A Gene Expression Based Proliferation Index as Independent Prognostic Factor in Multiple Myeloma.

Abstract BACKGROUND. The proliferation-rate of primary myeloma cells is a strong adverse prognostic factor in various trials, but not routinely assessed, partially due to effort in obtaining it. AIM. As gene-expression profiling is increasingly considered a standard diagnostics in myeloma, we investigated the possibility to develop a prognostically relevant gene-expression based proliferation index (GPI). PATIENTS AND METHODS. Gene expression was determined by Affymetrix DNA-microarrays in 784 samples including two independent sets of 233 and 345 CD138-purified myeloma cells from previously untreated patients. The GPI was derived by selecting genes associated with proliferation (in terms of gene ontology) differentially expressed in proliferating malignant (human myeloma cell lines) and benign (plasmablastic) cells compared to non-proliferating, non-malignant cells (normal plasma cells and memory B-cells). The GPI comprises the sum of the expression values of 50 genes (ASPM, AURKA, AURKB, BIRC5, BRCA1, BUB1, BUB1B, CCNA2, CCNB1, CCNB2, CDC2, CDC20, CDC25C, CDC6, CDCA8, CDKN3, CEP55, CHEK1, CKS1B, CKS2, DLG7, ESPL1, GINS1, GTSE1, KIAA1794, KIF11, KIF15, KIF20A, KIF2C, KNTC2, MAD2L1, MCM10, MCM6, MKI67, NCAPD3, NCAPG, NCAPG2, NEK2, NPM1, PAK3, PCNA, PGAM1, PLK4, PTTG1, RACGAP1, SMC2, SPAG5. STIL, TPX2, ZWINT). Proliferation of primary myeloma cells was assessed by propidium iodinestaining (n=67). Chromosomal aberrations were assessed by comprehensive iFISH using a set of probes for the chromosomal regions 1q21, 6q21, 8p21, 9q34, 11q23, 11q13, 13q14.3, 14q32, 15q22, 17p13, 19q13, 22q11, as well as the translocations t(4;14)(p16.3;q32.3) and t(11;14)(q13;q32.3). RESULTS. In the two groups, 39 and 32 percent of primary myeloma cells show a GPI above the median plus three standard deviations of normal bone marrow plasma cells, respectively. The GPI is significantly higher in advanced- compared to early-stage myeloma (P=.001) and in patients harboring a gain of 1q21 (n=95, P<0.001). It correlates significantly with proliferation as determined by propidium iodine in primary myeloma cells (rs=.52, P<.001, n=67). The GPI as continuous variable is significantly predictive for event-free survival (EFS, n=120, P<.001, n=345, P<.001, respectively) and overall survival (OAS, n=345, P<.001) in patients treated with high-dose chemotherapy, independent of serum-β2-microglobulin (B2M) or ISS-stage. A GPI above the median (GPIhigh) delineated significantly inferior EFS (n=168, 41.6 vs. 26 months, P=.04, HR 1.57, CI [1.02,2.42]; n=345, 68.6 vs. 45.2 months, HR 1.55, CI [1.16,2.09], P=.003) and OAS (n=345, P<.001) in two independent cohorts of patients undergoing high-dose chemotherapy. By using B2M above 3.5 mg/l and GPI as staging variables, four groups with difference in median EFS (n=345, B2M <3.5mg/l, GPIhigh/low 76.1 months; B2M < 3.5mg/l, GPIhigh 62.4 months, B2M ≥3.5mg/l, GPIlow 41.8 months, B2M ≥3.5mg/l, GPI 36.1 months, P<.001) and OAS can be delineated. CONCLUSION. The GPI represents a validated tool for the assessment of proliferation in multiple myeloma patients, allows a risk stratification in terms of proliferation either alone or in combination with B2M or ISS, respectively, and has the potential to be used within a risk adapted targeting of anti-proliferative treatment.

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CS1 Is Expressed on Myeloma Cells from Early Stage, Late Stage, and Drug-Treated Multiple Myeloma Patients, and Is Selectively Targeted by the HuLuc63 Antibody.

Blood ◽

10.1182/blood.v108.11.660.660 ◽

2006 ◽

Vol 108 (11) ◽

pp. 660-660 ◽

Cited By ~ 5

Author(s):

Susann Szmania ◽

Balaji Balasa ◽

Priyangi Malaviarachchi ◽

Fenguhuang Zhan ◽

Yongsheng Huang ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Protein Expression ◽

Nk Cells ◽

Late Stage ◽

Plasma Cells ◽

Nk Cell ◽

Early Stage ◽

High Dose ◽

Myeloma Cells

Abstract Introduction: One-third of multiple myeloma (MM) patients exhibit high-risk features such as abnormal cytogenetics, high LDH, amplification of CKS1-B or spiked expression of MAF, MAF-B or FGFR3. While not affecting complete response rates, median durations of event-free and overall survival, even with high-dose melphalan-based tandem autotransplants of such patients, do not exceed 24 mo and 36 mo, compared to 60 mo and ≥ 90 mo for the remainder. Monoclonal antibody (mAb)-mediated therapy may target a chemotherapy-resistant myeloma cell pool. CS1 (CD2 subset 1, CRACC, SLAMF7), a member of the CD2 family of cell surface glycoproteins, exhibits high-level expression on primary myeloma cells, indicating that CS1 is a potential target for treatment in MM. Methods: Gene expression was assessed using an Affymetrix GeneChip array. Protein expression was measured by flow cytometry, and immunohistochemistry (IHC), using HuLuc63, a novel humanized anti-CS1 mAb. HuLuc63-mediated lysis of myeloma cells via antibody dependent cellular cytotoxicity (ADCC) was measured by 51Cr-release. Results: CS1 mRNA was detected in >95% of CD138+ purified plasma cells from >95% of healthy donors, newly diagnosed myeloma patients, and those with relapsed myeloma (Fig. 1a). CS1 remained highly expressed in patients following VDTPACE treatment, albeit at a reduced level. CS1 expression was also high following bortezomib (Velcade®) treatment, with a subset of patients showing increased expression post-treatment. CS1 protein expression on primary myeloma cells was confirmed by flow cytometry, while IHC analysis of normal tissues revealed anti-CS1 staining primarily on CD138+ tissue plasma cells. Finally, we determined that HuLuc63 could induce killing of myeloma cells using purified allogeneic NK cells (Fig. 1b). Blocking the Fc receptor greatly reduced this activity indicating an ADCC mechanism. Killing of myeloma targets was also observed in autologous systems suggesting that HuLuc63 can overcome KIR-mediated NK cell inhibition of autologous NK cells. In summary, we observed high mRNA and protein expression of CS1 in myeloma from early stage, late stage, and treated patients, and showed enhanced lysis of myeloma cells in vitro with HuLuc63. Our data support the potential clinical utility of CS1-targeted therapy. HuLuc63 will be entering a phase I clinical trial for advanced myeloma patients. Figure 1a. CS1 mRNA is highly expressed in CD138+ purified plasa cells. Figure 1a. CS1 mRNA is highly expressed in CD138+ purified plasa cells. Figure 1b. CS1 antibody enhances killing of myeloma cells by allo - NK cells. Figure 1b. CS1 antibody enhances killing of myeloma cells by allo - NK cells.

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Prognostication by miRNome-Profiling in Multiple Myeloma

Blood ◽

10.1182/blood.v118.21.1822.1822 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1822-1822

Author(s):

Anja Seckinger ◽

Tobias Meißner ◽

Vladimir Benes ◽

Sabine Schmidt ◽

Jonathan Blake ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Bone Marrow ◽

Plasma Cells ◽

Proliferation Index ◽

Normal Bone Marrow ◽

Global Test ◽

Myeloma Cells ◽

Normal Bone ◽

Bone Marrow Plasma

Abstract Abstract 1822 INTRODUCTION. MicroRNAs are an abundant class of small non-protein-coding RNAs that function as negative gene regulators in diverse biological processes including cancer by affecting the stability and translation of mRNAs. METHODS. We determined expression of 559 miRNAs by miChip (Exiqon LNA Array probes V9.2) in CD138-purified myeloma cells from previously untreated patients (n=69), normal bone marrow plasma cells of healthy donors (pooled to n=3), and human myeloma cell lines (n=20). For normalization, an invariant-based method was applied. Gene expression profiling was performed using Affymetrix U133 2.0 DNA-microarrays. RESULTS. We found 29 miRNAs to be significantly up- and 35 down-regulated in myeloma cells vs. normal bone marrow plasma cells, respectively. Expression of 18 miRNAs was simultaneously significantly (P<.01) associated with event-free survival (EFS) and overall survival (OS), and allowed the delineation of prognostic groups. Of these, miRNAs miR-659 (located at 22q12.1) was significantly lower expressed in myeloma cells compared to normal bone marrow plasma cells. For this miRNA, low expression delineates a group with inferior EFS (median 19.7 months vs. not reached, P<.001) and OS (median 52.9 months vs. not reached, P<.001). This group shows a significantly higher gene expression based proliferation index. In contrast, high miR-590 expression (located at 7q11.23) delineates a group with inferior EFS (median 12.4 vs. 36 months, P<0.001) and OS (median 29.4 months vs. not reached, P<.001). By using Goeman's global test, a significant association of the predicted target gene signatures with survival could be found for both, EFS (miR-590-5p, P<.001; miR-659, P<.001) and OS (P=.009; P=.002). CONCLUSION. In conclusion, we demonstrate the prognostic relevance of miRNome profiling in multiple myeloma. Disclosures: No relevant conflicts of interest to declare.

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Bone Morphogenic Protein 6: A Prognostic Factor Expressed by Normal Plasma Cells and Multiple Myeloma Cells Inhibiting Their Proliferation and Angiogenesis Induction

Blood ◽

10.1182/blood.v112.11.2701.2701 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2701-2701

Author(s):

Anja Seckinger ◽

Tobias Meißner ◽

Jérôme Moreaux ◽

Hartmut Goldschmidt ◽

Axel Benner ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Overall Survival ◽

Cell Lines ◽

Plasma Cells ◽

Myeloma Cell ◽

High Dose Chemotherapy ◽

High Dose ◽

Myeloma Cells

Abstract BACKGROUND: Pathogenesis of multiple myeloma is partly attributed to an aberrant expression of proliferation-, pro-angiogenic and bone-metabolism modifying factors by malignant plasma-cells. AIM. Given the long and variable time-span from first diagnosis of early-stage plasma-cell dyscrasias to overt myeloma and the low proliferation rate of malignant plasma-cells, we hypothesize these to concomitantly express a novel class of anti-proliferative factors of potential prognostic relevance. Here, bone morphogenic proteins (BMPs) represent possible candidates, as they inhibit proliferation, stimulate bone formation, and have an impact on the survival of cancer patients. PATIENTS AND METHODS. We assessed expression of BMPs and its receptors by Affymetrix DNA-microarrays (n=434) including CD138-purified primary myeloma-cell-samples, normal bone-marrow plasma-cell-samples, polyclonal plasmoblasts-samples, human myeloma-cell-lines (HMCL), and whole bone-marrow. Presence and differential gene expression was determined by PANP-algorithm and empirical Bayes statistics. Event-free (EFS) and overall survival (OAS) were investigated for the 168 patients undergoing high-dose chemotherapy (HM-group) using Cox’s proportional hazard model. Findings were validated using the same strategy on an independent group of 345 patients from the Arkansas-group. For validation, quantitative real-time PCR and flow cytometry were performed. In vitro induction of angiogenesis was assessed using the AngioKit-assay. Effect of BMP6 on proliferation of HMCL was assessed by 3H-thymidine uptake. RESULTS. BMP6 is the only BMP expressed by normal- (13/14 samples) and malignant plasma-cells (228/233 samples). It is significantly lower expressed in proliferating non-malignant plasmablastic cells and human myeloma cell-lines. In vitro, BMP6 significantly inhibits proliferation of myeloma-cell-lines with an IC50 ranged from 0.08–2.15μg/ml, survival of primary myeloma-cells, and in vitro tubule formation down to the level of the negative control. High BMP6-expression in malignant plasma cells delineates significantly superior overall-survival for patients undergoing high-dose chemotherapy in both independent series of patients (n=168, P=.02 and n=345, P=.03, respectively, see below). CONCLUSION. With BMP6 we report for the first time the autocrine expression of a prognostically relevant anti-angiogenic and anti-proliferative factor and its receptors by normal and malignant plasma-cells. Figure Figure

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Symptomatic Outcome of CTDa In Multiple Myeloma Patients

KYAMC Journal ◽

10.3329/kyamcj.v11i3.49868 ◽

2020 ◽

Vol 11 (3) ◽

pp. 124-128

Author(s):

Zulfia Zinat Chowdhury ◽

Mohammad Ali ◽

AKM Mynul Islam ◽

Salina Haque ◽

Tamanna Bahar ◽

...

Keyword(s):

Multiple Myeloma ◽

Plasma Cells ◽

Cost Effective ◽

Complete Response ◽

High Dose Chemotherapy ◽

Albumin Level ◽

High Dose ◽

Female Ratio ◽

Stem Cell Rescue ◽

Bone Marrow Plasma

Background: Multiple Myeloma (MM) represents approximately 15% of all hematological malignancies. Despite the use of high-dose chemotherapy followed by stem cell rescue MM remains incurable at present. The goal is to control the disease as much as possible, providing the best quality of life to patients for the longest duration. Currently, CTDa (attenuated Cyclophosphamide, Thalidomide, Dexamethasone) is the best option of treatment as it is cost-effective, with no need for hospitalization with a good response. Objective: To find out the symptomatic responses and toxicities of CTDa in Multiple Myeloma patients. Materials and Methods: 25 patients of newly diagnosed MM patients were treated in the Haematology Department, Bangabandhu Sheikh Mujib Medical University (BSMMU) from July 2016 to July 2017. The mean age of the patients was 54 years, Male female ratio was 1.5:1 and most of the patients were farmers. After induction of 4 to 6 cycles of CTDa all patients were followed up at 6th and 12th weeks. At follow up we evaluated improvement of weakness, bone pain, Hb%, ESR, monoclonal protein, ß2microglobulin, bone marrow plasma cells and serum calcium and albumin level. Adverse effects, such as peripheral neuropathy, thromboembolic events, hyperglycemia, constipation, rash, and somnolence were also assessed. Results: Among 25 patients, complete response achieved only 13 patients (52%), where 20% and 16% of patients belonged to partial or no response respectively. The death occurred in 2 cases (12%). Conclusion: CTDa is a gentle approach to treat an especially frail group of patients, since virtually all patients ultimately relapse. KYAMC Journal Vol. 11, No.-3, October 2020, Page 124-128

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PTEN Gene Deletions in Patients with Multiple Myeloma.

Blood ◽

10.1182/blood.v104.11.4889.4889 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4889-4889

Author(s):

Xiao Ying Qi ◽

A. Keith Stewart ◽

Hong Chang

Keyword(s):

Multiple Myeloma ◽

Plasma Cells ◽

Progression Free Survival ◽

Myeloma Cell ◽

Suppressor Gene ◽

High Dose Chemotherapy ◽

Pten Gene ◽

Allelic Loss ◽

High Dose ◽

13Q Deletion

Abstract PTEN, a tumor suppressor gene, negatively regulates the anti-apoptotic action of akt phosphorylation. Allelic loss or mutation of this gene has been detected in many solid tumors and more recently in human myeloma cell lines (HMCLs). Expression of PTEN has resulted in growth inhibition and apoptosis of a HMCL, suggesting that it may play a role in the pathogenesis of multiple myeloma (MM). However, the PTEN status in tumor cells from patients with MM has not been determined. Using a triple staining method combining staining for cytoplasmic light chains and fluorescence in situ hybridization (FISH) with chromosome 10-centromere and PTEN-gene specific probes, we analyzed clonal plasma cells from 71 patients with MM, 10 with plasma cell leukemia (PCL) and 10 HMCLs. Hemizygous PTEN deletions were detected in 4 of 71 (5.6%) MM patients, 2 of 10 (20%) PCLs, and 2 of 10 (20%) HMCLs. The percentages of clonal plasma cells containing PTEN deletions ranged from 21–90% (median, 56%). Three of the 4 patients with PTEN deletions were detected at diagnosis with stage III disease (Duire-Salmon) and 1 was detected at relapse. Two patients had IgG kappa, 1 IgG lambda and 1 free lambda light chain. To correlate the PTEN status with other known genetic abnormalities in MM, we investigated 4 MM and 2 PCLs with PTEN deletions using FISH for chromosome13q, p53 status, translocations t(11;14), t(4;14) and t(14;16). One MM had a 13q deletion, 1 PCL had a t(11;14), and the other PCL had a t(14;16), a 13q deletion and a p53 deletion. All 4 MM patients with hemizygous PTEN deletions received melphalan based high-dose chemotherapy and autologous stem cell support. Their median overall survival (OS) was 48.1months, and progression free survival (PFS) was 42.8 months as compared to patients without PTEN deletions (OS, not reached, PFS, 25.8 months) (p=0.51 for OS, p=0.67 for PFS). Our results indicate that PTEN deletions are uncommon in MM patients and therefore unlikely represent a primary event for MM. PTEN deletions appear to occur in the advance stage of the disease, and are more frequently involved in PCL or HMCLs suggesting that deletions of PTEN may be associated with disease progression in a subset of MM.

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Expression of PRAME and WT1 in Multiple Myeloma Patients during High Dose Chemotherapy and Auto-SCT

Blood ◽

10.1182/blood.v112.11.5128.5128 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5128-5128 ◽

Cited By ~ 1

Author(s):

Andrey Misurin ◽

Tatyana Gaponova ◽

Larisa Mendeleeva ◽

Elena Parovichnikova ◽

Valeryi Savchenko

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Prognostic Factors ◽

Internal Control ◽

High Dose Chemotherapy ◽

High Dose ◽

Wt1 Gene ◽

Expression Levels ◽

The Moment ◽

Median Expression

Abstract High dose therapy enables further improvement in the outcome of multiple myeloma (MM) patients. However, it is still necessary to determine prognostic factors that may influence treatment results and provide additional criteria for the precise selection of treatment approaches. There are several tumor associated genes (MAGE, LAGE, GAGE, PRAME) that are over-expressed in different malignancies including MM. These genes are believed to modulate cancer properties and should be taken into account during treatment. Their significance as prognostic factors is under investigation. The aim of our study was to analyze the expression levels of PRAME and WT1 genes in MM patients during high dose chemotherapy following by auto-SCT. After having informed consent 25 primary MM patients were included into this study. The median age was 48 years (range, 31–62). All patients were treated by 3 cycles of VAD, Cyclophosphamide 6g/m2 + G-CSF to mobilize Stem cells, EDAP, melphalan 200 mg/m2 followed by auto-SCT. As second line therapy we used bortezomib+dexamethasone. Quantitative PRAME and WT1 gene expression analysis was performed by means of RQ-PCR. Results were normalized against expression of ABL gene which was used as internal control. Investigation was performed before treatment (n=25), after three VAD cycles (n=12), and before (n=5) and after auto-SCT (n=4). In primary MM patients: PRAME gene expression was found in 68% (n=17), WT1 in 24% (n=6) of patients, all of whom were PRAME-positive. Median expression levels were 0.1% (0.001–132%) for PRAME and 0.01% (0.002–0.07%) for WT1. PRAME and WT1 expression did not correlate with tumor bulk and was independent of the levels of M-protein, beta-2M and albumin. The expression of PRAME significantly decreased after 3 VAD cycles to 0.001–207% (n=8), at the moment of auto-SCT it was 0.05–6.1% (n=3) and after auto-SCT it was 0.013–4.9% (n=3). However for WT1, we observed increased of WT1 expression after 3 VAD cycles to 0.004-0.05% (n=4)and at the moment of auto-SCT it was 0.035–0.4% (n=3) and after auto-SCT – 0.019–2.03% (n=3). In the patients with high primary PRAME expression (>median expression) the frequency of CR+PR was significantly lower then in PRAME-negative primary patients and in patients with low (<median expression) primary PRAME expression (55% vs 84, p = 0.04). It was found also that WT1-positive primary patients were bad responders and they achieved only minimal response after 3 VAD cycles. It should be stressed that during treatment in a small number of initially negative PRAME and WT1 gene patients, we demonstrated detection by PCR. We detected the appearance of gene expression at low levels in 1 of 8 initially negative PRAME (6.1%) and in 5 of 19 initially negative WT1 (range of level 0.01–0.4%). The detection of gene expression did not correlate with disease status. All these patients achieve CR+ VGPR. In one of these secondary positive patients (acquired PRAME and WT1) relapse occurred. Conclusion: Expression of PRAME gene was found in 68% primary patients and the level of PRAME decreased with tumor reduction. High expression level of PRAME turned out to be a factor of unfavorable prognosis. Expression of WT1 was found in 24% of MM patients all of whom were PRAME-positive. WT1 expression increased during treatment in a small group of pts. Some initially negative pts acquired PRAME and WT1 expression during treatment, but clinical relevance of it is not clear so far.

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CD138 Regulates Myeloma Cell Survival, Proliferation, BM Engraftment and Tumor Organization

Blood ◽

10.1182/blood.v126.23.2974.2974 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2974-2974

Author(s):

David R Fooksman ◽

Amitabha Mazumder ◽

Mark McCarron

Keyword(s):

Plasma Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Early Stage ◽

Serum Starvation ◽

High Dose ◽

Rapid Reduction ◽

Myeloma Cells

Abstract Multiple myeloma is the 2nd most common blood cancer in adults with a median survival time of 5 years despite high-dose chemotherapy and bone marrow transplantation interventions. Syndecan-1 or CD138, is a heparan-sulfate coated glycoprotein, which is highly expressed on the surface of plasma cells and myeloma cells, important for adhesion and accumulating survival signals. Expression of CD138 is heterogeneous in myeloma tumors, in vivo and in vitro leading some to speculate it may distinguish stem-like subpopulations. While this role is highly disputed, we investigated the effect of CD138 expression on tumor pathology in vivo. To characterize CD138neg and CD138high subpopulations, we used GFP+ Vk*myc myeloma model from Leif Bergsagel, which develops myeloma tumors in BM and spleen of C57Bl/6 mice. We found CD138high populations were more proliferative in vivo based on EdU incorporation experiments. We transferred equal numbers of sorted subpopulations into hosts and found that CD138high cells generated larger tumors in the BM than CD138neg cells after 12 weeks. Analysis of these tumor-bearing mice revealed that all tumors contained both subpopulations, indicating that these two subsets are hierarchically equivalent. We find that in mice with small tumors, the majority of cells (80% or more) are CD138high cells, while in large tumors, the level drops (to 30-50% of tumor) with higher composition of CD138neg cells. We also find lower CD138 levels on myeloma cells found in the blood compared to BM. Using intravital two-photon time-lapse imaging in the tibial BM, we find that tumor cells from smaller, early stage tumors are physically arrested within the BM parenchyma, while in larger, more advanced tumors, myeloma cells are more motile and active. CD138neg cells were more apoptotic based on ex vivo Annexin V staining following serum starvation. Interestingly, serum starvation led to rapid reduction in CD138 surface expression. Taken together, we propose a model where CD138 expression regulates localization and survival in the BM niche, but is downregulated from the plasma membrane when tumor size outgrows the necessary resources, allowing myeloma cells to migrate and metastasize to distant new locations. Disclosures No relevant conflicts of interest to declare.

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Interleukin-6 gene expression in multiple myeloma: a characteristic of immature tumor cells

Blood ◽

10.1182/blood.v81.12.3357.3357 ◽

1993 ◽

Vol 81 (12) ◽

pp. 3357-3364 ◽

Cited By ~ 94

Author(s):

H Hata ◽

H Xiao ◽

MT Petrucci ◽

J Woodliff ◽

R Chang ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Tumor Cells ◽

Interleukin 6 ◽

Plasma Cells ◽

Receptor Gene ◽

Tyrosine Phosphatase ◽

Marker Genes ◽

Target Cells ◽

Myeloma Cells

Abstract Interleukin-6 (IL-6) has been suggested to play a major role in multiple myeloma. To investigate the source and target cells of IL-6 activity in multiple myeloma, expression of the cytokine and its receptor genes by myeloma plasma cells was studied. Tumor cells were sorted from bone marrow aspirates of myeloma patients using 4-parameter gating. Myeloma cells were identified as CD38high CD45negative- intermediate and by their light-scatter characteristics. Sorted cells contained only myeloma plasma cells. No contaminating cells were present as determined morphologically, by monoclonal cytoplasmic Ig analysis, and by polymerase chain reaction (PCR) amplification of marker genes. Myeloma cells from 45% of patients expressed IL-6. IL-6 receptor transcripts were found in 68% of the specimens. IL-6 gene expression correlated with expression of the IL-6 receptor gene (P < .005). Correlations observed between the expression of CD45, a protein tyrosine phosphatase expressed by B lymphocytes but not by plasma cells, and the expression of the IL-6 and IL-6-receptor genes (P < .0002 and P < .005, respectively) suggest that an autocrine IL-6 loop is functioning in myeloma in preplasma cells.

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Flow Cytometric Detection of Circulating Myeloma Cells Pretransplant in Patients with Multiple Myeloma: A Simple Risk Stratification System.

Blood ◽

10.1182/blood.v106.11.1164.1164 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1164-1164

Author(s):

David Dingli ◽

Grzegorz S. Nowakowski ◽

Angela Dispenzieri ◽

Martha Q. Lacy ◽

Suzanne R. Hayman ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Stem Cell ◽

Prognostic Factor ◽

Response To Therapy ◽

Independent Prognostic Factor ◽

High Dose ◽

Rapid Progression ◽

Myeloma Cells ◽

The Impact

Abstract Background: The presence of circulating myeloma cells (CMC) detected by flow cytometry at the time of diagnosis of multiple myeloma is associated with a shortened response to therapy and reduced overall survival (OS). We hypothesized that the presence of CMC at the time of stem cell collection prior to high dose therapy (HDT) and autologous stem cell transplantation (ASCT) would identifies a cohort of patients with a high risk of rapid progression. Methods: The Mayo Clinic myeloma transplant database was queried for patients who were mobilized using cyclophosphamide and hematopoietic growth factors. CMC was determined using flow cytometry by gating on a population of CD38 bright and CD45 negative cells. The impact of CMC on OS and time to progression (TTP) and its role in the context of established prognostic parameters was evaluated. Results: Of 246 patients with MM undergoing ASCT, 95 had CMC. Patients with CMC had significantly higher plasma cell labeling index, adverse cytogenetics, B2-M and resistant disease. Complete response (CR) rates post transplant were 32% and 36% for patients with and without CMC (p=0.5034). OS was 33.2 and 58.6 months (p=0.0052) while TTP was 14.1 and 22 months respectively (p=0.0005). Figure Figure On multivariate analysis, CMC remained an independent prognostic factor in a model that included cytogenetics and disease status at time of transplant (p=0.0314). A prognostic system based on the presence or absence of CMC and karyotype abnormalities was developed. Patients with neither, one or both parameters had a median, OS of 55, 48 and 21.5 months respectively (p<0.0001) while TTP was 22, 15.4 and 6.5 months for the same groups (p<0.0001). Conclusion: The presence of CMC at the time of HDT and ASCT is an independent prognostic factor. The combination of CMC and cytogenetics provides a simple yet powerful scoring system that stratifies patients and guides their management.

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Decreased Thymosin Beta 4 Expression Results in Poor Prognosis and Decreased Survival in Multiple Myeloma.

Blood ◽

10.1182/blood.v112.11.1703.1703 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1703-1703

Author(s):

Jo Caers ◽

Dirk Hose ◽

Ine Kuipers ◽

Tomas Jan Bos ◽

Els Van Valckenborgh ◽

...

Keyword(s):

Multiple Myeloma ◽

Plasma Cells ◽

Metastatic Potential ◽

Suppressor Gene ◽

Inhibitory Effect ◽

High Dose Chemotherapy ◽

Tumor Formation ◽

High Dose ◽

Thymosin Beta 4 ◽

Qrt Pcr

Abstract Thymosin beta 4 (Tβ4) is a small cytoplasmic and nuclear protein involved in different cellular processes including differentiation, migration and proliferation. It is overexpressed in several malignant cells, resulting in an increased angiogenic respons and metastatic potential of tumor cells. In the current study we wanted to evaluate the expression of Tβ4 in primary human multiple myeloma cells (MM) and murine 5TMM cells. Microarray analysis and qRT-PCR of 171 MM patients treated with high dose therapy and autologous stem cell transplantation, showed that MM cells display a decreased Tβ4 expression compared to plasma cells from healthy individuals. We investigated whether Tβ4 expression in MM cells influences the event free (EFS) and overall survival (OAS) of these patients. As Tβ4 was expressed in all MM cells, we compared survival of patients with Tβ4 expression in MM cells in the upper thirtile (Tβ4high MMC) against the lowest thirtile (Tβ4low MMC). Patients with Tβ4high MMC had an increased EFS (P< 0.05) and also their OAS tended to be longer. A similar observation was made in the 5TMM model where qRT-PCR and ELISA revealed a decreased expression of Tβ4 in BM samples from 5T33MM and 5T2MM diseased mice compared to control mice. To study the functionality of Tβ4, we overexpressed Tβ4 in 5T33MMvitro (5T33MMvt) cells by lentiviral transduction. These 5T33MMvt-Tβ4+ cells demonstrated a significantly decreased proliferative capacitity and an increased sensitivity to different anti-myeloma agents (bortezomib, melphalan, dexamethasone) compared to control 5T33MMvt cells. Furthermore, injection of 5T33MMvt-Tβ4+ cells in C57BlKaLwRij mice resulted in a significant decreased tumor formation (paraprotein concentration was 1.92 g/dl versus 3.74 g/dl for untransduced cells, p<0.05) and a prolonged survival compared to mice injected with untransduced 5T33MMvt cells (respectively 65.9 days versus 88.9 days, p< 0.05). In conclusion, we found a decreased Tβ4 expression in human and murine MM cells compared to normal plasma cells. In our cohort of MM patients treated with high-dose chemotherapy, low expression of TB4 identifies a group of patients with adverse prognosis while in the murine 5TMM33vt model, overexpression of Tβ4 resulted in an inhibitory effect on tumor formation suggesting a role of Tβ4 as a tumor suppressor gene in MM.

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