Exposure to Novel Agents Contribute to Extramedullary Spread of Multiple Myeloma Cells and Altered Expression of Adhesion Molecules: A Clinicopathologic Study of 91 Consecutive Autopsy Cases

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1832-1832
Author(s):  
Sohtaro Mine ◽  
Shotaro Hagiwara ◽  
Amane Tagashira ◽  
Toru Igari ◽  
Akiyoshi Miwa
Keyword(s):  
Multiple Myeloma ◽  
Adhesion Molecules ◽  
Conflicts Of Interest ◽  
Remission Rate ◽  
Novel Agents ◽  
Altered Expression ◽  
Myeloma Cells ◽  
Factors Associated ◽  
Patient Profile ◽  
Duration Of Illness

Abstract Abstract 1832 Background Recent development of novel agents such as bortezomib, lenalidomide, and thalidomide improved the remission rate and survival of multiple myeloma. However, the end stage of myeloma is still uncontrollable. The extramedullary disease (EMD) progression of myeloma was frequently observed in heavily treated patients, whereas few data exist about its incidence and predictive factors. It is well known that adhesion molecules play a role in disease progression and drug resistance. We investigated factors associated with the EMD progression based on the autopsy cases. In addition, the effect of the exposure to novel agents on the expression of adhesion molecules on myeloma cells was analyzed. Methods We reviewed autopsy reports and medical records of 91 multiple myeloma cases between 1979 and 2012 at the National Medical Center for Global Health and Medicine in Tokyo, Japan. The sites of myeloma cell invasion, infection, hemorrhage and renal complications were studied gross and microscopically. Patient profile, duration of illness, type of monoclonal gammopathy, clinical stage and history of treatment were studied. Durie & Salmon's criteria was used for diagnosis and staging. Factors associated with EMD were statistically analyzed using Student's t-test and the chi-square test. NCAM, VCAM, ICAM, and LFA-1 immunostaining in the bone marrow was performed. Results In 91 autopsy cases, 62.6% was male. Mean age and the duration of illness was 63.0 (38–85) years old and 40.6 months (1–156). Eighteen patients (19.8%), 23 (25.3%), and 15 (16.4%) were treated with novel agents, SCT and both respectively. EMD progression of myeloma cells was observed in 65 patients (71.4%). Frequent sites of EMD were spleen (48.9%), liver (37.8%), kidney (31.1%), lymph nodes (28.6%), lung (25.6%), pancreas (20.0%), and gastro-intestinal tract (18.9%). The incidence of EMD was significantly higher in patients treated with novel agents than in patients without novel agents (94.4% vs. 65.8%, p=0.016). The risk factors of EMD were novel agents, longer duration of illness, and adverse cytogenetic abnormalities. In the cases with novel agents, the expression of NCAM was significantly low (11.1% vs. 57.4%, p=0.049) compared to the cases without novel agents. However, the expression of VCAM was significantly higher in the cases with novel agents than the cases without novel agents (40.0% vs. 0%, p=0.01). Conclusion Multi-organ involvement of myeloma is not rare in autopsy cases of the disease. Exposure to novel agents may contribute to extramedullary spread of myeloma cells and altered expression of adhesion molecules. Further study on other adhesion molecules is needed. Disclosures: No relevant conflicts of interest to declare.

Activation of Rac1 through FARP1 Induces Dexamethasone Resistance in Multiple Myeloma Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2248-2248
Author(s):  
Atsuko Yamazaki ◽  
Masahiro Takeuchi ◽  
Tatsuzo Mishina ◽  
Miki Yamazaki ◽  
Chika Kawajiri ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Line ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Conflicts Of Interest ◽  
Novel Agents ◽  
Tumor Cell Death ◽  
Myeloma Cells ◽  
Dexamethasone Resistance ◽  
Number Of Cells

Abstract Recently, novel agents such as bortezomib and lenalidomide have been introduced for multiple myeloma (MM) treatment and have improved patients' survival drastically. However, dexamethasone remains a mainstay in the treatment of MM. Dexamethasone effectively induces tumor cell death when used for the initial treatment of MM. In addition, dexamethasone has a synergistic effect with novel agents and is hence used in combination with such agents. However, prolonged dexamethasone exposure may lead to drug resistance. To elucidate the mechanism of dexamethasone resistance, we generated a dexamethasone-resistant subline of the MM cell line RPMI8226. We cultured RPMI8226 cells with 1 µM dexamethasone for 7 weeks and established the dexamethasone-resistant cell line Dex-R. This cell line showed no difference in survival in the presence or absence of 1 µM dexamethasone. We then examined differences in gene expression between RPMI8226 and Dex-R cells using cDNA microarray. Expression of the FARP1 gene, which is a transforming growth factor beta (TGF-b) target gene in myeloma cells, was increased approximately 50-fold in Dex-R cells compared to that in RPMI8226 cells. In some myeloma patients who become chemoresistant, myeloma cells show high levels of FARP1 expression at the initial stage. FARP1 has a Rho-GEF domain and can associate with proteins on the cell membrane through the FERM domain. In the nervous system, FARP1 is involved in synaptogenesis via the activation of Rac1. Based on these observations, we hypothesize that Dex-R cells acquires dexamethasone resistance with an increase in the level of FARP1 expression via the activation of Rac1. To verify this hypothesis, we established inducible FARP1 knockdown Dex-R cells using the TET-ON lentiviral system. We cultivated these cells for 24 h with doxycycline and added 1 µM dexamethasone. A total of 48 h after adding dexamethasone, we measured cell viability using the MTS assay. We cultured Dex-R cells with a Rac1 inhibitor (NSC23766) and added dexamethasone 12 h later. FARP1 expression decreased to approximately 10% in FARP1 knockdown cells 24 h after the addition of doxycycline. Without dexamethasone, there was no difference in survival in the presence or absence of doxycycline. However, when cells were cultured with dexamethasone, the growth of FARP1 knockdown Dex-R cells was significantly inhibited compared with that of the control (Fig 1). Next, we examined the change in dexamethasone resistance on the addition of the Rac1 inhibitor. The number of cells increased after 96 h without dexamethasone. On the other hand, the number of cells significantly decreased when cultured with dexamethasone (Fig 2). These data suggest that resistance to dexamethasone in Dex-R cells was mitigated by the inhibition of Rac1. We conclude that the activation of Rac1 through FARP1 is one mechanism of dexamethasone resistance in MM. Disclosures No relevant conflicts of interest to declare.

Clinicopathological Analysis of CNS Involvement in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5326-5326
Author(s):  
Tomiteru Togano ◽  
Sohtaro MINE ◽  
Akiyoshi Miwa ◽  
Shotaro Hagiwara
Keyword(s):  
Spinal Cord ◽  
Multiple Myeloma ◽  
Peripheral Nerve ◽  
Central Canal ◽  
Novel Agents ◽  
Cell Leukemia ◽  
Cns Involvement ◽  
Myeloma Cells ◽  
Duration Of Illness ◽  
Clinicopathological Analysis

Abstract Background Extramedullary disease (EMD) is often observed in end stage of multiple myeloma (MM).However, central nervous system (CNS) involvement is very rare. Thus, little is known about CNS involvement of MM. We conducted clinicopathological analysis. Methods We reviewed medical records and pathological specimens of multiple myeloma cases between 1979 and 2015 at the National Center for Global Health and Medicine in Tokyo, Japan. Patient information and duration of illness, clinical finding including type of monoclonal protein, Durie & Salmon's (DS) staging, treatment and pathological findings were collected. CNS involvement was diagnosed pathologically. Results We found 15 cases of CNS involvement multiple myeloma. In 15 cases, 66.6 % was male. Mean age and the duration of illness were 56 years old and 35.7 months, respectively. DS stage was 3A (46 %), 2A (20 %), 2A (20 %), 1A (13 %). Monoclonal protein was IgG (46 %), IgA (33 %), BJP (0.6 %) and non-secretary (13 %). Lambda chain was 62 %. Manifestation at diagnosis CNS involvement was disturbance of consciousness (40 %), visual disturbance (20 %), dysphagia (13%), paresthesia of extremities (20 %), vomiting or nausea (13 %). Other organ involvement was spleen (60 %), pancreas (53 %), liver (53 %), adrenal gland (46 %), testis (31 %) and plasma cell leukemia (40 %). In two cases, peripheral nerve involvement was found. Seven of 15 patients were previously treated with novel agents such as bortezomib, thalidomide, and lenalidomide. Recent 5 years, the frequency of CNS involvement increased more than threefold (1.0 case/year), compared to 1979-2010 (0.3 case/year). In all cases, myeloma cells infiltrated dura mater or subarachnoid cavity. Infiltration in parenchyma of brain was seen in only 2cases. Myeloma cells were seen at loose section of connective tissue around vascular in most cases. Interestingly, myeloma cells were often infiltrated at peripheral nerve, spinal cord cavity and central canal without spinal cord. Conclusions In the era of novel agents, the incidence of CNS involvement in patients with multiple myeloma has increased. We found myeloma cells were often infiltrated at peripheral nerve, spinal cord cavity and central canal without spinal cord. Our study revealed that exposure to novel agents, lambda type MM, multi-organ involvement and plasma cell leukemia were associated with CNS involvement. Disclosures Hagiwara: Celgene Corporation: Membership on an entity's Board of Directors or advisory committees.

CD56 Expression Is the Potent Prognostic Marker of the Thalidomide Efficacy in the Patients with Multiple Myeloma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5142-5142
Author(s):  
Akio Mori ◽  
Yutaka Tsutsumi ◽  
Satoshi Hashino ◽  
Hiroe Kanamori ◽  
Makoto Ibata ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Adhesion ◽  
Adhesion Molecules ◽  
Prognostic Marker ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Myeloma Cells ◽  
Cd56 Expression

Abstract Thalidomide (Thal) alone or in combination with steroids achieves responses even in the setting of refractory multiple myeloma (MM), however, responses are still limited. The precise mechanism of Thal action is unknown, further, no distinct marker, which could prognosticate the efficacy of Thal, is known. Therefore, we evaluated the correlation between the efficacy of Thal and the potent prognostic factors in patients with refractory MM. Ten patients with refractory MM received Thal at doses of 50 or 100 mg per day and steroids, either dexamethasone (Dex) or prednisolone (PSL). Dex was administrated 20 mg per day, 4 days every 28 days, and PSL was administrated 10 mg per day. The median age was 71.5 years (range, 62–79 years) and 20 % were man, and all patients were diagnosed as clinical stage IIIA based on the Durie and Salmon classification. The therapeutic response was assessed according to the modified criteria of Southwest Oncology Group (SWOG). Among 10 patients, 7 patients were the responders; 2 had complete remission, 3 had partial remission, and 2 had minimal remission. There were no differences in the pretreatment characteristics of responders and nonresponders (age, sex, type and concentration of serum and/or urine monoclonal component, international prognostic index, presence of bone lesion, and chromosomal abnormalities). However, flow cytometric evaluation of the myeloma cells revealed that CD56, which is one of the adhesion molecules N-CAM, expressed more than 45 % in all responders, while those expressed less than 5 % in all nonresponders (84 ± 19 (±SD) % v/s 4 ± 2 %, P=0.017). Furthermore, CD56 expression of the myeloma cells was reduced from 84% to 70 ± 32 % after Thal therapy in all evaluated responders (P =0.048). These results suggest that CD56 expression of the myeloma cells could be the potent prognostic marker of the Thal efficacy. Moreover, it was reported that Thal reduced the expression of cell adhesion molecules, such as LFA-1 and ICAM-1, and abrogated the binding of MM cells to bone marrow stromal cells, that triggered the secretion of interleukin-6 and vascular endothelial growth factor. Taken together, it was suggested that Thal reduced the expression of CD56 and altered the MM cell adhesion to bone marrow stromal cells, and that could be one of the pathogenesis of anti-MM activity of Thal.

Serum/Glucocorticoid-Regulated Kinase 1 (SGK1) Is a Prominent Target Gene of the Transcriptional Response to Cytokines In Multiple Myeloma and Supports the Growth of Myeloma Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1915-1915
Author(s):  
Unn-Merete Fagerli ◽  
Thorsten Stühmer ◽  
Toril Holien ◽  
Randi Utne Holt ◽  
Ove Bruland ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Growth Factors ◽  
Cell Lines ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Transcriptional Response ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Growth And Survival ◽  
Stat3 Phosphorylation

Abstract Abstract 1915 Multiple myeloma is a paradigm for a malignant disease that exploits external stimuli of the microenvironment for growth and survival. A thorough understanding of the complex interactions between malignant plasma cells and their surrounding requires a detailed analysis of the transcriptional response of myeloma cells to environmental signals. We hypothesized that the intracellular signals evoked by cytokines converge and regulate transcription of a set of genes that are common targets for several growth factors and therefore constitute pivotal mediators of the tumor-promoting effects of autocrine or paracrine stimuli. To identify such targets, we determined the changes in gene expression induced by IL-6, TNFalpha, IL-21 or co-culture with bone marrow stromal cells in myeloma cell lines. Among a limited set of genes that were consistently activated in response to growth factors, a prominent transcriptional target of cytokine-induced signaling in myeloma cells was the gene encoding the serine/threonine kinase SGK1, which is a down-stream effector of PI3-kinase and highly homologous to AKT. We could demonstrate a rapid, strong and sustained induction of SGK1 in the cell lines INA-6, ANBL-6, IH-1, OH-2 and MM.1S as well as in primary myeloma cells. Pharmacologic inhibition of the JAK/STAT pathway abolished STAT3 phosphorylation and SGK1 induction. In addition, shRNA-mediated knock-down of STAT3 reduced basal and induced SGK1 levels, demonstrating the involvement of the JAK/STAT3 signaling pathway in SGK1 induction. Furthermore, down-regulation of SGK1 by shRNAs resulted in decreased proliferation and viability of myeloma cell lines. Our results indicate that SGK1 is a highly cytokine-responsive gene in myeloma cells promoting their growth and survival and represents an attractive candidate for further evaluation as a therapeutic target. Disclosures: No relevant conflicts of interest to declare.

Anti-CD38 Pretargeted Radioimmunotherapy Demonstrates Therapeutic Efficacy In a Human Multiple Myeloma Mouse Xenograft Model

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1842-1842
Author(s):  
Damian J. Green ◽  
Nural N. Orgun ◽  
Mark D. Hylarides ◽  
John M. Pagel ◽  
Donald K. Hamlin ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Plasma Cell ◽  
Nude Mice ◽  
Therapeutic Efficacy ◽  
Untreated Control ◽  
Novel Agents ◽  
Beta Particle ◽  
Control Groups ◽  
Myeloma Cells ◽  
Athymic Nude Mice

Abstract Abstract 1842 Multiple myeloma (MM) remains incurable despite improved response rates and improved progression free survival in the era of therapy with novel agents, including bortezomib, thalidomide, and lenalidomide. Disease persistence is presumably due to residual malignant plasma cell clones that evade or develop resistance to available therapies. The efficacy of radioimmunotherapy (RIT) in the treatment of hematologic malignancies is well established and the radiosensitivity of malignant plasma cells has been demonstrated in both preclinical and clinical settings. The ectoenzyme receptor CD38 is a plasma cell antigen that exhibits relatively specific, stable and uniform expression (95–100%) at a high epitope density on myeloma cells, making it an attractive target for antibody based therapies, including RIT. Pretargeted RIT (PRIT), using a multi-step streptavidin (SA)-biotin targeting system enhances the therapeutic index of delivered radiation. We have generated an anti-CD38 antibody (Ab)-SA synthetic chemical conjugate (OKT10-SA). The OKT10-SA construct binds with high avidity to myeloma cells while retaining full biotin-binding capability for radiolabeled DOTA-biotin. Blood, tumor and nonspecific organ uptakes of OKT10-SA were directly measured in biodistribution experiments involving athymic nude mice bearing human MM xenograft tumors. Groups of 5 mice with s.c. L363 human MM (IgG) xenograft tumors received 1.4 nmol (300 μg) of either OKT10-SA (anti-CD38 SA) or an IgG1 isotype matched control Ab BHV1-SA (bovine herpes virus-1) 22 hrs prior to synthetic biotin-acetyl-galactosamine clearing agent (CA; 5.8 nmol [50 μg]) and 24 hrs prior to trace labeled 111In-DOTA-biotin (1 μg). The CA removed >95% of both unbound OKT10-SA and BHV1-SA from the mouse circulation within 30 minutes of administration. Animals were euthanized and comprehensive tissue biodistributions were assessed 2, 24, 48 and 96 hrs after 111In-DOTA-biotin injection. Tumors excised from mice pretargeted with OKT10-SA contained 13.1 ± 1.9 % of the injected dose of 111In-DOTA-biotin per gram (% ID/g) after 2 hrs and 8.8 ± 2.8 % ID/g after 24 hrs compared to 2.4 ± 0.6 % ID/g after 2 hrs and 0.9 ± 0.4 % ID/g after 24 hrs in tumors excised from control mice pretargeted with BHV1-SA. Tumor-to-normal organ ratios of absorbed radioactivity were 8:1; 10:1; 8:1; and 6:1 respectively for blood, lung, liver and kidney in mice pretargeted with OKT10-SA; compared to 0.6:1; 0.9:1; 0.8:1 and 0.4:1 respectively, in control mice pretargeted with BHV1-SA. Therapy studies were then performed in athymic nude mice (n=9-10/group) bearing s.c. L363 human MM xenograft tumors. Reagent concentrations and time-points for administration of OKT10-SA, BHV1-SA and CA were identical to those reported for the biodistribution studies. The high energy beta particle emitter 90Yttrium (t1/2 = 64 hrs) was used as the therapeutic radionuclide. 90Y-DOTA-biotin (2 μg) was labeled with 400 μCi, 800 μCi, or 1200 μCi per mouse in 3 OKT10-SA groups and 3 control groups (untreated control; 800 μCi or 1200 μCi 90Y-DOTA-biotin following BHV1-SA). All mice in the untreated control and BHV1-SA control groups experienced exponential MM tumor growth and 78% of the untreated control animals required euthanasia within 17 days. All mice pretargeted with OKT10-SA demonstrated tumor shrinkage by day 6 at all dose levels (see figure). After 17 days, 90% of the OKT10-SA treated animals in the 400 μCi and 1200 μCi groups and 100% of the animals in the 800 μCi remained alive. One animal treated with 1200 μCi was euthanized on day 10 due to weight loss, however the remaining 9 animals from that group were 106 ±9% of initial body weight on day 17. Objective remissions were observed within 6 days in 100% of the mice treated with OKT10-SA followed by 1200 μCi of 90Y-DOTA-biotin, including 100% complete remissions (no detectable tumor in OKT10-SA treated mice compared to tumors that were 5240 ± 2495% of initial tumor volume in untreated control animals) by day 17. These studies represent the first application of both PRIT and CD38 targeted radioimmunotherapy in MM. Favorable OKT10-SA biodistribution findings correlate with early evidence of therapeutic efficacy. Tumor responses in this MM xenograft tumor model are encouraging, but long term toxicity and survival results are not yet mature. Future studies combining PRIT and novel agents are planned in xenograft and SCID-hu myeloma models. Disclosures: Gopal: Millenium. Wood:BD Biosciences: Research Funding.

A Novel Small Molecule with Epigenetic Activity Prolongs Survival in a Mouse Model of Multiple Myeloma Refractory to Standard Drugs

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5121-5121
Author(s):  
Sergei Vatolin ◽  
Khan Nazeer Shahper ◽  
Yvonne Parker ◽  
Daniel Lindner ◽  
Frederic J. Reu
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Chromatin Condensation ◽  
Xenograft Model ◽  
Myeloma Cells ◽  
Tail Vein ◽  
Tail Vein Injection ◽  
Mouse Xenograft Model ◽  
Number Of Patients

Abstract Abstract 5121 Multiple myeloma refractory to bortezomib, IMiDs™, and conventional therapies represents an unmet medical need. An increasing number of patients progress to this stage since treatment related mortality has decreased. To test promising compounds for activity in this setting we established an NSG mouse xenograft model with serial transplantation by tail vein injection of myeloma cells from a patient with IgG kappa myeloma relapsed and refractory to all standard drugs. Eight days after tail vein injection monoclonal human IgG can be detected in serum. Bone marrow engraftment in young (6–12 weeks) NSG mice after sublethal radiation (275cGy) is close to 100% (n=32). Untreated mice die within less than 2 months, usually with liver and spleen metastasis (anti-human CD138 flow cytometry). In a drug screen that used a novel method developed in our lab, chromatin condensation PCR, we identified a non nucleoside compound (4I3) that potently (1mM) reactivated expression of epigenetically silenced genes and displayed cancer-specific growth and survival inhibition in myeloma cell lines but not normal cells. Normal bone marrow cells continued to divide at doses 10x higher than required to kill 80% of myeloma cells. 4I3 suppressed DNMT1 protein but rapid cell kill (within 1–2 days) suggested additional mechanisms which we currently investigate. Given IV to mice after documentation of engraftment by IgG serum immunoblots, it prolonged survival in an ongoing experiment. Updated results will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

The Expression Of CD200 As a Prognostic Factor In Newly Diagnosed Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3082-3082
Author(s):  
Yi Li ◽  
Wenjun Wu ◽  
Jingsong He ◽  
Xiaoyan Han ◽  
Gaofeng Zheng ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Conflicts Of Interest ◽  
Progression Free Survival ◽  
Staging System ◽  
Newly Diagnosed ◽  
New Approach ◽  
Myeloma Cells ◽  
Targeting Therapy ◽  
Molecular Therapeutic

Abstract Introduction multiple myeloma (MM) is currently an incurable hematological malignancy. Discovering molecular therapeutic targets is a new approach to improve the outcome in the treatment of the malignant disease. As CD200 is a type Ⅰmembrane glycoprotein expressed on myeloma cells, we asked if the expression of CD200 could serve as a prognostic marker for MM patients. Our data indicated that the expression level of CD200 is indeed correlated with the prognosis of the MM patients. Methods bone marrow samples from 96 newly diagnosed MM patients from April 2011 to July 2013 were evaluated by flow cytometry, using PE-conjugated anti-CD200 mAb, FITC-conjugated anti-CD138 mAb, and PE-Cy7-conjugated anti-CD45 mAb. PE-or FITC-conjugated normal mouse IgG was used as isotype-matched controls. Results 96 MM patients were investigated in the present study, including 60 men and 36 women, with a median age of 63 years (range 34–86 years). 81/96(84%) MM patients were CD200 positive with a median Mean Fluorescence Intensity (MFI) of 127 as analyzed by flow cytometry, which was consistent with the previous studies. While in 15 of 96 patients, CD200 expression was undetectable. Among the CD200 positive ones 7.40% patients were classified as stage Ⅰ, 12.35% were stage Ⅱ, and 80.25% were stage Ⅲ according to Durie–Salmon staging criteria. 40.74% patients were stageⅠ, 22.22% were stage Ⅱ, and 37.04% were stage Ⅲ, according to the International Staging System (ISS). Analysis of the CD200 positive patients revealed the MFI<127 group had a better progression free survival (PFS) (p=0.046) (Fig 1A) and overall survival (OS) (p=0.069) compared to those with MFI≥127. In the patients with age ≥65 years old, PFS (p=0.023) (Fig 1B) and OS (p=0.044) (Fig 1C) were much shorter in the MFI≥127 group, compared to the MFI<127 ones. Conclusions Our study demonstrated that the expression and MFI of CD200 on primary multiple myeloma cells is correlated with the prognosis of the MM patients. The better PFS and OS were observed in the MFI<127 group compared to the patients with MFI≥127, especially in the patients with age≥65 years old. Improved PFS in CD200 positive ones is likely due to the immune suppression mediated by CD200. Our study suggests that targeting therapy against CD200 may become a new approach to the treatment of MM in clinical practice. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Fate of Ikaros in Multiple Myeloma Cells upon Treatment with Lenalidomide and Proteasome Inhibitor

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3281-3281
Author(s):  
Saravanan Ganesan ◽  
Nithya Balasundaram ◽  
Hamenth Kumar Palani ◽  
Ansu Abu Alex ◽  
Sachin David ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Proteasome Inhibition ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Ubiquitinated Proteins ◽  
Two Agents ◽  
Autophagy Pathway

Abstract Recent evidences suggests that the efficacy of Lenalidomide (LEN) depends upon its ability to degrade IKZF1 and IKZF3 proteins via cereblon dependent ubiquitin proteasome pathway [Science. 2014 Jan 17; 343(6168): 301-305]. Based on this model it would theoretically be antagonistic to combine LEN with proteasome inhibitors (PI). However, it is well recognized that there is significant synergism when LEN is combined with PI and this combination is routinely and effectively used in the clinic. The mechanism of synergy and the fate of IKZF1 and IKZF3 when these two agents are combined is poorly understood. We undertook a series of experiments to study the fate of IKZF1 when this combination of drugs was used in multiple myeloma cells. Combining LEN (1uM) along with bortezomib (BTZ; 1nM) a PI showed a significant kill on U266 cells (myeloma cell line) on day 5 post treatment (n=3; P=0.02) when compared to either of the agents alone. In an MTT assay, the synergism was well documented with a combination index of 0.5 (Fig 1a). Next we assessed the function of proteasome (chymotrypsin activity) when LEN was combined with PI. We observed that LEN alone does not interfere with proteasome activity. It was noted that BTZ alone at the concentration used (5 nM) was able to effectively inhibit the activity of proteasome (Fig 1b). It was also observed that combining these two agents does not interfere with BTZ action in inhibiting proteasome complex (Fig 1b). As a result of efficient proteasome inhibition, we observed an accumulation of ubiquitinated proteins in the BTZ and LEN + BTZ treated cells when compared to control and LEN alone treated cells (Fig 1c). Next, we looked for the fate of IKZF1 in U266 cells treated with LEN, BTZ and in combination of both the drugs. As reported, we observed a degradation of IKZF1 in U266 cells upon treatment with LEN. While we did not see any degradation of IKZF1 in BTZ alone treated cells. It was noted that in combination treated cells (LEN+BTZ) there was a degradation of IKZF1 (Fig 1c). In spite of significant proteasome complex inhibition, degradation of IKZF1 was observed which suggested a proteasome independent mechanism. It is well known that proteasome inhibition results in upregulation of the autophagy pathway which in turn can degrade the accumulated ubiquitinated proteins. We noted that upon treatment with BTZ or LEN+BTZ an induction of autophagy was observed, as evidenced by an increase in generation of LC3II bands on an immunoblot (Fig 1c). To support our hypothesis that IKZF1 is degraded by autophagy in the absence of proteasome complex, we pre-treated the U266 cells with an autophagy inhibitor (3-methyladenine) followed by treatment with LEN and BTZ and noted an accumulation of IKZF1 proteins (Fig 1d). We also observed a downregulation of IKZF1 target genes IRF4 and c-MYC by 12 and 24 hours in the combination treated cells (data not shown). Taken together this data demonstrates that there is (i) significant in-vitro synergism between the two agents (ii) the combination additively induces autophagy pathway (iii) IKZF1 protein can be degraded via this autophagy pathway in the presence of effective proteasome inhibition. While additional mechanisms of synergy between these two agents cannot be excluded, further enhancing autophagy pathway in these cells by drugs like sirolimus (autophagy inducer in myeloma cells) could potentially improve the synergy between these two drugs. Disclosures No relevant conflicts of interest to declare.

Phenotyping Studies of Clonotypic B Lymphocytes From Patients With Multiple Myeloma by Flow Cytometry

2009 ◽  
Vol 133 (10) ◽  
pp. 1594-1599 ◽  
Author(s):  
E. Joseph Conway ◽  
Jianguo Wen ◽  
Yongdong Feng ◽  
Albert Mo ◽  
Wan-Ting Huang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
B Lymphocytes ◽  
Light Chain ◽  
Peripheral Blood ◽  
Remission Rate ◽  
Light Chains ◽  
Immunoglobulin Light Chain ◽  
Peripheral Blood Stem Cells ◽  
Myeloma Cells

Abstract Context.—Clonotypic B lymphocytes, monoclonal B lymphocytes sharing identical, rearranged IGH-CDR3 sequences with the patient's myeloma cells, have been detected in the peripheral blood of patients with multiple myeloma. These cells have been postulated to act as a therapy-resistant tumor reservoir that drives recurrence. Objective.—To characterize clonotypic B lymphocytes for future investigation of their role in myeloma pathogenesis. Design.—Harvests of cryopreserved peripheral blood stem-cells from 20 myeloma patients were enriched for clonotypic B lymphocytes. Cytoplasmic immunoglobulin light chain and surface immunophenotype were analyzed by flow cytometry. IGH-CDR3 gene-rearrangement pattern was performed to determine clonality. Posttransplant remission rate was compared with the percentage of clonotypic B lymphocytes. Results.—Clonotypic B lymphocytes expressing CD34±, CD38+, CD184+, CD31±, CD50±, CD138−, CD19−, CD20−, and the same immunoglobulin light chain as the patients' known myeloma cells were identified in 12 of 20 patients (60%). Progenitor B lymphocytes expressing similar surface immunophenotype but opposite light chains were identified in the same patients. Polymerase chain reaction for IGH rearrangement showed clonal rearrangement pattern in clonotypic lymphocytes but not in B lymphocytes expressing light chains opposite to myeloma cells. There was no statistically significant correlation between the percentage of clonotypic B lymphocytes and response to autologous transplant. Conclusions.—Clonotypic B lymphocytes expressing CD34, but not CD19, were identified in stem cell harvests from patients with myeloma and could represent progenitor cells of neoplastic plasma cells. However, the same or similar immunophenotyping can be detected in both clonotypic B lymphocytes and benign progenitor B cells, suggesting clonality analysis might be needed to determine clonotypic B lymphocytes in patients with myeloma. Further studies are warranted to study the role of clonotypic B lymphocytes in the pathogenesis of myeloma.

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