Poor Prognosis In Multiple Myeloma Research Articles

Correlation between Clinical Factors and Prognosis in Newly Diagnosed Multiple Myeloma.

To explore the correlation between clinical factors and the prognosis of multiple myeloma (MM). A cohort study. At the Third Affiliated Hospital of Inner Mongolia Medical University, Mongolia from January 2014 to December 2018. The clinical data of 81 patients with newly diagnosed MM were collected retrospectively. The correlation of prognosis with immunophenotype and (FISH) Fluorescence in situ hybridization was subjected to univariate and multivariate analyses. Overall and progression-free survival was determined. The overall survival (OS) and progression-free survival (PFS) of patients with CD200+, CD81+, and CD27- were significantly shortened. CD200+ and CD27- were independent prognostic factors for OS and PFS in MM patients. The prognosis-related abnormal genes were analyzed, and univariate analysis revealed that OS and PFS were significantly shortened in patients with RB-1 deletion, CDKN2C deletion, and IGH rearrangement. CDKN2C deletion was an independent prognostic factor for OS and PFS in MM patients. CDKN2C deletion is an independent prognostic factor of MM. CD200+ is an independent risk factor for poor prognosis of MM. Key Words: Multiple myeloma, Prognostic analysis, Fluorescence in situ hybridization, Immunity, Immunophenotype.

Dkk1 Controls Cell-Cell Interaction through Regulation of Non-nuclear β-Catenin Pools.

SummaryDickkopf-1 (Dkk1) is a secreted Wnt antagonist with a well-established role in head induction during development. Numerous studies have emerged implicating Dkk1 in various malignancies and neurodegenerative diseases through an unknown mechanism. Using zebrafish gastrulation as a model for collective cell migration, we unveil such a mechanism, identifying a role for Dkk1 in control of cell connectivity and polarity in vivo, independent of its known function. We find that Dkk1 localizes to adhesion complexes at the plasma membrane and regions of concentrated actomyosin, suggesting a direct involvement in regulation of local cell adhesion. Our results show that Dkk1 represses cell polarization and integrity of cell-cell adhesion, independently of its impact on β-catenin protein degradation. Concurrently, Dkk1 prevents nuclear localization of β-catenin by restricting its distribution to a discrete submembrane pool. We propose that redistribution of cytosolic β-catenin by Dkk1 concomitantly drives repression of cell adhesion and inhibits β-catenin-dependent transcriptional output.

APOBEC3B Induction Following DNA Damage Response Modulates the Survival and Treatment Response in Human Multiple Myeloma

Apolipoprotein B mRNA editing catalytic polypeptide-like 3B (APOBEC3B, A3B) is one of 7-membered DNA cytosine deaminase family, causing cytosine-to-uracil (C-to-U) deamination in single-stranded DNA and promoting mutations in multiple human cancers including multiple myeloma (MM). High APOBEC3B expression is found in a significant portion of MM patients with MAF overexpression among t(14;16) and t(14;20). A3B upregulation is further associated with poor prognosis in MM, suggesting its role in the MM pathophysiology. However, approximately 23% MM patients with high APOBEC3 activity are associated with MAF/MAFA/MAFB translocations, the remainder of patients with high APOBEC3 carry neither translocations nor overexpression of these genes. Besides, studies are lacking on how A3B is regulated and the role of A3B in drug responses in MM. We here defined new mechanisms controlling A3B expression and further characterized its impact on treatment responses to current anti-MM therapies. Using qRT-PCR, A3B transcript is significantly higher than other members of the APOBEC3 gene family in MM cell lines (n=19) and MM patients, indicating that A3B may play a major role in MM. Using immunoblotting analysis, A3B protein expression was further confirmed in MM cell lines with various levels (n=10). Importantly, A3B mRNA upregulation by 1.34-42.64 folds was observed in CD138-purified cells from majorities of MM patients (83.3%) when compared to PBMC from the same individual (n=12). In MM cell lines without MAF/MAFA/MAFB translocation as a study model, higher A3B protein expression is associated with higher DNA damage levels as evidenced by higher γ-H2AX. These results suggest that A3B expression might be influenced by DNA damage levels in MM cells. Following a short time treatment of gamma-irradiation to cause DNA damages, A3B expression in viable MM cells was enhanced in a dose-dependent manner. We next treated MM cells (n=5) with common anti-MM drugs such as Melphalan (Mel) and Bortizomib (btz), both of which induce DNA damages, followed by examination of changes in A3B and γ-H2AX. Under sublethal treatment conditions of Mel or btz, A3B was consistently induced at both mRNA and protein levels in multiple MM cell lines regardless of the baseline A3B expression. Significantly, A3B was upregulated and associated with increased γ-H2AX in patient MM cells treated with Mel or btz under sub-lethal doses. Since DNA damages activate the ATR/ATM pathway, we next investigated whether these kinases mediate A3B induction following treatments with these compounds in MM cells. The presence of ATM or ATR inhibitors blocked A3B upregulated by these DNA damage-inducing treatments in MM cell lines (n=3), indicating an ATM/ATR-dependent pathway for A3B changes. Next, gene-specific CRISPR knock out (KO) and inducible-shRNA knockdown (KD) were used to determine the functional impact of perturbation of A3B in proliferation and survival of MM cells. Both KO and KD of A3B decreased growth and viability of MM cell lines regardless of sensitive or resistant to dexamethasone or lenalidomide. Using LIVE/DEAD fixable Aqua Stain and annexin V-based flow cytometric analysis, A3B inhibition enhanced growth arrest followed by apoptosis in MM cells. Significantly, A3B KD by its shRNA in RPMI8226 MM cells enhanced sensitivity to pomalidomide. Taken together, these data indicate that increased A3B level plays a critical role in MM cell survival and drug responses. DNA damages triggered by IR, Mel, or btz further enhance A3B expression via ATM/ATR pathway, which in turn increases subclonal diversity leading to drug resistance. The role of A3B in disease pathophysiology and progression, coupled with its function in mediating treatment response, suggest potential utility of targeting A3B in MM. Disclosures Munshi: Celgene: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Takeda: Consultancy. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.

P53 Pathway Activation Mediated High c-MAF Expression Is Associated with Overall and Post-Progression Survival in Multiple Myeloma

Background: Chromosomal abnormalities are strongly associated with prognosis of multiple myeloma (MM). Among them, t (14;16) resulting in high expression of c-MAF, and t (14;20) resulting in high expression of MAFB, lead to poor prognosis. However, clinical significance and mechanisms underlying high c-MAF and MAFB expression without these translocations have not been fully elucidated. Methods: A total of 96 MM and 38 MGUS patients, 10 controls, and 9 MM cell lines were included in this study. RNA was extracted from purified CD138+ plasma cells from bone marrow (BM) mononuclear cells. c-MAF, MAFB, p53, and p21 RNA expressions were determined by RQ-PCR. Their expression levels were normalized against ACTB levels and calculated with 2-ΔΔCt value. Inhibition studies using BRD4 inhibitor JQ1, and MYC inhibitor10058-F4, and MM cell lines expressing doxycycline-inducible p53 (Tet-on p53) or c-MAF knockdown (KD) with siRNA were used for in vitro studies. Results: c-MAF expression in MM patients was higher than in MGUS patients and control (median level 0.043, 0.025, 0.002, p<0.001), but MAFB expression did not differ between MM, MGUS, and control (p=0.371). Although c-MAF expression level was significantly higher in MM with t (14;16) (p=0.0018), high c-MAF expression was observed in patients without the translocations, suggesting a role of c-MAF in MM progression. Other cytogenetic abnormalities such as del 17p, or t (4;14), did not affect c-MAF expression. Overall survival (OS) of MM patients with high c-MAF expression was significantly inferior compared to the patients with low c-MAF expression (HR 2.46, p=0.002, 2 years survival rate 42.9% vs. 72.7%, median 20.3 months vs. not reached), but progression-free survival (PFS) did not differ (p=0.551). Instead, post-progression survival (PPS) was significantly shorter in the patients with high c-MAF expression (HR 4.67, p<0.001, two years survival 0% vs. 60.2%, median 3.6 months vs. 49.3 months) suggesting that c-MAF confers drug resistance to residual disease. MAFB expression did not affect OS, PFS, and PPS. Reduction of cyclin D2 expression by c-MAF KD in vitro and a positive correlation between c-MAF and cyclin D2 expression (r=0.29, p<0.001) in vivo in patients, support previous reports describing that MM proliferation is induced by c-MAF via cyclin D2. c-MAF was not correlated with APOBEC3B expression. c-MAF expression was positively correlated with MDM2 and MYC (r=0.387, p=0.03 and r=0.221, p=0.019, respectively) but not with p53 and PUMA. A tendency of positive correlation was also observed with p21/CDK1NA (r=0.304, p=0.085). p53 overexpression from the Tet-on p53 and p53 stabilization by nutlin-3 increased c-MAF expression in three cell lines with t (14;16). c-MAF KD together with p53 overexpression significantly suppressed the proliferation, implying synthetic lethality. These findings suggest that c-MAF expression is upregulated in response to proliferation arrest induced by the p53 pathway; suppressing this responsive c-MAF expression abrogates cell growth more efficiently. JQ1 and CPI203 increased c-MAF expression in cell lines with t (14;16), suggesting that c-MAF expression is not controlled by super-enhancers. Conclusion: Although the mechanism of high expression of c-MAF and MAFB has previously been reported in IgH translocation, the relationship between c-MAF and drug resistance remains to be determined. The induction of c-MAF expression by tumor suppressor gene p53 is suggested to be a mechanism underlying poor prognosis of MM with t (14;16), and suppressing c-MAF expression with chemotherapeutic drugs might prevent emergence of drug resistance in residual tumor cells. Disclosures Handa: Ono: Research Funding.

Influence of Cytogenetics in Patients with Relapsed Refractory Multiple Myeloma Treated with Oral Selinexor and Dexamethasone: A Post-Hoc Analysis of the STORM Study

Influence of Cytogenetics in Patients with Relapsed Refractory Multiple Myeloma Treated with Oral Selinexor and Dexamethasone: A Post-Hoc Analysis of the STORM Study

Epstein-Barr virus infection is associated with clinical characteristics and poor prognosis of multiple myeloma.

The aim of the present study was to evaluate the relationship of Epstein–Barr virus (EBV) infection and multiple myeloma (MM) and its impact on clinical characteristics and prognosis. Fresh peripheral blood mononuclear cells (PBMCs) from 139 MM patients who had been diagnosed and treated from January 2010 to May 2018 and 50 PBMC samples from healthy donors were obtained. PCR was carried out for detection of EBV-DNA. The results indicated a significantly higher EBV-DNA concentration among 139 MM patients compared with healthy controls (P<0.05). Correlation analysis showed that the expression of EBV-DNA was positively correlated with the serum free light chain ratio (sFLCR) and progressive disease (PD)/relapse (P<0.05). Especially, in EBV-DNA high-expression MM patients, EBV-DNA concentration for patients with sFLCR ≥100 was higher than that of patients with sFLCR <100. EBV-DNA concentration was higher in patients with disease PD/relapse than those without disease PD/relapse. In univariate analysis, the progress free survival (PFS) was inferior in MM patients with high expression of EBV-DNA, high lactate dehydrogenase (LDH), and high-risk according to mSMART and International Myeloma Working Group (IMWG), stage III according to R-ISS staging, extramedullary lesions, and genetic changes (P<0.05). However, in multivariate analysis, LDH, poor karyotype, R-ISS staging, and mSMART were independent prognostic factors for PFS. Taken together, our studies suggest that an association exists between EBV infection and clinical characteristics of MM patients, and EBV infection appears to have a slight impact on the prognosis of MM. However, the results require further validation in other independent prospective MM cohorts.

High Expression Levels of Long Noncoding RNA Small Nucleolar RNA Host Gene 18 and Semaphorin 5A Indicate Poor Prognosis in Multiple Myeloma

Background: The aim of this study was to detect the expression of long noncoding RNA small nucleolar RNA host gene 18 (SNHG18) andsemaphorin 5A (SEMA5A) genes in multiple myeloma (MM) patients and to explore the correlation of the expression of these genes with the clinical characteristics and prognosis of MM patients. Methods: Forty-seven newly diagnosed MM, 18 complete remission MM, 13 refractory/relapse MM, and 22 iron deficiency anemia (serving as control) samples were extracted at the Department of Hematology, Second Affiliated Hospital of Xian Jiaotong University between January 2015 and December 2016. The clinical features of the MM patients are summarized. Real-time quantitative PCR was performed to analyze the relative expression levels of the SNHG18 and SEMA5Agenes. The clinical characteristics and overall survival (OS) of the MM patients were statistically analyzed while measuring different levels of SNHG18 and SEMA5Agene expression. At the same time, the correlation between the expression of SNHG18 and SEMA5A was also analyzed. Results: The analysis confirmed that SNHG18 and its possible target gene SEMA5A were both highly expressed in newly diagnosed MM patients. After analyzing the clinical significance of SNHG18 and SEMA5A in MM patients, we found that the expression of SNHG18 and SEMA5A was related to the Durie-Salmon (DS), International Staging System (ISS), and Revised International Staging System (R-ISS) classification systems, and the Mayo Clinic Risk Stratification for Multiple Myeloma (mSMART; p < 0.05). Moreover, we observed a significant difference in OS between the SNHG18/SEMA5A high expression group and the low expression group. We found a positive correlation between SNHG18 and SEMA5A expression (r = 0.709, p < 0.01). Surprisingly, the expected median OS times of both the SNHG18 and SEMA5Ahigh expression groups were significantly decreased, which was in contrast to those of both the SNHG18 and SEMA5Alow expression groups and the single-gene high expression group (p < 0.05). Conclusion: High expression of both SNHG18 and SEMA5A is associated with poor prognosis in patients with MM.

PB2149 NUMBER OF INVOLVED BONE REGIONS IS A SIMPLE AND USEFUL PREDICTIVE MARKER OF EARLY DISEASE PROGRESSION AND DEATH IN NEWLY DIAGNOSED MULTIPLE MYELOMA PATIENTS

Background:The number or size of focal lesions (FL) on whole‐body magnetic resonance imaging (MRI) or positron emission tomography integrated with computed tomography (PET‐CT) was shown to be associated with poor prognosis in multiple myeloma (MM) patients. However, it is complicated to determine the number or amount of FL on MRI or PET‐CT imaging and patients cannot always undergo such medical imaging examination at diagnosis for many reasons. Therefore, developing simple method to estimate the amount of tumor burden from imaging data may be useful to predict prognosis of MM patients under various conditions.Aims:The aim of this study was to assess whether the number of involved bone regions at diagnosis could become a predictive marker of disease progression and survival in newly diagnosed MM patients.MethodsClinical and imaging data of patients newly diagnosed with MM and immediately treated with chemotherapy from January 2013 to August 2018 at the institution were evaluated retrospectively. All imaging data had been interpretated by experienced radiologists. Each bone lesion was sorted according to 8 anatomical regions: skull, rib including clavicula, scapula, sternum, spine, ilium, upper limb, and lower limb. Factors associated with progression‐free survival (PFS) or overall survival (OS) were statistically analyzed using log‐rank test and Cox regression model.Results:Eighty‐three evaluated patients had a median age of 69 years old (yo) (range: 45‐92). 9% of them were classified as revised International Staging System (R‐ISS) stage 3. As imaging tools for evaluation of bone disease, CT and additional skeletal x‐ray were conducted in all cases, MRI (of not the whole‐body, usually of the spine) in 80%, PET‐CT in 45% and bone scintigraphy in 16%. Median number of involved bone regions was 2 (range: 0‐8). 9% of patients had no bone disease and 46% had bone lesions in at least 3 different regions. The most involved region was spine (77%) following rib (46%), ilium (38%), lower limb (27%), sternum (26%), skull (24%), upper limb (21%), and scapula (13%). Only 3 patients had extramedullary disease (EMD). 95% of patients were initially treated with therapeutic regimen containing bortezomib or lenalidomide, 34% combined with radiation, and 6% followed by surgery. Autologous transplantation was performed in only 11 patients (13%). With a median follow‐up of 26 months (range: 0‐72 months), 2‐year PFS and OS for all patients were 48% and 78%, respectively. The presence of bone lesions in at least 3 regions (PFS; P=. 035, OS; P=. 018), advanced age (&gt;65 yo, PFS; P=. 039, &gt;74 yo, OS; P=. 007), low hemoglobin level (&lt;10 g/dL, PFS; P=. 030, OS; P=. 058), decreased renal function (creatinine clearance &lt;40 ml/min, PFS; P=. 032, OS; P=. 017), high collected serum calcium level (&gt;11 mg/dL, PFS; P=. 020, OS; P=. 027), R‐ISS stage 3 (PFS; P=. 002, OS; P=. 004) and EMD (PFS; P=. 023, OS; P&lt;. 0001) adversely affected both PFS and OS. In a multivariate analysis, the presence of bone lesions in at least 3 regions (HR: 2.74, P=. 004), advanced age (&gt;65 yo, HR: 3.05, P=. 002) and EMD (HR: 33.0, P=. 029) were significantly associated with inferior PFS. Also the presence of bone lesions in at least 3 regions (HR: 3.04, P=. 025) and EMD (HR: 51.7, P=. 004) were independent risk factors in OS.Summary/Conclusion:Our results suggest that the presence of bone lesions in at least 3 different regions at diagnosis is a beneficial factor for predicting early progression and death, which can be easily evaluated using conventional imaging system at almost any institutions.

The presence of bone marrow fibrosis is associated with poorer prognosis in newly diagnosed multiple myeloma treated with immunomodulatory agents and/or proteasome inhibitors.

e19514 Background: The treatment of newly diagnosed multiple myeloma (NDMM) has evolved significantly with the advent of the immunomodulatory agents (IMiDs) and proteasome inhibitors (PIs). While the presence of bone marrow fibrosis (BMF) has previously been associated with poor prognosis in multiple myeloma (MM), these studies were small and conducted prior to the widespread use of IMiDs and PIs. Here, we determined the incidence of BMF in NDMM patients and correlated the degree of BMF with prognosis in a population enriched for IMiD and/or PI exposure. Methods: Bone marrow biopsies from 306 MM patients seen at Duke between 2003 and 2013 were screened for BMF using a reticulin stain. Samples were scored as absent, mild, moderate, or severe fibrosis based on the degree and intensity of staining. The association between presence and degree of BMF to progression free survival (PFS) and overall survival (OS) was calculated using Kaplan-Meier analysis. Results: Of the 306 patients evaluated, 248 (81.0%) were treated with an IMiD, 241 (78.8%) were treated with a PI, and 217 (70.9%) received both. Additionally, 160 (52.3%) patients went on to receive an autologous stem cell transplant (HSCT). A total of 193 patients (63.1%) were evaluable for BMF. Of these, 96 (49.7%) had detectable BMF, while 97 (50.3%) had no BMF. The degree of BMF was mild in 60 patients (62.5%), and moderate or severe in 34 patients (35.4%). Median PFS in patients without BMF was 30.4 months, and 21.8 months in patients with BMF present (log-rank p = 0.02). Median OS was 61.1 months in patients without BMF, and 46.3 months in patients with BMF (log-rank p = 0.048). Patients with moderate or severe BMF had a particularly poor prognosis with a PFS of only 18.8 months and an OS of 32.7 months. Conclusions: Our study represents the largest dataset to date examining the incidence of BMF in MM patients, and is the only one to examine the association of BMF with prognosis in the era of novel therapies and widespread use of HSCT. Our data suggests that BMF is common in NDMM, and MM patients with BMF (particularly those with more extensive BMF) have a poorer prognosis even when treated with IMiDs and PIs. These data emphasize the importance of determining the presence and degree of BMF at time of MM diagnosis, and suggest a role for adjunctive therapies that target BMF in MM patients with co-existing BMF.

Trabectedin triggers direct and NK-mediated cytotoxicity in multiple myeloma

BackgroundGenomic instability is a feature of multiple myeloma (MM), and impairment in DNA damaging response (DDR) has an established role in disease pathobiology. Indeed, a deregulation of DNA repair pathways may contribute to genomic instability, to the establishment of drug resistance to genotoxic agents, and to the escape from immune surveillance. On these bases, we evaluated the role of different DDR pathways in MM and investigated, for the first time, the direct and immune-mediated anti-MM activity of the nucleotide excision repair (NER)-dependent agent trabectedin.MethodsGene-expression profiling (GEP) was carried out with HTA2.0 Affymetrix array. Evaluation of apoptosis, cell cycle, and changes in cytokine production and release have been performed in 2D and 3D Matrigel-spheroid models through flow cytometry on MM cell lines and patients-derived primary MM cells exposed to increasing nanomolar concentrations of trabectedin. DNA-damage response has been evaluated through Western blot, immunofluorescence, and DNA fragmentation assay. Trabectedin-induced activation of NK has been assessed by CD107a degranulation. miRNAs quantification has been done through RT-PCR.ResultsBy comparing GEP meta-analysis of normal and MM plasma cells (PCs), we observed an enrichment in DNA NER genes in poor prognosis MM. Trabectedin triggered apoptosis in primary MM cells and MM cell lines in both 2D and 3D in vitro assays. Moreover, trabectedin induced DDR activation, cellular stress with ROS production, and cell cycle arrest. Additionally, a significant reduction of MCP1 cytokine and VEGF-A in U266-monocytes co-cultures was observed, confirming the impairment of MM-promoting milieu. Drug-induced cell stress in MM cells led to upregulation of NK activating receptors ligands (i.e., NKG2D), which translated into increased NK activation and degranulation. Mechanistically, this effect was linked to trabectedin-induced inhibition of NKG2D-ligands negative regulators IRF4 and IKZF1, as well as to miR-17 family downregulation in MM cells.ConclusionsTaken together, our findings indicate a pleiotropic activity of NER-targeting agent trabectedin, which appears a promising candidate for novel anti-MM therapeutic strategies.

PiRNA-823 delivered by multiple myeloma-derived extracellular vesicles promoted tumorigenesis through re-educating endothelial cells in the tumor environment.

Extracellular vesicles (EVs) can carry a wide array of RNAs in the tumor microenvironment, and are crucial for communication between tumor and surrounding stromal cells, including endothelial cells. Piwi-interacting RNAs (piRNAs) are important regulators implicated in the pathogenesis of multiple myeloma (MM). However, little is understood about the role of piRNA-823 in intercellular communication between MM and endothelial cells. In this study, we found that piRNA-823 mainly accumulated in EVs from peripheral blood of MM patients and EVs derived from MM cells (MM-derived-EVs). Increased piRNA-823 expression was associated with late stages and poor prognosis of MM. The MM-derived-EVs effectively transferred piRNA-823 to EA.hy926 endothelial cells. The piRNA-823 mimic and inhibitor were designed to upregulate or to suppress the endogenous function of piRNA-823. Transfection with piRNA-823 mimic or treatment with MM-derived-EVs significantly promoted the proliferation, tube formation, and invasion of EA.hy926 cells by enhancing the expression of VEGF, IL-6, and ICAM-1 and attenuating apoptosis. EA.hy926 cells transfected with piRNA-823 mimic or pre-treated with MM-derived-EVs promoted the growth of xenograft MM in mice. In contrast, the transfection with piRNA-823 inhibitor or treatment with EVs from piRNA-823 inhibitor-transfected-MM cells had diametrically opposite effects. Our findings demonstrated that piRNA-823 carried by MM-derived-EVs is essential for the re-education of ECs toward a unique environment amenable to the growth of MM cells by altering its biological characteristics. Our findings may pave the way for the development of new piRNA-mediated prognostic stratification and therapeutic strategies for MM.

High subclonal fraction of 17p deletion is associated with poor prognosis in multiple myeloma

AbstractDeletions of chromosome 17p (del17p) that span the TP53 gene are associated with poor outcome in multiple myeloma (MM), but the prognostic value of del17p cancer clonal fraction (CCF) remains unclear. We applied uniform cytogenetic assessments in a large cohort of newly diagnosed MM (NDMM) patients carrying varying levels of del17p. Incremental CCF change was associated with shorter survival, and a robust CCF threshold of 0.55 was established in discovery and replication data sets. After stratification on the 0.55-CCF threshold, high-risk patients had statistically significantly poorer outcomes compared with low-risk patients (median progression-free survival [PFS] and overall survival [OS], 14 and 32 vs 23.1 and 76.2 months, respectively). Analyses of a third data set comprising whole-exome sequencing data from NDMM patients identified presence of TP53 deletions/mutations as a necessary requirement for high-risk stratification in addition to exceeding the del17p CCF threshold. Meta-analysis conducted across 3 data sets confirmed the robustness of the CCF threshold for PFS and OS. Our analyses demonstrate the feasibility of fluorescence in situ hybridization– and sequencing-based methods to identify TP53 deletions, estimate CCF, and establish that both CCF threshold of 0.55 and presence of TP53 deletion are necessary to identify del17p-carrying NDMM patients with poor prognosis.

High expression of CHML predicts poor prognosis of multiple myeloma.

Multiple myeloma is a hematological tumor with a malignant proliferation of myeloma cells. Although the survival time after treatment has improved, the recurrence rate of MM is still high. Choroideremia-like (CHML) protein is essential for the prenylation modification of various Rab proteins and it exerts biological effects on vesicle trafficking and signal transduction. However, little is identified about the relationship between CHML gene and MM. We integrated gene expression profiles of 1907 MM patients (1959 MM samples) from the 7 datasets. The relationship between CHML gene expression level and event-free survival (EFS), overall survival (OS), ISS stage, molecular subtype, relapse, therapy was analyzed. The differential gene exression profile of CHML-high MM group and CHML-low MM group and possible pathway related to CHML were conducted. Our data showed that EFS (P < 0.0001) and OS (P < 0.0001) in MM patients with high expression of CHML were lower than those with low CHML expression. The gene expression level of CHML was increased in subtypes of MM with poor prognosis, especially in proliferation subtype (P < 0.001). Cell division pathway (P < 0.01) was high enriched of the differential expressed genes of CHML-high group vs CHML-low group. CHML gene can be considered as an independent factor to evaluate the prognosis of MM. High expression of CHML is associated with poor survival, which is related to cell proliferation and cell division of myeloma cells.

Lack of Spleen Signal on Diffusion Weighted MRI is associated with High Tumor Burden and Poor Prognosis in Multiple Myeloma: A Link to Extramedullary Hematopoiesis?

Due to the low frequency of abnormalities affecting the spleen, this organ is often overlooked during radiological examinations. Here, we report on the unexpected finding, that the spleen signal on diffusion-weighted MRI (DW-MRI) is associated with clinical parameters in patients with plasma cell dyscrasias.Methods: We investigated the spleen signal on DW-MRI together with clinical and molecular parameters in 295 transplant-eligible newly diagnosed Multiple Myeloma (NDMM) patients and in 72 cases with monoclonal gammopathy of undetermined significance (MGUS).Results: Usually, the spleen is the abdominal organ with the highest intensities on DW-MRI. Yet, significant signal loss on DW-MRI images was seen in 71 of 295 (24%) NDMM patients. This phenomenon was associated with the level of bone marrow plasmacytosis (P=1x10-10) and International Staging System 3 (P=0.0001) but not with gain(1q), and del(17p) or plasma cell gene signatures. The signal was preserved in 72 individuals with monoclonal gammopathy of undetermined significance and generally re-appeared in MM patients responding to treatment, suggesting that lack of signal reflects increased tumor burden. While absence of spleen signal in MM patients with high risk disease defined a subgroup with very poor outcome, re-appearance of the spleen signal after autologous stem cell transplantation was seen in patients with improved outcome. Our preliminary observation suggests that extramedullary hematopoiesis in the spleen is a factor that modifies the DW-MRI signal of this organ.Conclusions: The DW-MRI spleen signal is a promising marker for tumor load and provides prognostic information in MM.

High Levels of APOBEC3B Gene Expression Contribute to Poor Prognosis in Multiple Myeloma Patients

Introduction: Poor prognosis and drug resistance in multiple myeloma (MM) is associated with increased mutational load. APOBEC3B is a major contributor to mutagenesis, especially in myeloma patients with t(14;16) MAF subgroup. It was shown recently that presence of the APOBEC signature at diagnosis is an independent prognostic factor for progression free survival (PFS) and overall survival (OS). We hypothesized that high levels of APOBEC3B gene expression at diagnosis may also have a prognostic impact in myeloma. To consider APOBEC3B as a potential target for therapy more studies are necessary to understand how APOBEC3B expression is regulated and how APOBEC3B generates mutations.Methods: Gene expression profiling (GEP, U133 Plus 2.0) of MM patients was performed. APOBEC3B gene expression levels were investigated in plasma cells of healthy donors (HD; n=34), MGUS (n=154), smoldering myeloma (SMM; n=219), MM low risk (LR; n=739), MM high risk (HR; n=129), relapsed MM (RMM; n=74), and primary plasma cell leukemia (pPCL; n=19) samples. The samples from relapse were taken on or after the progression/relapse date but within 30 days after progression/relapse from Total Therapy trials 3, 4, 5 & 6. GEP70 score was used to separate samples into LR and HR groups. We also investigated APOBEC3B expression in different MM molecular subgroups and used logrank statistics with covariate frequency distribution to determine an optimal cut off APOBEC3B expression value. Gene expression was compared in cases with low expression of APOBEC3B (log2<7.5) and high expression of APOBEC3B (log2>10), and an optimal cut-point in APOBEC3B expression was identified with respect to PFS. To explore the role of MAF and the non-canonical NF-ĸB pathway we performed functional studies using a cellular model of MAF downregulation. TRIPZ lentiviral shRNA MAF knockdown in the RPMI8226 cell lines was used to explore MAF-dependent genes. NF-ĸB proteins, p52 and RelB, were investigated in the nuclear fraction by immunoblot analysis.Results: Expression of APOBEC3B in HD control samples (log2=10.9) was surprisingly higher than in MGUS (log2=9.51), SMM (log2=9.09), and LR (log2=9.40) and was comparable to HR (log2=10.4) and RMM (log2=10.6) groups. Expression levels of APOBEC3B were gradually increased as disease progressed from SMM to pPCL. The high expression of APOBEC3B in HD places plasma cells at risk of APOBEC induced mutagenesis where the regulation of APOBEC3B function is compromised. The correlation between APOBEC3B expression and GEP70 score in MM was 0.37, and there was a significant difference in APOBEC3B expression between GEP70 high and low risk groups (p=0.0003). An optimal cut-point in APOBEC3B expression of log2=10.2 resulted in a significant difference in PFS (median 5.7 yr vs.7.4 yr; p=0.0086) and OS (median 9.1 yr vs. not reached; p<0.0001), between high and low expression. The highest APOBEC3B expression was detected in cases with a t(14;16). We analyzed t(14;16) cases with the APOBEC mutational signature and compared them to t(14;16) cases without the APOBEC signature and found elevated MAF (2-fold) and APOBEC3B (2.7-fold) gene expression in samples with the APOBEC signature. No APOBEC signature was detected in SMM cases, including those with a t(14;16). High APOBEC3B levels in myeloma patients was associated with overexpression of genes related to response to DNA damage and cell cycle control. Significant (p<0.05) increases of NF-κB target genes was seen in high APOBEC3B cases: TNFAIP3 (4.4-fold), NFKB2 (1.7-fold), NFKBIE (1.9-fold), RELB (1.4-fold), NFKBIA (2.0-fold), PLEK (2.5-fold), MALT1 (2.5-fold), WNT10A (2.4-fold). However, in t(14;16) cases there was no significant increase of NF-κB target genes except BIRC3 (2.5-fold) and MALT1 (2.0-fold). MAF downregulation in RPMI8226 cells did not lead to changes in NF-κB target gene expression but MAF-dependent genes were identified, including ETS1, SPP1, RUNX2, HGF, IGFBP2 and IGFBP3. Analysis of nuclear fraction of NF-ĸB proteins did not show significant changes in expression of p52 and RelB in RPMI8226 cells after MAF downregulation.Conclusions: Increased expression of APOBEC3B is a negative prognostic factor in multiple myeloma. MAF is a major factor regulating expression of APOBEC3B in the t(14;16) subgroup. NF-ĸB pathway activation is most likely involved in upregulation of APOBEC3B in non-t(14;16) subgroups. DisclosuresDavies:TRM Oncology: Honoraria; MMRF: Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; ASH: Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy. Morgan:Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Research Funding; Takeda: Consultancy, Honoraria.

Matrix Metalloproteinase and Tissue Inhibitor of Metalloproteinases Is Associated with Multiple Myeloma Progression, Prognosis and Extramedullary Plasmacytoma

Backgrounds and Aims: Matrix metalloproteinase (MMP) is endopeptidase enzyme degrading extracellular matrix, and tissue inhibitor of metalloproteinases (TIMP) is negative regulator of MMP. MMP is well known to be involved in metastatic mechanism of cancer cell and oncogenesis. However expression and role of MMP and TIMP has not been well established in multiple myeloma (MM). Therefore we examined whether expression of MMP and TIMP was involved in progression and prognosis of MM and extramedullary plasmacytoma (EMP) formation.Materials and Methods: Purified bone marrow plasma cells by using anti-CD138 antibody and magnetic beads obtained from 151 MM, 64 MGUS, 18 control and 5 EMP were subjected to the study after informed consent. The study was approved by IRB following Declaration of Helsinki. Whole transcriptome by next generation sequencer (NGS) using Illumina Next Seq 500 was performed in part of the samples to select genes to be studied, then expression level of MMP and TIMP determined by RQ-PCR Delta Ct value normalized with ACTB was used for analysis. MM cell lines KMS11, KMS12PE, KMS12BM, KMM1, RPMI8226, MM1S were used for the in vitro study.Results: We selected TIMP-1, 2and MMP14, 24 based on transcriptome analysis data comparing MM and EMP. The expression level of TIMP1 and MMP24 was significantly higher in MM (median delta Ct: 0.033 for TIMP1, 0.00025 for MMP24) than in MGUS (median delta Ct: 0.013 for TIMP1, 0.00006 for MMP24) (p=0.005, p=0.001), however TIMP2, MMP14 level did not differ in between MM and MGUS. Interestingly, TIMP1, 2 and MMP14, MMP24 were expressed with strikingly higher levels in EMP than in MM with 20 times, 60 times, 300 times, 500 times respectively (p=0.01, p=0.02, p=0.004, p=0.004). Both TIMP1 and 2 expression were higher in the MM patients with high risk cytogenetic karyotype t(4;14), t(14;16), del 17p than in the patients without such karyotype (p=0.006, p=0.008), however the levels of MMP14 and 24 did not differ in between cytogenetic risk groups. Positive correlations were found in between TIMP1 and 2, MMP14 and 24 in both MM and MGUS group respectively (r=0.34, p<0.001, r=0.49, p<0.001 in MM, r=0.32, p=0.008, r=0.63, p<0.001 in MGUS). Since TIMP2 and MMP14 were higher in EMP, the effect of recombinant TIMP2, siRNA-TIMP2 and MMP14 inhibitors marimastat/ilomastat were tested in MM cell lines KMM1, KMS11, KMS12PE and RPMI8226. However, these interventions did not change proliferation rates of these cell lines. The cell lines were treated with doxorubicin or bortezomib to study if these agent can change TIMP and MMP expression. Doxorubicin significantly increased expression level of MMP14, 24 and TIMP1, 2, but bortezomib increased only MMP14, 24. In the newly diagnosed MM (NDMM) patients (n=77), median time of overall survival (OS) of the patients with high TIMP1 (more than median value) was significantly shorter (2.7 years vs not reached) and 3 year OS rate was inferior (40% vs 65%) (p=0.0095). Progression free survival (PFS) tended to be inferior for high TIMP1 group, but the difference did not reach statistical significance (p=0.221). OS and PFS were not different according to either TIMP2, MMP14 or MMP24.Conclusions: Our results suggest that TIMP1, 2 and MMP14, 24 were associated with EMP formation. Among those factors, TIMP1 is the one which may play a key role for MM progression and chemo-resistance based on the results revealing its upregulation by antineoplastic agents and association with poor prognosis of MM patients. Our results is consistent with a previous report describing that high serum TIMP1 concentration was associated with poor prognosis of MM. TIMP is recently shown to play another role besides negative regulator for MMP, so further study to elucidate its specific role for chemo-resistance contributes to develop novel therapy targeting TIMP and MMP pathway. DisclosuresTsukamoto:Kyowa-Kirin: Research Funding; Chugai: Research Funding; Eisai: Research Funding; Pfizer: Research Funding. Handa:Celgene: Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Lack of a Spleen Signal on Diffusion Weighted MRI Is Associated with High Tumor Burden and Poor Prognosis in Multiple Myeloma

▪Background: Whole body medical imaging is an integral component of Multiple Myeloma (MM) evaluation as it reveals bone disease and/or focal lesions. While established imaging techniques, such as PET-CT, are powerful in delineating focal pathologies, they suffer from low specificity when it comes to assessment of diffuse bone marrow (BM) signals. Especially in patients treated with G-CSF, discrimination between malignant and non-malignant BM signals is barely possible. Diffusion-weighted MRI with background suppression (DWIBS) is a novel functional MRI technique which measures water diffusion in vivo. Basically, diffusion of water is more restricted in tissues with high cellularity, making DWIBS a promising tool to investigate malignant diffuse signals. Initially, we searched for an internal reference to classify the level of BM infiltration. We selected the spleen, since it is the abdominal organ with the highest restriction on DWIBS. Unexpectedly, we observed significant heterogeneity in the spleen signal itself including a subgroup of MM patients with total loss of the signal on DWIBS. Perplexingly, these patients suffered from high tumor burden and poor outcome. Here, we report on our strategy to elucidate this phenomenon and to evaluate its clinical value.Methods: We investigated 295 transplant-eligible newly diagnosed MM patients and 72 cases with monoclonal gammopathy of undetermined significance (MGUS). This study was approved by the institutional review board (#205415). DWIBS was performed on a 1.5 Tesla Philips Achieva scanner. The spleen signal was assessed on DWI and ADC maps by two experienced investigators in consensus read. The Kaplan-Meier method was used for survival analyses. Molecular characterization included fluorescence in situ hybridization, and gene expression profiling of CD138-enriched plasma cells (PCs). Differential gene expression was performed using a threshold of 2-fold expression difference. Wilcoxon or Fisher's exact tests were used to compare the median of a continuous variable or the distribution of discrete variables across groups, respectively. Correlation coefficients were determined using Spearman's rank correlation.Results: Absence of the spleen signal on DWIBS was a frequent phenomenon in MM, seen in 71/295 (24%) patients. In all of these patients asplenia was excluded using alternative imaging techniques. Lack of a signal was highly positively associated with tumor-load parameters, such as the degree of BMPC infiltration (P=1x10-10) or International Staging System (ISS) 3 (P=0.0001). In contrast, it was not associated with age, gender, or the tumor progression markers gain(1q) and del(17p). Patients with absence of spleen signal experienced unfavorable outcome (hazard ratio of 1.8 for PFS and OS). In order to further investigate the biological underpinnings of this phenomenon we performed a differential gene expression analysis of purified CD138 MM cells. No differentially expressed genes were found between patients with and without spleen signal, suggesting that the absence of the spleen signal mainly reflected increased tumor burden rather than specific tumor features. As a proof of concept, we addressed the spleen signal in individuals with MGUS, and longitudinally in MM patients who presented with absence of the spleen signal at diagnoses. Indeed, in all 78 individuals with MGUS the signal was preserved, and the majority of MM patients showed re-appearance of the spleen on DWIBS during treatment as the tumor burden declined. Interestingly, re-appearance of the spleen was helpful to distinguish between malignant and non-malignant hyperintensities in the BM, making the spleen signal a promising parameter for MM follow-up investigations.Conclusions: Due to the low frequency of abnormalities affecting the spleen, this organ is often overlooked during abdominal examinations using cross-sectional imaging techniques. Here we show that the spleen signal on DWIBS provides clinically useful information on tumor burden in MM. Moreover, it opens the avenue to distinguish between malignant and reactive BM hypercellularity on imaging in patients receiving treatment. Our observation strongly supports the hypothesis, that the spleen signal is suppressed by a high BMPC involvement. Yet, the reasons for this phenomenon are still elusive. [Display omitted] DisclosuresRoy Choudhury:University of Arkansas for Medical Sciences: Employment, Research Funding. Epstein:University of Arkansas for Medical Sciences: Employment. Barlogie:Dana Farber Cancer Institute: Other: travel stipend; International Workshop on Waldenström's Macroglobulinemia: Other: travel stipend; ComtecMed- World Congress on Controversies in Hematology: Other: travel stipend; European School of Haematology- International Conference on Multiple Myeloma: Other: travel stipend; Celgene: Consultancy, Research Funding; Millenium: Consultancy, Research Funding; Myeloma Health, LLC: Patents & Royalties: : Co-inventor of patents and patent applications related to use of GEP in cancer medicine licensed to Myeloma Health, LLC; Multiple Myeloma Research Foundation: Other: travel stipend. Davies:Amgen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Morgan:Janssen: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

MiR-221/222 Promote Dexamethasone Resistance of Multiple Myeloma through Inhibition of Autophagy By Targeting ATG12

Dexamethasone (Dex) is the most widely used chemotherapeutic drug in the treatment of multiple myeloma (MM). Inherent or acquired resistance to Dex is broadly associated with poor prognosis in MM. Many microRNAs are aberrantly expressed in MM, including miR-221/222, which have been reported to act as oncogenes in many cancer types. Recently, accumulating evidence has shown that miR-221/222 are involved in the development of chemoresistance in a variety of cancers. However, there is still a lack of valuable data regarding the role of miR-221/222 in the chemoresistance of MM.Here, we first evaluated the expression levels of miR-221/222 in plasma cells (PCs) from MM patients by qRT-PCR analysis. The results showed that miR-221/222 were markedly upregulated in PCs from newly diagnosed MM patients compared to healthy donors, and they were further upregulated in PCs from patients with relapsed MM. In addition, we found that the expression levels of miR-221/222 were inversely correlated with Dex-sensitivity of human MM cell lines (HMCLs). Importantly, enforced expression of miR-221/222 dramatically reduced the sensitivity of Dex-sensitive HMCLs to Dex, while inhibition of miR-221/222 re-sensitized Dex-resistant HMCLs to Dex.Previous studies have shown that Dex-induced cell death in lymphoid leukemia is mediated through initiation of autophagy. To study whether autophagy was involved in Dex-induced cell death in MM cells, HMCLs were exposed to Dex, and then autophagy in these cells was evaluated by the transmission electron microscopy and western blot analysis. The results showed that Dex induced the occurrence of autophagy in Dex-sensitive HMCLs, but not in Dex-resistant HMCLs. Moreover, pharmacological inhibitors of autophagy could significantly reduce Dex-induced cell death in Dex-sensitive HMCLs. These results reveal that autophagy is critical for the induction of cell death following Dex treatment in MM.MicroRNAs have been reported to play an important role in regulating autophagy. We therefore examined whether miR-221/222 can regulate autophagy in MM cells. Low miR-221/222 expressing MM.1S (Dex-sensitive) or high miR-221/222 expressing MM.1R (Dex-resistant) cells were transfected with agomir-221/222 or antagomir-221/222, respectively, and then the level of autophagy was evaluated. The results showed that overexpression of miR-221/222 reduced the level of autophagy in MM.1S cells, while inhibition of miR-221/222 elevated the level of autophagy in MM.1R cells. Using microRNA target prediction bioinformatics tools and dual-luciferase reporter assay, we confirmed that autophagy-related gene 12 (ATG12) was a novel target gene of miR-221/222. Indeed, miR-221/222 could negatively regulate the expression of ATG12 at both the mRNA and protein levels in MM cells. In addition, knockdown of ATG12 by siRNA markedly reduced the autophagy-inducing and Dex-sensitizing activity of miR-221/222 antagomirs in MM.1R cells. Of note, in MM.1S cells, Dex treatment could further decreased the expression of miR-221/222, accompanied by upregulated expression of ATG12, whereas silencing the expression of ATG12 could significantly inhibited Dex-induced autophagy and cell death. Thus, these results suggest that ATG12 is a key player in miR-221/222-mediated autophagy inhibition and Dex-resistance.Next, we evaluated whether miR-221/222 could regulate autophagy and Dex-sensitivity of MM cells invivo. NOD/SCID mice were subcutaneously injected with MM.1R cells to establish Dex-resistant MM xenografts. Combined treatment with antagomir-221/222 plus Dex showed a remarkable reduction of tumor size compared to antagomir-221/222 or Dex alone (397.6±55.08 mm3 VS 895.8±72.44 mm3 VS 987.3±68.49 mm3). Immunohistochemistry and western blot analysis of the retrieved xenografted tumors showed that combination treatment with antagomir-221/222 plus Dex induced upregulation of ATG12, as well as extended autophagy with increased p62 degradation and Beclin-1 expression.In conclusion, our data reveal that upregulation of miR-221/222 promotes Dex resistance of MM cells through inhibition of autophagy by targeting ATG12. Therefore, miR-221/222-ATG12 autophagy-regulatory axis may potentially be applied in glucocorticoid resistance prediction and treatment. DisclosuresNo relevant conflicts of interest to declare.

Profilin 1 induces drug resistance through Beclin1 complex-mediated autophagy in multiple myeloma.

Autophagy plays an important role in multiple myeloma (MM) for homeostasis, survival and drug resistance, but which genes participate in this process is unclear. We identified several cytoskeleton genes upregulated in MM patients by gene expression profiling (GEP) datasets; in particular, patients with high profilin 1 (PFN1) expression had poor prognosis in MM. In vitro, overexpressed PFN1 promotes proliferation and bortezomib (BTZ) resistance in MM cells. Further study indicated overexpression of PFN1 significantly promoted the process of autophagy and induced BTZ resistance in MM. Otherwise, knockdown of PFN1 blocked autophagy and sensitized MM to BTZ. Co‐immunoprecipitation in MM cells indicated that PFN1 could bind Beclin1 complex and promote the initiation of autophagy. Inhibition of autophagy by blocking the formation of Beclin1 complex could reverse the phenotype of BTZ resistance in MM. Our findings suggested that PFN1 could promote autophagy through taking part in Beclin1 complex and contribute to BTZ resistance, which may become a novel molecular target in the therapy of MM.

ARNT/HIF‐1β links high‐risk 1q21 gain and microenvironmental hypoxia to drug resistance and poor prognosis in multiple myeloma

1q21 gain is a common cytogenetic abnormality featuring high‐risk multiple myeloma (HRMM). However, the molecular mechanism underlying the adverse prognostic effect of 1q21 gain remains largely unclear. Here, we report that ARNT/HIF‐1β, a 1q21 gene, is highly expressed in HRMM and induced by microenvironmental hypoxia, which confers drug resistance and correlates with inferior outcome. Analysis of the gene expression profile database revealed that ARNT expression was upregulated in MM and increased with disease progression or in HRMM subtypes (particularly 1q21 gain), while correlated to shorter overall survival. In a cohort of 40 MM patients, qPCR further validated that ARNT expression was higher in MM patients than normal donors. MM cells carrying 1q21 gain or acquired drug resistance displayed a robust increase in HIF‐1β protein level. Hypoxia induced HIF‐1β expression via a NF‐κB‐dependent process. Notably, HIF‐1β overexpression impaired bortezomib sensitivity, whereas shRNA knockdown of ARNT reversed hypoxia‐mediated drug resistance. Together, these findings suggest that ARNT/HIF‐1β might represent a novel biomarker for risk stratification and prognosis of HRMM patients, as well as a potential therapeutic target for overcoming 1q21 gain‐ or microenvironment‐mediated and acquired drug resistance in MM.