To overcome these limitations, we developed a novel radiopharmaceutical L-[ 18F]Fluorohomoleucine (FHL) targeting LAT1 amino acid transporter. LAT1 has been significantly correlated with the proliferative activity of MM cells, poor prognosis and response to treatment in MM patients, presenting an appealing molecular target for assessing disease activity. Methods: FHL was synthesized as described by Nodwell et al. (J Nucl Med, 2019) and FDG was obtained from BC Cancer. LAT1 expression in multiple myeloma (MM) cell lines was confirmed by flow cytometry. Cell uptake assays were conducted using luciferase-transfected MM1S, L363, NCI H929 and AMO1 MM cells, and 2-amino-bicyclo[2.2.1]heptanes-2-carboxylic acid (BCH) was used as a blocking agent. For in vivo studies, male NRG mice were injected either with 1 x 10 6 MM cells intravenously (i.v.) via the caudal vein or subcutaneously (SQ) with 1 x 10 7 cells. Tumor engraftment was monitored weekly on an in vivo bioluminescence imager Perkin Elmer's IVIS Ilumina 5, starting at 2 weeks post-injection (p.i.). Biodistribution and PET/CT imaging studies were conducted at 4-6 weeks p.i. Results: All MM cells tested exhibited LAT1-specific FHL uptake in vitro with L363 and NCI H929 demonstrating the highest tracer uptake and average blocking efficacy over 83% and 93%, respectively. We were able to observe weekly progression of disease in the i.v. cohort using IVIS imaging. Biodistribution and PET imaging studies revealed FHL accumulation in the SQ xenografts, pancreas and excretion via the urinary tract. The SQ tumor uptake values were 4.53±0.82 %ID/g in AMO1 xenografts, 5.55±0.82 %ID/g in NCI H929, and 6.37±0.70 %ID/g in L363 tumors. In the i.v. cohort, the mouse intravenously injected with L363 cells demonstrated accumulation of radioactivity in the skull and vertebrae, as anticipated from the IVIS images. Furthermore, preliminary data suggests that FHL was able to detect a more extensive bone and lymphatic involvement than FDG in MM xenografts. Conclusion: We have demonstrated the ability of FHL for in vivo imaging of three human multiple myeloma tumors in SQ murine models, with the tumor uptake ranging from 4.53±0.82 %ID/g in AMO1 xenografts to 6.37±0.70 %ID/g in L363 tumors. Work is underway to establish the utility of this tracer in detecting disease dissemination in the orthotopic murine models of multiple myeloma, with promising preliminary results presented herein.

High prevalence of Wilms tumor 1 expression in multiple myeloma and plasmacytoma: A cohort of 142 Asian patients' samples.

Wilms tumor 1 (WT1) is a promising target antigen for cancer immunotherapy. However, WT1 protein expression and its clinical correlation in multiple myeloma (MM) patients are still limited. We, therefore, investigated WT1 expression in 142 bone marrow and plasmacytoma samples of MM patients at different stages of the disease by immunohistochemistry. The correlations between WT1 expression and clinical parameters or treatment outcomes were evaluated. The overall positive rate of WT1 expression was 91.5%; this high prevalence was found in both bone marrow and plasmacytoma samples, regardless of the disease status. Cytoplasmic WT1 expression was correlated with high serum free light chain ratio at presentation. However, no significant association between WT1 expression and treatment outcome was observed. This study confirms the high prevalence of WT1 expression in an Asian cohort of MM, encouraging the development of immunotherapy targeting WT1 in MM patients, particularly in those with extramedullary plasmacytoma or relapsed disease.

Tasquinimod suppresses tumor cell growth and bone resorption by targeting immunosuppressive myeloid cells and inhibiting c-MYC expression in multiple myeloma

BackgroundImmunotherapy emerged as a promising treatment option for multiple myeloma (MM) patients. However, therapeutic efficacy can be hampered by the presence of an immunosuppressive bone marrow microenvironment including myeloid cells....

The Landscape of Small Nucleolar RNA Expression in Multiple Myeloma

Background Small nucleolar RNAs (snoRNAs) are non-coding RNAs involved in the maturation of ribosome RNAs and various other biological processes. Emerging evidence demonstrates that snoRNAs play an essential role in tumor initiation and progression. However, their role in multiple myeloma (MM) pathogenesis remains poorly characterized. To date, the profiling of small RNAs in MM has mainly focused on microRNAs and approaches are confined to PCR- or array-based techniques. Here, we used next generation sequencing (NGS) to explore the snoRNA expression patterns of MM patients and to identify the association between snoRNAs and mRNAs, molecular subgroups of MM and clinical parameters. Methods Purified CD138+ cells from 71 newly diagnosed MMs and 4 refractory/relapsed MMs were included. Three CD19+ B cell samples from healthy donors were used as control. Total RNA was isolated with the AllPrep DNA/RNA kit (Qiagen, Germany). The mRNA and snoRNA NGS libraries were prepared using the Illumina TruSeq stranded mRNA and the NEBNext multiplex small RNA library kit, respectively. Sequencing of mRNA and snoRNA was performed on an Illumina NextSeq and NovaSeq 6000 PE 100 S1 platform, respectively. Paired-end RNA-seq reads were mapped to the hg19 reference genome using STAR. Gene expressions were quantified using featureCounts. Differentially expressed mRNAs and snoRNAs were identified using DESeq2. The Kaplan-Meier method was used for survival analyses. Progression-free survival (PFS) time was measured from enrollment to relapse or death from any cause, whichever occurred first. Results Using NGS, we observed expression of 625 full-length snoRNAs. Compared to normal B cells high levels of snoRNA expression were detected in MM plasma cells. Looking for associations between snoRNA expression and molecular subgroups of MM, we found an upregulation of 50 snoRNAs in patients with a hyperdiploid karyotype. The pattern of upregulation supported a regulatory mechanism in this molecular subgroup that goes beyond a pure copy-number effect. Even in patients with multiple trisomies the overwhelming majority (82%) of upregulated snoRNAs were located on chromosome 15, including the SNORD115 family on 15q11. In patients with gain of 1q21, all snoRNAs located on 1q25 were upregulated and the highest expression level of those snoRNAs appeared in patients with amp1q, indicating a copy-number effect. SNORD78, which was recently shown to impact outcome in prostate cancer and lung cancer, was significantly overexpressed in gain1q patients and defined a subgroup with inferior PFS (p =0.019) and overall survival (p = 0.011). SCARNA22, which was upregulated in patients with a t(4;14), was another snoRNA with a negative impact on PFS (31.06 months vs unreached, p = 0.007). To further characterize MM with upregulation of prognostically relevant snoRNAs, we analyzed bulk RNAseq of CD138-enriched MM cells and performed a pathway enrichment analysis. Comparing SNORD78 high expression group with the low group, we found 537 upregulated genes, e.g.SH2D2A, and CDK1, and 229 downregulated genes. Differentially expressed genes were enriched for cell cycle and chromosome segregation. For SCARNA22, we found 963 up- and 219 down-regulated genes. These showed enrichment for cell adhesion (upregulated in SCARNA22high) and protein targeting to the membrane or the endoplasmic reticulum (downregulated in SCARNA22high). Conclusion In this study we provide first insights into the expression patterns of snoRNAs in MM by deep sequencing. We identified snoRNAs dysregulated in MM subgroups, with two of them being associated with poor outcome. This study also shows that expression of particular snoRNAs is associated with specific chromosome gains, which is a common element in MM. Disclosures No relevant conflicts of interest to declare.

Bioinformatics Analysis of the Key Genes and Pathways in Multiple Myeloma.

ObjectiveTo study the differentially expressed genes between multiple myeloma and healthy whole blood samples by bioinformatics analysis, find out the key genes involved in the occurrence, development and prognosis of multiple myeloma, and analyze and predict their functions.MethodsThe gene chip data GSE146649 was downloaded from the GEO expression database. The gene chip data GSE146649 was analyzed by R language to obtain the genes with different expression in multiple myeloma and healthy samples, and the cluster analysis heat map was constructed. At the same time, the protein-protein interaction (PPI) networks of these DEGs were established by STRING and Cytoscape software. The gene co-expression module was constructed by weighted correlation network analysis (WGCNA). The hub genes were identified from key gene and central gene. TCGA database was used to analyze the expression of differentially expressed genes in patients with multiple myeloma. Finally, the expression level of TNFSF11 in whole blood samples from patients with multiple myeloma was analyzed by RT qPCR.ResultsWe identified four genes (TNFSF11, FGF2, SGMS2, IGFBP7) as hub genes of multiple myeloma. Then, TCGA database was used to analyze the survival of TNFSF11, FGF2, SGMS2 and IGFBP7 in patients with multiple myeloma. Finally, the expression level of TNFSF11 in whole blood samples from patients with multiple myeloma was analyzed by RT qPCR.ConclusionThe study suggests that TNFSF11, FGF2, SGMS2 and IGFBP7 are important research targets to explore the pathogenesis, diagnosis and treatment of multiple myeloma.

P-075: CD200 expression in multiple myeloma is regulated by P53 and exerts its function as immune checkpoint on T cells via DOK2

P-075: CD200 expression in multiple myeloma is regulated by P53 and exerts its function as immune checkpoint on T cells via DOK2

CD43 is an adverse prognostic factor in newly diagnosed multiple myeloma

Currently, the expression pattern and prognostic value of CD43 expression in multiple myeloma (MM) remain unknown. 109 newly diagnosed MM patients were recruited and CD43 expression was determined by multiparameter flow cytometry, of which 77 (70.6%) were CD43 positive. Patients with positive CD43 expression were more likely to present with, hemoglobin < 85 g/L (p = 0.008), International Staging System (ISS) stage III (p = 0.044), 13q14 deletion (p = 0.034) and more monoclonal plasma cells (p = 0.003). Patients with CD43 positive had significantly poor treatment response (p = 0.021), progression-free survival (PFS) (p = 0.012), and overall survival (OS) (p = 0.023) than those without CD43. The poorer prognosis of CD43-positive patients was retained in multivariate analysis (p = 0.005 for PFS; p = 0.013 for OS). Our study indicated that CD43 was an independent adverse prognostic factor in multiple myeloma.

Abstract 5352: Genome-wide CRISPR-cas9 screening identifies KDM6A as a modulator of CD38 expression in multiple myeloma: Therapeutic implications

Abstract Daratumumab (Dara) is the first-in-class monoclonal antibody targeting CD38 which triggers both direct and immune mediated cytotoxicity in preclinical in vitro and in vivo models of multiple myeloma (MM) in the bone marrow (BM) milieu. Importantly, Dara combination therapies can achieve high frequency and extent of response in both relapsed/refractory and newly diagnosed MM. However, relapse of MM is commonly observed due, at least in part, to downregulation of CD38 on MM cells. Here we utilize genome-wide CRISPR-cas9 knockout (KO) screening to identify novel regulators mediating CD38 expression and Dara sensitivity in MM cells. We identified 46 different sgRNAs to be positively correlated with MM cytotoxicity triggered by Dara and NK cells treatment. We further performed second round screening using the cell population with lowest 5% CD38 expression after CRISPR lentiviral library transduction. Importantly, KDM6A was the top ranked of the overlapping genes from these two screenings. Next, we individually KO of KDM6A in MM cells and found that both mRNA and protein levels of CD38 were significantly decreased, as well as the cell surface CD38 expression detected by flow cytometry. Re-introduction of KDM6A into KDM6A KO cells restored the expression level of CD38 and Dara-induced cytotoxicity, indicating that KDM6A positively regulates CD38 expression in MM cells. Since KDM6A regulates demethylation of lysine 27 at histone H3 (H3K27) to activate gene expression, we next examined whether CD38 expression was altered by methylation status of H3K27. From our ChIP-seq and ChIP Q-PCR data, we identified that H3K27me3 level was highly enriched on the promotor area of CD38 in KDM6A KO cells compared to control cells. These data indicate that KDM6A modulates CD38 expression by regulating H3K27me3 level on the CD38 promotor, thereby mediating sensitivity of MM cells to Dara-induced cytotoxicity. H3K27me3 is regulated by the balance of demethylase (KDM6A) and methyltransferase (EZH2). However, downregulating H3K27me3 on the CD38 promoter by enhancing KDM6A activity is challenging. Therefore, we next explored whether EZH2 inhibitor can restore CD38 expression and overcome Dara resistance in KDM6A KO cells. Indeed, H3K27me3 level on the CD38 promotor area was significantly decreased by EZH2 inhibitor. Moreover, EZH2 inhibitor treatment upregulated both CD38 mRNA and protein levels in KDM6A KO cells. Importantly, EZH2 inhibitor also restored sensitivity of KDM6A KO cells to Dara-mediated NK cell cytotoxicity. Taken together, our studies demonstrate that KDM6A regulates H3K27me3 level on CD38 promotor area and CD38 expression in MM. Moreover, we validate that combination treatment with EZH2 inhibitor and Dara can overcome Dara resistance in preclinical MM models, providing the rationale for combination clinical trials to overcome Dara resistance and improve patient outcome. Citation Format: Jiye Liu, Lijie Xing, Teru Hideshima, Kenneth Carl Anderson. Genome-wide CRISPR-cas9 screening identifies KDM6A as a modulator of CD38 expression in multiple myeloma: Therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5352.

FTO promotes Bortezomib resistance via m6A-dependent destabilization of SOD2 expression in multiple myeloma.

Drug resistance is the main reason for the failure of Bortezomib (Bort) in the treatment of multiple myeloma (MM), which seriously affects the efficacy of Bort. Therefore, the exploration of Bort resistance to treat MM will be very beneficial. Thus, this study aims to study the function and mechanism of Fat mass and obesity associated (FTO) on the Bort resistance of MM. In the present study, we demonstrated that FTO promotes Bort resistance via m6A-dependent destabilization of SOD2 expression in MM. These findings may provide a substantial evidence for the Bort resistance in MM.

Evaluation of the Prognostic Significance of Cereblon Protein Expression in Multiple Myeloma.

Considerable progress has been made in the treatment of multiple myeloma (MM) patients with the development of various new agents that increased survival rates over the past fifteen years. Cereblon (CRBN) plays an important role in mediating the antitumor effects of immunomodulatory drugs (IMiDs) among these new agents. The aim of our study is to investigate immunohistochemically (IHC) cereblon protein expression status in MM. Immunohistochemically, CRBN expression and its relationship with various prognostic factors were evaluated in bone marrow biopsies of 96 patients with MM in a single centre. Cytoplasmic and nuclear CRBN expression was detected in all neoplastic cells. While a complete or partial response to treatment was obtained in 45 patients, the disease was stable in 13 and progressive in 17 patients. Survival was longer in those treated with IMiD-containing regimens (p = 0.044). Both the survival rate (p = 0.013) and the survival time were significantly increased (p = 0.023) in those who received the treatment protocol containing protease inhibitors. A significant relationship was found between the treatment protocol and treatment response in the chi-squared analysis (p = 0.008). Although the longest survival time - though not statistically significant - was detected in the group treated with protease inhibitors (log rank, p = 0.217). The survival analysis revealed the presence of a relationship between IgG and IgA positivity and survival. In this study, the survival time of the patients who received treatment regimens containing protease inhibitors and IMiD was longer, independent of the presence of strong nuclear CRBN expression. The survival rate was significantly higher in those who used IMiD and protease inhibitors in combination. Since the survival rate was found to be increased in IgG positive cases and we thought that evaluation of immunoglobulin tissue expression in MM cases can provide prognostic prediction.

Annotating CD38 Expression in Multiple Myeloma with [18F]F-Nb1053.

Noninvasive diagnosis of multiple myeloma (MM) is a clinical challenge. CD38 is an established biomarker for MM, and the development of CD38-targeted radiotracers may improve the management of MM. By taking the advantages of bioorthogonal click chemistry, a nanobody (i.e., Nb1053-LLQS) specific for CD38 was successfully labeled with 18F. The diagnostic efficacy and specificity of the developed tracer (i.e., [18F]F-Nb1053) were evaluated by immuno-positron emission tomography (immunoPET) imaging in disseminated MM.1S-bearing models. [18F]F-Nb1053 was developed with high radiochemical purity (>98%) and excellent immunoreactivity. [18F]F-Nb1053 immunoPET successfully delineated disseminated MM lesions in preclinical MM models. The uptake in the humerus, femur, and tibia was 1.42 ± 0.50%ID/g, 1.35 ± 0.53%ID/g, and 1.48 ± 0.67%ID/g (n = 6), respectively. Tumor uptake of [18F]F-Nb1053 decreased after daratumumab premedication, indicating the superior specificity of the reported probe. This work successfully developed a novel CD38-specific probe [18F]F-Nb1053 that may potentially optimize the management of MM upon clinical translation.

CD43 Expression Is an Adverse Prognostic Factor in Newly Diagnosed Multiple Myeloma

CD43 expression is an adverse prognostic factor in newly diagnosed multiple myelomaIntroductionCD43 is an abundant, heavily-glycosylated, cell surface protein expressed on bone marrow hematopoietic stem cells and most white blood cells. Previous studies have found that CD43 also expressed in some types of lymphoma cells and associated with adverse outcomes. However, the prognosis value of CD43 expression in multiple myeloma (MM) remain unknown.Patients and MethodsA total of 109 MM patients, newly diagnosed in Nanfang Hospital of Southern Medical University from January 2017 to December 2019, were included in the study. CD43 was detected by flow cytometre as follows: 2 ml of heparin anticoagulated bone marrow was collected from the patients at the time of diagnosis，1×10 6 cells were detected, and a gate was set for identifying abnormal plasma cells characterized by CD138 expression and CD38. CD43 positive was defined as more than 20% of the plasma cells expressed CD43.ResultsA total of 109 patients with newly diagnosed MM were enrolled in the study, including 77 patients (70.6%) for CD43 positive and 32 patients (29.4%) for CD43 negative . The median age was 58 years, and the male-female ratio was 1.7:1. Patients in the CD43 positive group were more likely to have international staging system (ISS) stage III (67.5% VS 46.9%, P= 0.044), hemoglobin < 85g/L (64.9% VS 37.5%, P= 0.008), 13q deletion (31.4% VS 10.4%), and higher percentage of bone marrow monoclonal plasma cells detected by flow cytometry (5.5% VS 1.4%, P=0.003).Most patients enrolled in the study received bortezomib-based treatment. The very good partial response (VGPR) or better rate after 4 induction cycles was significantly lower in the CD43 positive group than CD43 negative group (35.1% VS 56.3%, P=0.041), and the overall response rate (ORR) in the CD43 positive group was lower than that in CD43 negative group (75.3% VS 84.4%, P=0.299), but no significantly difference.The median follow-up time was 22 months. Patients with CD43 positive had significantly lower PFS (median PFS 24 months VS not reached, P =0.012), and OS (median OS not reached, P = 0.023) than those with CD43 negative. Multivariate analysis indicated that CD43 positive expression was an independent poor risk factor for PFS (HR 2.517 95%CI 1.178-5.376, P = 0.017) and OS (HR 3.664 95%CI 1.100-12.075, P = 0.034).ConclusionOur study showed that CD43 expression was an adverse prognosis for multiple myeloma. DisclosuresNo relevant conflicts of interest to declare.

Cdc37 Expression in Multiple Myeloma and Its Role in Cell Proliferation

To investigate the expression of cell division cycle protein 37 (Cdc37) in multiple myeloma (MM) and its effect on MM cell proliferation. The expression of Cdc37 mRNA in CD138+ cells derived from 63 newly diagnosed MM patients and 8 healthy people were detected by real-time quantitative PCR (RT-qPCR). Cdc37 was down-regulated by lentivirus in MM cell line NCI-H929. CCK-8 assay and soft agar clone formation assay were conducted to explore the role of Cdc37 on MM proliferation in vitro. To further verify the effect of Cdc37 on MM cell proliferation in vivo, NOD/SCID mice subcutaneous tumorigenesis model was established. Flow cytometry was carried out to explore the role of Cdc37 on cell cycle. Cell cycle associated proteins and NF-κB pathway were detected by Western blot (WB). Cdc37 was highly expressed in newly diagnosed CD138+cells compared with healthy people. After Cdc37 suppression by shRNA lentivirus infection in NCI-H929 cells, the proliferation of MM cells were decreased in vitro and in vivo. Compared with the control group, the ratio of cells arrested in G0/G1 phase significantly increased in NCI-H929-Cdc37 shRNA cells, the expression of cyclin D1 decreased, while the expression of p21 and p53 was significantly up-regulated. Meanwhile, the activation of NF-κB signaling pathway was hampered in NCI-H929-Cdc37 shRNA cells. Cdc37 is highly expressed in newly diagnosed MM patients. Inhibition of Cdc37 results in decreased proliferation activity and G0/G1 arrest in NCI-H929 cells. The possible mechanism may be to inhibit the activation of NF-κB signaling pathway.

PHD finger protein 19 expression in multiple myeloma: Association with clinical features, induction therapy outcome, disease progression, and survival.

BackgroundPHD finger protein 19 (PHF19), also known as polycomb‐like protein 3 (PCL3), promotes the progression of multiple myeloma (MM) and drug resistance; however, its role in the management of MM remains unclear. Therefore, we aimed to elucidate the correlation between PHF19 expression and treatment response, disease progression, and survival of patients with MM.MethodsPlasma cells derived from the bone marrow of 101 patients with de novo MM were collected prior to induction therapy, as were plasma cells derived from the bone marrow of 30 healthy donors. PHF19 expression in plasma cells was analyzed using quantitative reverse transcription polymerase chain reaction. Furthermore, the response to induction therapy, progression‐free survival (PFS), and overall survival (OS) were assessed.ResultsPHF19 expression tends to be upregulated more often in MM patients than in healthy donors (p < 0.001) and can accurately predict MM risk (area under curve [AUC], 0.916; 95% confidence interval [CI], 0.869–0.962). Furthermore, elevated PHF19 expression was correlated with higher International Staging System (ISS) (p = 0.036) and revised ISS stages (p = 0.035). In addition, MM patients who achieved complete response (CR) exhibited reduced PHF19 compared to those who did not (p = 0.028). Moreover, increased PHF19 expression was correlated with unfavorable PFS (p = 0.006) and OS (p = 0.027) rates. Furthermore, the results of multivariate Cox analysis also revealed that PHF19 high expression was independently associated with a reduced PFS rate (hazard ratio: 2.025, p = 0.028).ConclusionIncreased PHF19 expression is correlated with poor induction therapy response and unfavorable long‐term prognosis of MM.

CD56 expression in multiple myeloma: Correlation with poor prognostic markers but no effect on outcome

CD56 or neural cell adhesion molecule (NCAM) is a membrane glycoprotein expressed on neural cells, muscle tissues and myeloma cells. Expression of CD56 has been studied in patients with multiple myeloma (MM) with controversial results. The scope of this study was to examine the expression of CD56 in MM patients at diagnosis and investigate its association with clinicopathologic parameters. We retrospectively collected and analyzed data from 109 patients with MM diagnosed over the last decade (January 2010 to June 2020). Expression of CD56 was assessed by immunohistochemistry in bone marrow biopsies and investigated its association with a variety of clinicopathological parameters. For the statistical analysis χ2 test and Mann-Whitney U tests were used to compare categorical and continuous variables in CD56+ and CD56- patients, respectively. Statistical analysis was performed using SPSS 21.0 for Windows (SPSS, Chicago, IL). Based on the expression of CD56 the patient population was divided to CD56+ patients and CD56- patients; Sixty-eight patients were CD56 + and 41 patients were CD56–. Absence of CD56 expression was associated with unfavorable prognostic parameters such as elevated lactate dehydrogenase (LDH) and β2-microglobulin levels, advanced stage according to the International Staging System (ISS) and clonal bone marrow plasma cell infiltration ≥ 60%, but no effect on outcome, while the expression of CD56 was associated with well differentiated neoplastic plasma cells. Our study confirmed that lack of CD56 expression is a possible marker of poor prognosis in patients with MM. The detection of CD56 expression by either immunohistochemistry or flow cytometry is simple and cheap, and it could be incorporated in future prognostic or predictive scores. Prospective studies are needed in order to evaluate the role of expression of CD56 as a predictive biomarker in the era of novel regimens.

MAF and NF-κB Signaling Pathways Regulate Expression of APOBEC3B in Multiple Myeloma

IntroductionAPOBEC mutagenesis is a source of mutations in ~50% of human cancers, resulting from the over-expression of APOBEC proteins, including APOBEC3B (A3B). High expression of A3B is associated with poor prognosis and drug resistance in many cancers. In multiple myeloma, the chromosomal translocation t(14;16) leads to the over-expression of the transcription factor MAF through juxtaposition with the IGH super-enhancer. We have shown that in this poor prognosis sub-group A3B is also over-expressed and there is an enrichment for APOBEC signature mutations and a higher mutational load. It is not clear whether over-expression of A3B results from direct activation by MAF or indirectly through downstream pathways. In other cancers, mitosis, cell-cycle related gene expression, and the PKC/NF-κB signaling pathway may also affect expression of A3B.MethodsGene expression profile (GEP, U133 Plus 2.0) of 50 human myeloma cell lines (HMCLs) and 1310 myeloma patients was performed. mRNA levels of MAF and the AID /APOBEC family was investigated by qPCR in HMCLs. Western blotting and a DNA cytosine deaminase activity assay were used to detect A3B protein and analyze A3B enzymatic activity which results in mutagenesis, respectively. The effect of MAF on expression of A3B was investigated in inducible TRIPZ lentiviral shRNA c-MAF knockdown in HMCLs with high expression of MAF. Bortezomib, a proteasome inhibitor, and phorbol 12-myristate 13-acetate (PMA), a PKC dependent activator of the NF-κB signaling pathway, was used to investigate A3B expression in TRIPZ lentiviral shRNA MAF (MF KO) and A3B (A3B KO) knockdown HMCLs. Localization of A3B in regular HMCLs and cell lines with over-expression of A3B (generated using ViraPower T-Rex lentiviral expression system) was investigated by immunofluorescent analysis (IFA) using laser confocal and high resolution microscopy.ResultsBy microarray, high levels (log2>10) of A3B mRNA were found in approximately half of multiple myeloma patients. The majority of t(14;16) MAF patients had high levels of A3B. Increased expression of A3B was also detected in 72% of HMCLs. In t(14;16), MAF, and t(14;20), MAFB, HMCLs there was a strong positive correlation between MAF and A3B mRNA expression (r2= 0.51 and r2=0.43, respectively). On the basis of levels of MAF expression two groups of HMCLs were selected. Group 1 with high expression of MAF (MF) with the presence of a t(14;16) ARP1, CAG, JJN3, OCMY5, MM1S, and RPMI8226. Group 2 had low expression of MAF (non-MF) and was represented by cell lines with t(11;14) - U266, XG1 and KMS12BM; t(4;14) - KMS28PE and H929; or t(14;20) SACHI. We found a strong positive correlation (r2=0.68) between MAF and A3B mRNA expression in MF cell lines and no such correlation in the non-MF group (r2=0.09). Upon down-regulation of MAF by shRNA, JJN3 and RPMI8226 showed reduced expression of A3B protein (-33% and -29%, respectively) by 72 hours with accompanying reduction in cytosine deaminase activity (-23% and -26%, respectively). There was a positive correlation between expression of MAF protein and A3B protein in JJN3 MAF KO r2= 0.21, in RPMI8226 MAF KO r2=0.50; and a positive correlation between MAF protein and DNA cytosine deaminase activity in JJN3 MAF KO r2=0.21 and in RPMI8226 MAF KO r2=0.34. Bortezomib reduced expression of MAF and A3B and DNA cytosine deaminase activity in JJN3 and RPMI8226 cell lines. PMA increased the expression of A3B mRNA in HMCLs within 6 hours of treatment and lasted for 7 days after treatment. The highest sensitivity of A3B to PMA was detected in JJN3 and U266 cell lines, with increases of 4.4 and 8.2-fold, respectively, after 6 hours of treatment. Protein levels of A3B were increased 2.4-fold in both cell lines but DNA cytosine deaminase activity was increased (27%) only in JJN3 cells after PMA treatment.ConclusionsKnock down of MAF results in decreased expression and activity of A3B, indicating that MAF is a regulator of A3B expression in multiple myeloma. Expression of A3B in t(14;16) cases is dependent upon MAF expression but can also be increased through activation of the PKC/NF-κB signaling pathway, resulting in increased DNA damage. Understanding the mechanisms responsible for mutagenesis in the t(14;16) subgroup through A3B, which results in new clone formation and drug resistance in multiple myeloma will be highly beneficial in designing new therapeutic strategies. DisclosuresDavies:Seattle Genetics: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Morgan:Bristol Myers: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria.

This Is a Title in Title Case: SDF1,CXCR4 and MVD Play Important Roles in Multiple Myeloma

The myeloid microenvironment of patients with multiple myeloma plays an important role in regulating the occurrence, development, invasion, secretion and angiogenesis of myeloma cells. Chemokines SDF-1 and its receptor CXCR4 form SDF1-CXCR4 biologicalaxis in bone marrow microenvironment. The role and mechanism of SDF1-CXCR4 biologicalaxis in multiple myeloma remain to be studied.We collected 144 cases of hematology bone marrow from January 2013 to June 2017 in Southwest Medical University Affiliated Hospital and the Third People's Hospital of Chengdu, of which 104 cases were multiple myeloma patients, and 40 cases were normal as control. The relationship between SDF1-CXCR4 expression and its clinical features were analyzed. Through these analysis we tried to learn the role of SDF1-CXCR4 in the development, progression and prognosis of multiple myeloma, and hoped to provide evidence for the diagnosis and treatment of multiple myeloma.As a result, the positive expression of SDF1 and CXCR4 in bone marrow biopsy of patients with multiple myeloma was significantly higher than that in the normal control group. More importantly, there was a positive correlation between the expression of SDF1 and CXCR4, indicating that SDF1-CXCR4 in bone marrow microenvironment plays an important role in the occurrence of multiple myeloma.The density of microvessel density (MVD) in the myeloma was also significantly higher than that in the normal control group. The expression of SDF1-CXCR4 in the myeloma group was positively correlated with the expression of MVD. This study demonstrates that multiple myeloma cell growth and microvascular proliferation are significantly correlated in microenvironment. It is suggested that the secretion of chemokine SDF1-CXCR4 in the bone marrow microenvironment of multiple myeloma may promote microvascular proliferation and the growth of tumor cells, which may be the pathogenesis.Multiple myeloma clinical features were analyzed according to SDF1-CXCR4 and MVD expression levels. We found that SDF1-CXCR4 and MVD expression levels were not correlated with gender, age and immunoglobulin typing, but were significantly correlated with multiple myeloma staging in bone marrow tissue of MM patients. But we found that in the later stage of SDF1, the expression level of SDF1、CXCR4 and MVD is higher. This finding further indicates that chemokine SDF1-CXCR4 secretion and microvascular proliferation play important roles in progression of multiple myeloma.The Kplan-Meier analysis on overall survival (OS) and progression-free survival (PFS) was performed according to the levels of SDF1-CXCR4 and MVD expression in multiple myeloma. We discovered that the OS rate and PFS rate were lower in the patients with higher expression of SDF1, CXCR4 and MVD , which indicates that chemokine SDF1-CXCR4 secretion and microvascular proliferation also play important roles in prognosis of multiple myeloma.Our findings reveal that chemokine SDF1-CXCR4 secretion and microvascular hyperplasia in bone marrow microenvironment are essential in the occurrence, development and prognosis of multiple myeloma. The higher secretion of chemokine SDF1-CXCR4 suggests the worse progression and poor prognosis. Chemokine SDF1-CXCR4 is expected to become a new target for MM treatment. DisclosuresNo relevant conflicts of interest to declare.

POS-267 HYPERTENSION IN PERITONEAL DIALYSIS Renal Expression in Multiple Myeloma: Experience in a Department of Internal Medicine

Kidney expression is common during multiple myeloma (MM) and affects the prognosis of the disease. The objective of our study is to describe the epidemiologic, clinical, biological, histologic, therapeutic, and progression aspects of renal involvement in MM in the internal medicine setting.

Aminopeptidase Expression in Multiple Myeloma Associates with Disease Progression and Sensitivity to Melflufen.

Simple SummaryThe aims of this study were to investigate aminopeptidase expression in multiple myeloma and to identify the aminopeptidases responsible for the activation of the peptide–drug conjugate melflufen in multiple myeloma. We observed a differential expression of aminopeptidases between relapsed/refractory and newly diagnosed multiple myeloma patients. A higher expression of the aminopeptidase genes XPNPEP1, RNPEP, DPP3, and BLMH in multiple myeloma plasma cells was associated with shorter patient overall survival. The peptide–drug conjugate melflufen was particularly active towards plasma cells from relapsed/refractory multiple myeloma patients. Melflufen could be hydrolyzed to its active form by the aminopeptidases LAP3, LTA4H, RNPEP, and ANPEP, all of which are expressed in multiple myeloma. These results indicate critical roles for aminopeptidases in disease progression and the activity of melflufen in multiple myeloma.Multiple myeloma (MM) is characterized by extensive immunoglobulin production leading to an excessive load on protein homeostasis in tumor cells. Aminopeptidases contribute to proteolysis by catalyzing the hydrolysis of amino acids from proteins or peptides and function downstream of the ubiquitin–proteasome pathway. Notably, aminopeptidases can be utilized in the delivery of antibody and peptide-conjugated drugs, such as melflufen, currently in clinical trials. We analyzed the expression of 39 aminopeptidase genes in MM samples from 122 patients treated at Finnish cancer centers and 892 patients from the CoMMpass database. Based on ranked abundance, LAP3, ERAP2, METAP2, TTP2, and DPP7 were highly expressed in MM. ERAP2, XPNPEP1, DPP3, RNPEP, and CTSV were differentially expressed between relapsed/refractory and newly diagnosed MM samples (p < 0.05). Sensitivity to melflufen was detected ex vivo in 11/15 MM patient samples, and high sensitivity was observed, especially in relapsed/refractory samples. Survival analysis revealed that high expression of XPNPEP1, RNPEP, DPP3, and BLMH (p < 0.05) was associated with shorter overall survival. Hydrolysis analysis demonstrated that melflufen is a substrate for aminopeptidases LAP3, LTA4H, RNPEP, and ANPEP. The sensitivity of MM cell lines to melflufen was reduced by aminopeptidase inhibitors. These results indicate critical roles of aminopeptidases in disease progression and the activity of melflufen in MM.