scholarly journals PHF19 Promotes Drug Resistance through EZH2 Inactivation in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4495-4495 ◽  
Author(s):  
Tengteng Yu ◽  
Lanting Liu ◽  
Shuaishuai Zhang ◽  
Mu Hao ◽  
Lugui Qiu
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Drug Resistance ◽  
Western Blotting ◽  
Poor Prognosis ◽  
Post Treatment ◽  
Bone Lesions ◽  
Newly Diagnosed ◽  
Polycomb Repressive Complex

Abstract Background: Multiple myeloma (MM) is a plasma cell malignancy characterized by mass growth of clonal plasma cells in the bone marrow, the secretion of abnormal monoclonal immunoglobulins and lytic bone lesions. Although current therapies have improved the outcome of patients, MM remains an incurable disease with a high rate of relapse and refractory. The survival of MM patients ranged from less than one month to more than 10 years. MM cells survival are more dependent on bone marrow microenvironment which induced drug-resistance of anti-MM agents. Cytogenetic aberrations of MM cell, such as chromosomal translocation, deletion and amplification induced oncogenes activation and tumor suppressor genes inactivation. Furthermore, epigenetic changes, such as histone methylation, have been suggested as a mediation of chemotherapy resistance in several cancer types, including MM. PcG (Polycomb group) proteins are conserved transcriptional repressors and essential to regulate cell fate.There were two main families of chromatin-modifying complexes, PRC1 (Polycomb repressive complex 1) and PRC2-4. In Drosophila, PRC2 contains the H3K27 histone methytransferase E (Z) whose trimethylation activity towards PcG target genes is stimulated by PCL (polycomb-like) protein. Three PCL paralogues have been identified: PHF1, MTF2 and PHF19. In this study, we found out that PHF19 is overexpressed and was related with relapse and drug resistance in multiple myeloma. Materials and methods: Gene Expression Profile Assay (GEP) was conducted in sequential patient samples including newly diagnosed, post-treatment and relapsed. 51 newly diagnosed and relapsed paired samples, 19 newly diagnosed and post-treatment paired specimens, 9 patients have newly diagnosed, post-treatment and relapsed samples at different time points were elucidated in this study. MM relapsed and drug-resistance highly correlated genes were screened and the prognosis and survival analysis were conducted. Real time quantitative PCR (RQ-PCR) and Western blotting were used to analysis the expression of the gene in MM cell lines and MM patients. The candidate gene was overexpressed in MM cells by lentiviral infection. The cell proliferation and drug sensitivity of MM cells and proteins in correlated signaling pathways were detected. Results: GEP assay showed that 56 genes expression in MM resistant clones after treatment and relapse have significantly increased, with 20 genes closely associated with the poor prognosis. Among them, PHF19, a component of the Polycomb repressive complex 2 (PRC2) family,significantly increased in relapsed and refactory patients and myeloma cell lines.The progression-free survival (PFS) and overall survival (OS) were significantly shortened in MM patients with overexpressed PHF19. PHF19 overexpression (OE) promoted the proliferation and inhibited apoptosis of MM cells. The sensitivity to doxorubicin and vincristine was significantly reduced in the PHF19 OE cells. Western blotting showed that the phosphorylation of EZH2 was significantly increased in PHF19 OE cells, while H3K27me3 level was significantly down-regulated. Overexpressed PHF19 through activating NF-κB signaling pathway induced persistent expression of EZH2 and their downstream anti-apoptotic gene, such as IGF1, BCL2 and HIF1α, which induced cell proliferation and drug resistance. Thus targeting PHF19, inhibiting the phosphorylation of EZH2 and sustaining histone H3K27 methylation level may be a potential therapeutic target in relapsed or refractory myeloma patients. Conclusion s: PHF19 expression was obviously increased in MM relapse and drug resistance patients with poor prognosis. Inhibition PHF19 and counteraction EZH2 phosphorylation should be combined to improve chemotherapy induced hypermethylation of H3K27, which may be a new therapeutic strategies in relapsed or refractory myeloma. Disclosures No relevant conflicts of interest to declare.

scholarly journals Incidental Discovery of Multiorgan Extramedullary Plasmacytomas in the Setting of Newly Diagnosed Multiple Myeloma and Delayed Hemolytic Transfusion Reaction

2017 ◽  
Vol 2017 ◽  
pp. 1-4
Author(s):  
Joselle Cook ◽  
Steven Song ◽  
Anthony Ventimiglia ◽  
Carol Luhrs
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Poor Prognosis ◽  
Transfusion Reaction ◽  
Newly Diagnosed ◽  
Hemolytic Transfusion Reaction ◽  
Delayed Hemolytic Transfusion Reaction ◽  
Incidental Discovery ◽  
Extramedullary Plasmacytomas

Extramedullary plasmacytomas (EMPs) are defined by the presence of clonal plasma cell proliferation outside of the bone marrow, portending an overall poor prognosis. This case highlights extramedullary plasmacytomas as an unusual presenting manifestation of multiple myeloma. Through incidental discovery during a delayed hemolytic transfusion reaction workup, EMPs were found in the liver, spleen, and possibly the lung. Though rare at presentation, this case emphasizes that the presence of EMPs should be considered at the outset as it not only impacts the treatment regimen for such patients but also considerably affects prognosis.

High Trip13 and Low P31 Comet Cause Drug Resistance and Poor Prognosis In Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3820-3820
Author(s):  
Yi Tao ◽  
Zhimin Gu ◽  
Ye Yang ◽  
Hongwei Xu ◽  
Xiaojing Hu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Lines ◽  
Poor Prognosis ◽  
Caspase 3 ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Newly Diagnosed ◽  
Caspase 9 ◽  
Over Expression

Abstract Background We have recently established that increased chromosomal instability (CIN) signature is linked to drug resistance and poor outcome in multiple myeloma (MM) and other cancers. Thyroid Hormone Receptor Interactor 13 (Trip13), one of the 56 drug-resistant genes, plays a key role in chromosomal recombination and structure development during meiosis and has been reported to be increased in some malignancies including lung cancer, prostate cancer and breast cancer. In this study, we investigated how important Trip13 is in myelomagenesis and progression. Materials and Methods Gene expression profiling (GEP) was analyzed on plasma cells from 22 healthy donors, 44 patients with monoclonal gammopathy of undetermined significance (MGUS), 351 patients with newly diagnosed multiple myeloma, and 9 human myeloma cell lines, as well as on 36 sequential samples at diagnosis, pre-1st, pre-2nd and post-2nd autologous stem cell transplantation (ASCT). Over-expression and knock-down experiments of Trip13 were performed on myeloma cell lines by lentivirus transfection. Cell viability was assessed by trypan exclusion assay. Western blots were used to detect the expression of Trip13, P31 comet, caspase-8, caspase-9, caspase-3 and PARP, and checkpoint related proteins MAD2 and CDC20 in Trip13 overexpressed or Trip13 shRNA-transfected myeloma cells. Results Sequential GEP samples showed that Trip13 expression increased in 8 of 9 patients after chemotherapy and ASCT compared to the samples at diagnosis strongly suggesting that increased Trip13 is associated with drug resistance. Trip13 was already significantly increased in MGUS patients, newly diagnosed MM patients and MM cell lines compared with normal plasma cells. Furthermore, Trip13 was significantly higher in high-risk MMs than in low-risk MMs and increased Trip13 was linked to an inferior event-free survival (EFS) (p<0.01) and overall survival (OS) (p<0.01) in 351 newly diagnosed MMs. In contrast, the Trip13-interacting gene P31 comet was down-regulated in high-risk MMs and high expression of P31 was associated with good outcome. Interestingly, patients with high Trip13 and low P31 comet have the worst outcome compared to patients with only one of these, suggesting the interaction of Trip 13 and p31 has a synergistic effect on MM progression. Transfection of Trip13 into ARP1 and OCI-My5 cells significantly increased cell proliferation, while knock-down Trip13 in OCI-My5, H929, RPMI8226 cells inhibited cell growth and induced MM cell apoptosis with increases of cleaved caspase-8, caspase-9, caspase-3 and PARP. Mechanistic studies showed that Trip13 over-expression decreased P31comet and MAD2 expression by western blotting, but increased CDC20. Conclusions The association of increased Trip13 and decreased p31 is a good biomarker for MM drug resistance and poor prognosis. Our results also show Trip13 and P31 comet could be potential targets to overcome drug resistance in MM. Disclosures: No relevant conflicts of interest to declare.

siRNA Knockdown of MMSET Disrupts Cell Cycle and Induces Apoptosis in t(4;14) Myeloma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 245-245
Author(s):  
Jose Brito ◽  
Brian Walker ◽  
Faith Davies ◽  
Julie Irving ◽  
Gareth J. Morgan
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Western Blotting ◽  
Protein Level ◽  
Poor Prognosis ◽  
Critical Role ◽  
In Vivo Studies ◽  
Expression Array

Abstract Multiple myeloma (MM) is characterised by a clonal expansion of plasma cells in the bone marrow. The recurrent immunoglobulin translocation t(4;14)(p16;q32) occurs in 15% of MM patients and is associated with poor prognosis, through an unknown mechanism. The t(4;14) translocation up-regulates fibroblast growth factor receptor 3 (FGFR3) and multiple myeloma SET (MMSET) genes. While the role of FGFR3 in myelomagenesis has been shown in various in vitro and in vivo studies, the up-regulation of MMSET at the protein level and its involvement in the pathogenesis of t(4;14) myeloma is still elusive. Moreover, about 30% of MM tumours with the t(4;14) translocation are reported to lack expression of FGFR3 due to the loss of der(14). Interestingly, the poor prognosis associated with the t(4;14) translocation in such patients lacking FGFR3 expression remains unchanged. These findings suggest that MMSET, if up-regulated at the protein level may be involved in the pathogenesis of t(4;14) myeloma and be a target for therapeutic manipulation. To assess the involvement of MMSET in the pathogenesis of t(4;14) MM we initially evaluated the expression of MMSET variants in a panel of human MM cell lines with and without the t(4;14) translocation by western blotting. Several isoforms corresponding to the expected molecular weight of MMSET II, MB4-2II, MB4-3II and REIIBP isoforms were expressed in MM cells, the REIIBP being exclusively up-regulated in t(4;14)-positive cell lines. In an attempt to determine the biological function of each MMSET isoform, western blotting was performed on nuclear and cytosol fractions isolated from t(4;14)-positive cell lines. It was found that MMSET II, MB4-2II and MB4-3II localise to the nucleus and not in the cytoplasm, but in contrast, REIIBP is found in both cellular fractions. To further determine the involvement of MMSET in the t(4;14) pathogenesis, MMSET expression was knocked down using siRNAs in t(4;14)-positive and t(4;14)-negative MM cell lines and its effect on cell proliferation, cell cycle and apoptosis was assessed by MTT, propidium iodide and annexin-V staining, respectively. It was found that the knockdown of MMSET expression significantly impairs cell proliferation (p&lt;0.05, two-way ANOVA) and led to a reduction of t(4;14)-positive cells in the S and G2/M phases of the cell cycle compared to t(4;14)-negative cells. In addition, the knockdown of MMSET expression induced apoptosis in t(4;14)-positive cells when compared with t(4;14)-negative cells. Lastly, in order to determine the targets of MMSET in cells carrying the t(4;14) translocation, expression array analysis on cells in which MMSET was knocked down by siRNAs was performed. It was found that several key genes involved in cell cycle control are deregulated by the knockdown of MMSET expression. Our data suggest that MMSET up-regulation may be playing a critical role in the oncogenic behaviour of MM cells carrying the t(4;14) translocation and validates MMSET as a therapeutic target in t(4;14) MM.

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

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e19514-e19514
Author(s):  
Barry Paul ◽  
Gavin Loitsch ◽  
Daniel Feinberg ◽  
Ian Barak ◽  
Zhiguo Li ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Poor Prognosis ◽  
Proteasome Inhibitors ◽  
Cell Transplant ◽  
Newly Diagnosed ◽  
Bone Marrow Fibrosis ◽  
Bone Marrow Biopsies ◽  
Immunomodulatory Agents ◽  
Marrow Fibrosis

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.

scholarly journals The Role of PET-MRI in Multiple Myeloma Patients

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 12-12
Author(s):  
Laurent Garderet ◽  
Driss Kharroubi-Lakouas ◽  
Damien roos Weil ◽  
Nathalie Jacque ◽  
Maya Ouzegdouh Ouarab ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Bone Lesion ◽  
Bone Marrow Infiltration ◽  
Bone Lesions ◽  
Newly Diagnosed ◽  
Advisory Committees ◽  
Imaging Protocol ◽  
Mri Scans

Introduction Low-dose CT scans and magnetic resonance imaging (MRI) using T2- and T1-weighted contrasted enhanced images are the standard methods of bone staging for newly diagnosed and relapsed multiple myeloma (MM) patients. Fluoro-desoxy-glucose positron emission tomography (18F-FDG PET) is also a validated procedure for the imaging of MM bone lesions and the evaluation of extramedullary plasmacytoma. Furthermore, PET CT is useful to determine the prognosis and to assess the minimal residual disease. A new hybrid technology combining PET and MRI provides metabolic and anatomic information simultaneously. We evaluated the results obtained using this new examination. Materials and Methods Thirty-nine MM patients were prospectively included in this single center observational study. Fifty-one PET-MRI scans were performed in 4 patient populations: 1/ newly diagnosed smoldering MM (NDSMM, n =10); 2/ newly diagnosed symptomatic MM (NDMM, n=18); 3/ MM patients with long term follow-up (FU, n=17); 4/ MM patients with biochemical relapse (Rel, n=6). The imaging protocol consisted of T1-weighted, DIXON and diffusion-weighted (b=50 and b=800) axial sequences from the head to the knees simultaneously acquired by PET, followed by coronal and sagittal T1-weighted and T2-weighted sequences centered on the full spine and basin. MRI was considered to be positive in the case of diffuse bone marrow infiltration (DI), diffuse patchy bone marrow infiltration (DPI), focal bone marrow lesions (FL) or extra-medullary disease (EMD). PET was considered to be positive when FDG-avid focal bone lesions, a diffuse medullary area and/or FDG-avid EMD were observed. Medullary compression was also evaluated using MRI. Results The characteristics of the patients are summarized in Table 1. They were classified according to the ISS scale (I, n=18; II, n=7; III, n=3; missing, n=11) and none had high-risk cytogenetics or extra-medullary disease. The hybrid PET-MRI technique provided good image quality in all cases with no artefacts. No medullary compression was detected by MRI. Among the overall 51 PET-MRI examinations, 38 (74%) were positive for MRI and 27 (53%) for PET. Table 2 summarizes the bone marrow results obtained using MRI and PET in the four groups of patients. EMD was not detected by either MRI or PET. In NDSMM patients, MRI was positive and PET was negative in 30%, while none were positive only for PET. In NDMM, MRI was positive and PET was negative in 22% of cases, while 5% were positive only for PET (Figure 1 shows a negative PET and a positive MRI scan in the same patient). In FU patients, 35% of MRI scans remained positive with PET negative, while 6% were PET-positive and MRI-negative. In the case of relapsed patients, MRI and PET scans were in 100% agreement. Conclusions Using PET-MRI, in addition to a search for medullary compression, MRI can provide additional bone marrow information with respect to PET in about 22 to 30% of cases, particularly in NDSMM and NDMM. In a single examination, PET-MRI can detect bone, as well as potential extra-medullary lesions and medullary compression, without irradiation. The baseline PET examination is useful to evaluate the prognosis. Legend to Figure 1: PET and MRI scans in an asymptomatic myeloma patient. PET is negative but MRI reveals a bone lesion in the left iliac crest (arrow). Disclosures Leblond: Janssen: Honoraria; Lilly: Honoraria; Astra Zeneca: Honoraria; Roche: Honoraria; Amgen: Honoraria; Gilead: Honoraria; Beigene: Honoraria; Beigene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria.

scholarly journals Treatment Approaches of Multiple Myeloma

2021 ◽  
Author(s):  
Minyahil Alebachew Woldu ◽  
Atalay Mulu Fentie ◽  
Tamrat Assefa Tadesse
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Plasma Cells ◽  
Clinical Investigation ◽  
Malignant Neoplasm ◽  
Bone Destruction ◽  
Organ Damage ◽  
Autologous Stem Cell Transplant ◽  
Bone Lesions ◽  
Newly Diagnosed

Multiple Myeloma (MM) is the most common malignant neoplasm of plasma cells that accumulate in bone marrow, leading to bone destruction and marrow failure. Clinical investigation of MM requires the evaluation of bone marrow for plasma cell infiltration, and detection and quantification of monoclonal protein in the serum or urine, and evidence for end-organ damage (i.e., hypercalcemia, renal insufficiency, anemia, or bone lesions). The overall goal of treatment of MM is to improve survival. The treatment landscape and clinical outcome of MM have changed in the last two decades, with an improved median survival of 8–10 years. Management of MM involves induction, consolidation, and maintenance therapy. Currently, Autologous stem cell transplant (ASCT) is considered as the standard care of treatment for newly diagnosed fit MM patients. Multiple combinations of proteasome inhibitors (PIs) and immunomodulatory drugs (IMIDs) such as Thalidomide, lenalidomide, and pomalidomide have been under evaluation in ASCT-eligible and ineligible settings, and studies are still ongoing. For patients with ASCT-eligible newly diagnosed MM, induction therapy with triple drugs should contain an IMiD, a PI, and a corticosteroid, usually lenalidomide-bortezomib-dexamethasone. For ASCT-ineligible patients on lenalidomide with dexamethasone (Rd), with addition of bortezomib or daratumumab can be considered.

SCIO-469, a Potent and Selective Inhibitor of p38a MAPK, Normalizes the Bone Marrow Microenvironment and Inhibits Multiple Myeloma Cell Proliferation in In Vitro and In Vivo Models.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1501-1501 ◽  
Author(s):  
Aaron N. Nguyen ◽  
Mamatha Reddy ◽  
Margaret Henson ◽  
Elizabeth G. Stebbins ◽  
Gilbert O’Young ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Drug Resistance ◽  
Critical Role ◽  
Bone Destruction ◽  
Great Promise ◽  
In Vivo Models

Abstract Despite recent advances in the treatment of multiple myeloma (MM), this disease remains incurable. Accumulating evidence suggest that the bone marrow (BM) microenvironment of MM plays a critical role in tumor growth, survival, and drug resistance. A key aspect of this tumor-supportive environment is elevated levels of cytokines and other soluble factors. Most prominent among these is IL-6, which acts as a survival factor for MM cells and promotes their proliferation, migration, and drug resistance. Other mediators also implicated in the disease are VEGF and TNFa. The p38 MAPK is activated by a multitude of signals, including pro-inflammatory cytokines (e.g., TNFa and IL-1ß) and environmental stress. Furthermore, p38 activation has been shown to be important for the synthesis and secretion of IL-6, VEGF, and TNFa. Consequently, inhibition of p38 is postulated to reduce the production of these factors implicated in MM and to have therapeutic benefit by suppressing the tumor-supportive state of the BM microenvironment. Here, we demonstrate that SCIO-469, a specific and potent inhibitor of p38a MAPK, strongly inhibits MM cell proliferation by affecting MM cells directly as well as the BM microenvironment. SCIO-469 directly inhibits MM cell proliferation in long term culture. Importantly, SCIO-469 potently inhibits IL-6 and VEGF secretion from BM stromal cells (BMSC). To examine the effect of inhibiting BMSC-derived factors important in MM, we measured MM cell proliferation using transwell plates that separate BMSC from MM cells via a porous membrane. In transwell plates containing only MM cells, MM cell proliferation was modest and was inhibited by SCIO-469. In contrast, the presence of BMSC in transwell inserts dramatically increased the proliferation of MM cells over the course of the study. This result suggests that factors (e.g., IL-6) secreted by BMSC greatly stimulate MM cell proliferation. When SCIO-469 was added to these transwell cultures containing BMSC, MM cell proliferation was inhibited significantly. Consistent with these results, we show that levels of IL-6 under these conditions mirror exactly the proliferation of MM cells; IL-6 level is high in vehicle-treated cultures and is suppressed in SCIO-469-treated cultures. Finally, in a mouse xenograft plasmacytoma model of MM, we show that p38 inhibition significantly inhibited the increase in MM tumor volume. Collectively, our data indicate that SCIO-469 is a suppressor of the BM microenvironment and an effective inhibitor of MM cell proliferation in vitro and in vivo. Since SCIO-469 also inhibits secretion of osteoclast-stimulating factors (RANKL, IL-11, and MIP1a) in the microenvironment, SCIO-469 may not only inhibit MM cell survival but may also alleviate bone-related pathologies (bone destruction and osteolytic lesions) commonly associated with MM. Therefore, SCIO-469 may offer great promise for an improved outcome for patients with MM.

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