Detection and quantitation of malignant cells in the peripheral blood of multiple myeloma patients

Abstract One of the distinguishing features of multiple myeloma (MM) is the proliferation of plasma cells that home to the bone marrow (BM). However, there still remains some uncertainty concerning the presence of related malignant cells in the peripheral blood of myeloma patients. Using consensus oligonucleotide primers, we amplified the third complementary determining region (CDR3) of rearranged immunoglobulin heavy chain alleles from MM marrow samples by polymerase chain reaction (PCR). From the sequences of the products, we derived allele-specific oligonucleotides (ASO), and these were used in subsequent amplification reactions to detect malignant clones in the peripheral blood of myeloma patients. This method is highly specific and sensitive to 1 malignant cell in the background of 10(5) normal cells. Using this method we detected circulating malignant cells in 13 of 14 previously untreated MM patients. Furthermore, by applying ASO-PCR to artificial titrations of initial BM DNA sample into normal peripheral blood lymphocyte (PBL) DNA we were able to generate standard curves and quantitate the amount of tumor in the patient PBL. We observed a wide variation in the amount of circulating tumor between patients. In addition, we found that the incidence of circulating tumor cells was independent of BM tumor burden and stage of disease. The detection and quantitation of circulating tumor cells in the PBL of MM patients may offer an alternative assessment of the disease and may be an important consideration in the use of peripheral stem cells in bone marrow transplantation.

Download Full-text

Detection and quantitation of malignant cells in the peripheral blood of multiple myeloma patients

Blood ◽

10.1182/blood.v80.7.1818.bloodjournal8071818 ◽

1992 ◽

Vol 80 (7) ◽

pp. 1818-1824 ◽

Cited By ~ 1

Author(s):

D Billadeau ◽

L Quam ◽

W Thomas ◽

N Kay ◽

P Greipp ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Peripheral Blood ◽

Alternative Assessment ◽

Plasma Cells ◽

Malignant Cell ◽

Malignant Cells ◽

Normal Peripheral Blood

One of the distinguishing features of multiple myeloma (MM) is the proliferation of plasma cells that home to the bone marrow (BM). However, there still remains some uncertainty concerning the presence of related malignant cells in the peripheral blood of myeloma patients. Using consensus oligonucleotide primers, we amplified the third complementary determining region (CDR3) of rearranged immunoglobulin heavy chain alleles from MM marrow samples by polymerase chain reaction (PCR). From the sequences of the products, we derived allele-specific oligonucleotides (ASO), and these were used in subsequent amplification reactions to detect malignant clones in the peripheral blood of myeloma patients. This method is highly specific and sensitive to 1 malignant cell in the background of 10(5) normal cells. Using this method we detected circulating malignant cells in 13 of 14 previously untreated MM patients. Furthermore, by applying ASO-PCR to artificial titrations of initial BM DNA sample into normal peripheral blood lymphocyte (PBL) DNA we were able to generate standard curves and quantitate the amount of tumor in the patient PBL. We observed a wide variation in the amount of circulating tumor between patients. In addition, we found that the incidence of circulating tumor cells was independent of BM tumor burden and stage of disease. The detection and quantitation of circulating tumor cells in the PBL of MM patients may offer an alternative assessment of the disease and may be an important consideration in the use of peripheral stem cells in bone marrow transplantation.

Download Full-text

Multiple myeloma: circulating lymphocytes that express plasma cell antigens

Blood ◽

10.1182/blood.v64.2.352.bloodjournal642352 ◽

1984 ◽

Vol 64 (2) ◽

pp. 352-356

Author(s):

GJ Ruiz-Arguelles ◽

JA Katzmann ◽

PR Greipp ◽

NJ Gonchoroff ◽

JP Garton ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Plasma Cell ◽

Peripheral Blood ◽

Plasma Cells ◽

Peripheral Blood Lymphocytes ◽

Tumor Burden ◽

B Cell Differentiation ◽

Blood Lymphocytes ◽

Bone Marrow Plasma

The bone marrow and peripheral blood of 14 patients with multiple myeloma were studied with murine monoclonal antibodies that identify antigens on plasma cells (R1–3 and OKT10). Peripheral blood lymphocytes expressing plasma cell antigens were found in six cases. Five of these cases expressed the same antigens that were present on the plasma cells in the bone marrow. Patients that showed such peripheral blood involvement were found to have a larger tumor burden and higher bone marrow plasma cell proliferative activity. In some patients, antigens normally found at earlier stages of B cell differentiation (B1, B2, and J5) were expressed by peripheral blood lymphocytes and/or bone marrow plasma cells.

Download Full-text

Myeloma Cell Associated Therapeutic Protein Discovery Using Single Cell RNA-Seq Data

Blood ◽

10.1182/blood-2020-142205 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 4-5

Author(s):

Lijun Yao ◽

Reyka G Jayasinghe ◽

Tianjiao Wang ◽

Julie O'Neal ◽

Ruiyang Liu ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Cell Surface ◽

B Cell ◽

Tumor Cells ◽

Plasma Cell ◽

Tissue Specificity ◽

Plasma Cells ◽

Expression Data ◽

Intracellular Proteins

Multiple myeloma (MM) is a hematological cancer of the antibody-secreting plasma cells. Despite therapeutic advancements, MM remains incurable due to high incidence of drug-resistant relapse. In recent years, targeted immunotherapies, which take advantage of the immune system's cytotoxic defenses to specifically eliminate tumor cells expressing certain cell surface and intracellular proteins have shown promise in combating this and other B cell hematologic malignancies. A major limitation in the development of these therapies lies in the discovery of optimal candidate targets, which require both high expression in tumor cells as well as stringent tissue specificity. In an effort to identify potential myeloma-specific target antigens, we performed an unbiased search for genes with specific expression in plasma and/or B cells using single-cell RNA-sequencing (scRNAseq) of 53 bone marrow samples taken from 42 patients. By comparing >40K plasma cells to >97K immune cells across our cohort, we were able to identify a total of 181 plasma cell-associated genes, including 65 that encode cell-surface proteins and 116 encoding intracellular proteins. Of particular interest is that the plasma cells from each patient were shown to be transcriptionally distinct with unique sets of genes expressed defining each patient's malignant plasma cells. Using pathway enrichment analysis, we found significant overrepresentation of cellular processes related to B-Cell receptor (BCR) signaling, protein transport, and endoplasmic reticulum (ER) stress, involving genes such as DERL3, HERPUD1, PDIA4, PDIA6, RRBP1, SSR3, SSR4, TXNDC5, and UBE2J1. To note, our strategy successfully captured several of the most promising MM therapeutic targets currently under pre-clinical and clinical trials, including TNFRSF17(BCMA), SLAMF7, and SDC1 (CD138). Among these, TNFRSF17 showed very high plasma cell expression, with concomitant sharp exclusion of other immune cell types. To ascertain tissue specificity of candidate genes outside of the bone marrow, we analyzed gene and protein expression data from the Genotype-Tissue Expression (GTEx) portal and Human Protein Atlas (HPA). We found further support for several candidates (incl. TNFRSF17,SLAMF7, TNFRSF13B (TACI), and TNFRSF13C) as being both exclusively and highly expressed in lymphoid tissues. While several surface candidates were not found to be lymphocyte-restricted at the protein level, they remain relevant considerations as secondary targets for bi-specific immunotherapy approaches currently under development. To further investigate potential combinatorial targeting, we examine sample-level patterns of candidate co-expression and mutually-exclusive expression using correlation analysis. As the majority of our detected plasma cell-specific genes encode intracellular proteins, we investigated the potential utility of these epitopes as therapeutic targets via MHC presentation. Highly expressed candidates include MZB1, SEC11C, HLA-DOB, POU2AF1, and EAF2. We analyzed protein sequences using NetMHC and NETMHCII to predict high-affinity peptides for common class-I and class-II HLA alleles. To correlate MHC allelic preference with candidate expression in our cohort, we performed HLA-typing for 29 samples using Optitype. To support our scRNAseq-driven findings, we cross-referenced gene expression data with 907 bulk RNA-sequencing samples, including 15 from internal studies and 892 from the Multiple Myeloma Research Foundation (MMRF), as well as bulk global proteomics data from 4 MM cell lines (TIB.U266, RPMI8226, OPM2, MM1ST) and 4 patients. We see consistent trends across both cohorts, with high positive correlation (Pearson R ranging between 0.60 and 0.99) for a majority of genes when comparing scRNA and bulk RNA expression in the same samples. Our experimental design and analysis strategies enabled the efficient discovery of myeloma-associated therapeutic target candidates. In conclusion, this study identified a set of promising myeloma CAR-T targets, providing novel treatment options for myeloma patients. Disclosures Goldsmith: Wugen Inc.: Consultancy. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Adhesion molecule immunophenotype of bone marrow multiple myeloma plasma cells impacts the presence of malignant circulating plasma cells in peripheral blood

International Journal of Laboratory Hematology ◽

10.1111/ijlh.13387 ◽

2020 ◽

Author(s):

Indrė Klimienė ◽

Mantas Radzevičius ◽

Rėda Matuzevičienė ◽

Katažina Sinkevič‐Belliot ◽

Zita Aušrelė Kučinskienė ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Adhesion Molecule ◽

Peripheral Blood ◽

Plasma Cells

Download Full-text

Rituximab in CD20 Positive Multiple Myeloma: A Prospective Study from the IFM Group.

Blood ◽

10.1182/blood.v108.11.3577.3577 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3577-3577

Author(s):

Philippe Moreau ◽

Laurent Voillat ◽

Lotfi Benboubker ◽

Charles Dumontet ◽

Claire Mathiot ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Tumor Cells ◽

Stable Disease ◽

Plasma Cells ◽

Single Agent ◽

Stage I ◽

Minor Response ◽

Cd20 Expression ◽

Anti Cd20

Abstract Multiple myeloma (MM) is a heterogenous disease. A strong association between small mature plasma cell morphology, t(11;14) and CD20 expression has been described in approximately 10% of the patients with MM (Robillard et al, Blood 2003). In this subgroup of patients with MM expressing CD20, rituximab (anti-CD20 chimeric monoclonal antibody) could target the antigen and could have a clinical impact. Thus we conducted a prospective phase II trial of 4 weekly IV infusions of 375 mg/m2 rituximab in patients with MM expressing CD20 on at least 33% of tumor cells. From 07/2004 to 02/2006, 14 consecutive patients, median age 65 years, with either stage I MM never pretreated (n = 7) or stage III MM in relapse or refractory after a median of 2 lines of therapy (n= 7) were treated. Immunophenotype using flow cytometry revealed that a median of 75% of tumor cells were expressing CD20 (range, 33–100%) at the onset of therapy. Responses were evaluated 3 months after therapy according to EBMT criteria. At the reference date of June 1st, 2006, a single patient, who had relapsed 8 months after a double autologous SCT, experienced a minor response, ongoing 18 months after rituximab therapy. At the time of rituximab therapy, 100% of its plasma cells were expressing CD20, and 3 months after treatment, bone marrow examination showed 0.6% of plasma cells, none of them expressing CD20. Five patients (1 with relapsed MM and 4 with stage I MM) experienced stable disease, ongoing for 3, 4, 4, 11 and 12 months, respectively. Three patients with stage I MM had stable disease but subsequently progressed 10, 11, and 15 months after therapy, respectively. The last 5 patients with relapsed MM did not respond to anti-CD20 therapy, despite partial clearance of CD20+ plasma cells in the bone marrow in 2 cases. Conversely in the 3 latter cases, the percentage of CD20+ plasma cells did not decrease despite rituximab therapy. Several factors have been described to explain resistance against rituximab in a variety of B-cell malignancies such as the level of CD20 expression, dissociated action of complement-dependent cytotoxicity and antibody-dependant cellular cytotoxicity, polymorphism in FcgammaRIIIA receptor, and may be inadequate dose schedule. These mechanisms could explain the marginal activity of rituximab as single-agent in CD20+ MM.

Download Full-text

Spontaneous Tumoural Regression in a Patient with t(4;14) Translocation Multiple Myeloma. A Case Report.

Blood ◽

10.1182/blood.v110.11.4777.4777 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4777-4777

Author(s):

Noemi Puig ◽

Christine Chen ◽

Joseph Mikhael ◽

Donna Reece ◽

Suzanne Trudel ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Case Report ◽

Bone Marrow Biopsy ◽

Peripheral Blood ◽

Low Dose ◽

Plasma Cells ◽

Protein Electrophoresis ◽

Urine Collection ◽

Normal Limits

Abstract INTRODUCTION Despite recent advances, multiple myeloma continues to be an incurable malignancy, with a median overall survival (OS) of 29–62 months. A shortened survival is seen in myeloma patients having a t(4;14) translocation either with standard or high-dose chemotherapy (median OS 26 and 33 months, respectively). CASE REPORT A 60 year-old female was found to have a high ESR (121mm/h) and low hemoglobin (113g/L) in December 2005. Further work-up led to the diagnosis of stage 1A (Durie-Salmon) multiple myeloma on the basis of the following investigations: a protein electrophoresis showed IgG 12.2g/L, IgA 23.4g/L and IgM 0.33g/L with an IgA-kappa paraprotein; a bone marrow biopsy revealed 20–30% infiltration with atypical plasma cells, kappa restricted; IGH-MMSET fusion transcripts were detected by RT-PCR, consistent with the presence of t(4;14) positive cells in the specimen; a metastatic survey showed generalized osteopenia throughout the axial skeleton and multiple subtle permeative lucencies in the proximal humeral diaphyses bilaterally. A 24-hour urine collection showed 0.05g/L proteinuria with no Bence-Jones proteins detected. Her peripheral blood counts were as follows: hemoglobin 118g/L (MCV 91fL), platelets 275 bil/L and white blood cells 6.6 bil/L with 3.9 neutrophils and 1.8 lymphocytes. Her electrolytes and calcium were within normal limits but she had a slightly elevated creatinine at 107umol/L (normal <99). Her b2-microglobulin, C-reactive protein and albumin were all normal at 219nmol/L (normal ≤219), 4mg/L (normal ≤12) and 36g/L (36–50) respectively. No active therapy was recommended apart from monthly PAMIDRONATE for permeative lucencies. Her past medical history was significant for an IgA cryoglobulinemia diagnosed in 1985 when she presented with arthritis, purpura and Raynaud’s phenomenon. Her cryocrit has been ranging from 0–25% over the years; most recently still at 5%. She did not require any treatment until 1989 when she was started on low dose-steroids. Her flares consist mainly of lower limbs arthritis and purpura and they have been treated with intermittent PREDNISONE 5–7.5mg per day. A progressive drop in her M-protein has been documented since June 2006 with her most recent protein electrophoresis revealing no paraprotein, quantitative IgG is 7.7g/L, IgA 2.23g/L and IgM 0.63g/L. A bone marrow biopsy has shown less than 5% plasma cells. Her peripheral blood counts and biochemistry remained within normal limits and her skeletal survey is unchanged. A 24-hour urine collection shows no significant proteinuria (0.07g/L). Her free light chains assay revealed kappa 13.8mg/L and lambda 11.0mg/L with a ratio kappa/lambda 1.3. CONCLUSIONS We have documented tumoural regression in a patient with IgA-kappa multiple myeloma and t(4;14) only receiving intermittent low dose PREDNISONE and monthly PAMIDRONATE. This exceptional phenomenon has been well described with other malignancies such as testicular germ cell tumours, hepatocellular carcinomas and neuroblastomas; however, to the best of our knowledge, only in 2 cases of multiple myeloma. The unusual nature of this finding is highlighted by the presence of the t(4;14) in the plasma cells, known to be associated with more aggressive disease. The underlying mechanisms, speculated to be immunological for most of the other cancers, remain completely unknown in this case.

Download Full-text

High Dimensional Immune Profiling in Smoldering Multiple Myeloma Identifies Novel Organizing Features of the Tumor Microenvironment

Blood ◽

10.1182/blood-2019-130554 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4384-4384

Author(s):

Hearn Jay Cho ◽

Deepak Perumal ◽

Adeeb H Rahman ◽

Seunghee Kim-Schultze ◽

Jennifer Yesil ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Tumor Microenvironment ◽

Tumor Cells ◽

Research Funding ◽

Mononuclear Cells ◽

Plasma Cells ◽

High Dimensional ◽

Integrated Analysis ◽

Symptomatic Disease

Multiple myeloma (MM) is a malignancy of plasma cells that arises from premalignant Monoclonal Gammopathy of Undetermined Significance (MGUS) and often progresses through an asymptomatic Smoldering (SMM) phase lasting months or years before manifesting clinical symptoms warranting therapy. Current research indicates that the tumor microenvironment (TME) in the bone marrow may play a significant role in governing progression to symptomatic disease. Therefore, understanding of the interactions between malignant plasma cells and the TME in early disease states is critical in the pursuit of therapies that will prevent progression to symptomatic disease. We performed high dimensional genomic and immunologic analysis of bone marrow specimens from 73 subjects with SMM. We performed RNA-seq on the malignant plasma cells isolated by anti-CD138 magnetic bead positive selection, mass cytometry (CyTOF) and T cell receptor sequencing (TCR Seq) of CD138-depleted bone marrow mononuclear cells, and proteomics, seromic, and grand serology analysis of bone marrow plasma. These samples and assays provided a broad view of the tumor cells and the cellular and soluble components of the TME. Each of these assays identified self-organizing clusters of subjects, indicating that subgroups of SMM patients shared common characteristics in the tumor or TME populations. We then applied novel bioinformatic methods to compare data from pairs, trios, quartets, and quintets of assays to identify communities of subjects with similar immunologic and genomic characteristics. Integrated analysis of CyTOF, proteomic, and TCR Seq resolved three distinct communities with a high degree of significance. These communities shared distinct cellular and proteomic features that suggested early adaptive, activated adaptive, or innate immune characteristics. These results suggest that the continuum from MGUS to MM does not consist of a single pathway in either the tumor cells or the TME, and that complex interactions ultimately determine progression. This suite of assays (CyTOF, proteomics, and TCR Seq) may be applicable in translational and clinical studies to understand key tumor and immune determinants of SMM and lead to rationally designed therapy to replicate these conditions to prevent progression to symptomatic disease. Disclosures Cho: Genentech: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; The Multiple Myeloma Research Foundation: Employment; Takeda: Research Funding; BMS: Consultancy; Agenus: Research Funding; GSK: Consultancy. Adams:Janssen Pharmaceuticals R&D: Employment, Other: Own Stock. Parekh:Foundation Medicine Inc.: Consultancy; Celgene Corporation: Research Funding; Karyopharm Inc.: Research Funding.

Download Full-text

Mobilization of tumor cells and hematopoietic progenitor cells into peripheral blood of patients with solid tumors [see comments]

Blood ◽

10.1182/blood.v83.3.636.636 ◽

1994 ◽

Vol 83 (3) ◽

pp. 636-640 ◽

Cited By ~ 323

Author(s):

W Brugger ◽

KJ Bross ◽

M Glatt ◽

F Weber ◽

R Mertelsmann ◽

...

Keyword(s):

Breast Cancer ◽

Bone Marrow ◽

Tumor Cell ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Peripheral Blood ◽

Stage Iv ◽

Breast Cancer Patients ◽

Cell Recruitment ◽

Stage Iv Breast Cancer

Abstract Peripheral blood progenitor cells (PBPCs) are increasingly used for autografting after high-dose chemotherapy. One advantage of PBPCs over the use of autologous bone marrow would be a reduced risk of tumor-cell contamination. However, the actual level of tumor cells contaminating PBPC harvests is poorly investigated. It is currently not known whether mobilization of PBPCs might also result in mobilization of tumor cells. We evaluated 358 peripheral blood samples from 46 patients with stage IV or high-risk stage II/III breast cancer, small cell (SCLC) or non- small cell (NSCLC) lung cancer, as well as other advanced malignancies for the detection of epithelial tumor cells. Monoclonal antibodies against acidic and basic cytokeratin components and epithelial antigens (HEA) were used in an alkaline phosphatase-anti-alkaline phosphatase assay with a sensitivity of 1 tumor cell within 4 x 10(5) total cells. Before initiation of PBPC mobilization, circulating tumor cells were detected in 2/7 (29%) patients with stage IV breast cancer and in 2/10 (20%) patients with extensive-disease SCLC, respectively. In these patients, an even higher number of circulating tumor cells was detected after chemotherapy with VP16, ifosfamide, and cisplatin (VIP) followed by granulocyte colony-stimulating factor (G-CSF). This approach has previously been shown to be highly effective in mobilizing PBPCs. In the 42 patients without circulating tumor cells during steady state, tumor cells were mobilized in 9/42 (21%) patients after VIP+G-CSF induced recruitment of PBPCs. The overall incidence of tumor cells varied between 4 and 5,600 per 1.6 x 10(6) mononuclear cells analyzed. All stage IV breast cancer patients and 50% of SCLC patients were found to concomitantly mobilize tumor cells and PBPCs. Kinetic analyses showed two patterns of tumor cell recruitment depending on the presence or absence of bone marrow disease: (1) early after chemotherapy (between days 1 and 7) in patients without marrow infiltration, and (2) between days 9 and 16 in patients with marrow infiltration, ie, within the optimal time period for the collection of PBPCs. We show that there is a high proportion of patients with circulating tumor cells under steady-state conditions, and in addition a substantial risk of concomitant tumor cell recruitment upon mobilization of PBPCs, particularly in stage IV breast cancer patients with bone marrow infiltration. The biologic and clinical significance of this finding is unknown at present.

Download Full-text

Clinical and Prognostic Impact of (11;14)(q13;q32) Translocation on Patients with Multiple Myeloma

Blood ◽

10.1182/blood-2019-130809 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5507-5507

Author(s):

Daisuke Miura ◽

Kentaro Narita ◽

Ayumi Kuzume ◽

Rikako Tabata ◽

Toshiki Terao ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Tumor Cells ◽

Clinical Characteristics ◽

Mononuclear Cells ◽

Plasma Cells ◽

Stage Iii ◽

Prognostic Impact ◽

Cd20 Expression ◽

The Impact

Introduction. Translocations involving chromosome 14 at band q32, the immunoglobulin heavy chain (IgH) locus, are considered to be the most important initiating events for the development of multiple myeloma (MM). Among the IgH translocations in MM, t(11;14)(q13;q32) is the most frequently reported, and associated with a lymphoplasmacytic morphology. This translocation have been traditionally considered as standard-risk chromosomal abnormality compared to other translocations such as t(4;14) or t(14;16), although some controversies on the prognostic impact of this translocation still remain. This study aimed to clarify the clinical and prognostic impact of t(11;14) in Japanese patients in relation to other clinical variables such as immunophenotype of the tumor cells, other cytogenetic abnormalities, and use of stem cell transplantation (SCT). Patients and methods. Among the 244 consecutive patients with newly diagnosed MM, treated at Kameda Medical Center between April 2009 and July 2019, 234 patients, having cytogenetic analysis data (including t(11;14), t(4;14), t(14;16), and del(17p) by interphase fluorescence in situ hybridization (iFISH)) fully available, were included in this study. Data regarding the patients' clinical and laboratory characteristics, including the International Staging System (ISS), immunophenotype of the tumor cells, baseline circulating plasma cells (CPCs), treatment responses, disease progression, and survival status, were collected. iFISH was performed with CD138-purified bone marrow plasma cells, and the cut off values for translocation were ≥ 10% and for del(17p) ≥ 20%. Using multicolor flow cytometry, surface marker analysis of bone marrow samples and quantification of pre-treatment CPCs on peripheral blood mononuclear cells were simultaneously performed. CPCs were reported as the percentage of total mononuclear cells. Patients were considered to be negative for clonal CPCs at a sensitivity of 10−4 (0.01%) clonal plasma cells for all events evaluated. Results. The incidence of patients harboring t(11;14) was 24.4% (n = 57); t(11;14) was detected significantly high in light-chain-only subtypes (P < 0.001). We compared clinical characteristics of patients carrying t(11;14) with others. Myeloma cells with t(11;14) were associated with negative expression of CD56 (P < 0.001), CD117 (P = 0.046), and CD200 (P = 0.006), and positive expression of CD20 (P = 0.01) and CD81 (P = 0.035). Patients with t(11;14) were associated with positive CPCs (P = 0.011). In order to focus on the impact of t(11;14), we divided the patients into 4 groups: (A) no specific cytogenetic abnormality listed above (n = 137), (B) t(11;14) group (n = 57), (C) t(4;14) or t(14;16) group (n = 29), and (D) del(17p) only (n = 10), and the clinical characteristics and survival of the patients were compared across the three groups (A), (B), and (C). Almost all the patients (> 95%) in this cohort received bortezomib-based therapy. Median progression-free survival (PFS) and overall survival (OS) of patients in (A), (B), and (C) groups were 55.6, 34.2, and 30.2 months (m) (A vs. B, P = 0.036, and A vs. C, P = 0.031), and not reached, 51.2, and 79.8 m (A vs. B, P = 0.11, and A vs. C, P = 1.00), respectively. However, patients harboring t(11;14) were further divided into CD20-positive and -negative groups, the latter having poor prognosis (36.1 vs. 26.7, P = 1.0 for median PFS, and not reached vs. 44.2, P = 0.029 for median OS). Compared to other groups, patients without CD20 expression had significantly shorter OS (vs. A, vs. B, P = 0.024, 0.035, respectively), whereas those with CD20 expression tended to have longer OS, without statistical significance (Figure 1).Univariate analysis revealed ISS stage III, creatinine > 2.0 mg/dL, use of SCT, t(11;14) without CD20 expression, and age ≥ 70 years to be associated with shorter OS, whereas multivariate analysis demonstrated ISS stage III, use of SCT, and t(11;14) without expression CD20 (HR 1.88; 95% CI 1.10-3.21; P = 0.021) to be independent prognostic factors for poor OS. Conclusions. Our findings demonstrated that patients harboring t(11;14) had distinct clinical and immunophenotypic characteristics, two subsets of the disease entities with a clearly different survival according to CD20 expression. Disclosures Matsue: Ono Pharmaceutical: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Novartis Pharma K.K: Honoraria; Celgene: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria.

Download Full-text

Preferential Inhibition of Malignant Cell Growth by CDDO in Waldenström Macroglobulinemia.

Blood ◽

10.1182/blood.v108.11.2528.2528 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2528-2528

Author(s):

Sherine F. Elsawa ◽

Anne J. Novak ◽

Marina Konopleva ◽

Michael Andreeff ◽

Thomas E. Witzig ◽

...

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

Cell Death ◽

B Cells ◽

Cell Growth ◽

Peripheral Blood ◽

Malignant Cell ◽

Dependent Manner ◽

Malignant Cells ◽

Dose Dependent

Waldenström macroglobulinemia (WM) is a B cell disorder with a highly variable clinical outcome, where some patients remain asymptomatic, while others have significant symptoms and require therapeutic intervention. Clinical symptoms include infiltration of lymphoplasmacytic cells into the bone marrow, production of a monoclonal IgM protein, anemia, lymphadenopathy, and serum hyperviscosity. Despite the introduction of multiple chemotherapeutic regimens over the past several decades, WM remains an incurable disease. 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) and its methyl ester derivative (CDDO-Me) and imidazolide derivative (CDDO-Im) are synthetic triterpenoids derived from oleanolic acid. These compounds have been shown to induce apoptosis of several tumor cell types including breast cancer, lung cancer, ovarian cancer, melanoma, osteosarcoma, leukemia, and multiple myeloma cells. The goal of this study was to evaluate the potential role of synthetic triterpenoids in WM. Preliminary studies on malignant B cells indicated that CDDO-Im induced the greatest amount of cell death and we therefore used this derivative of CDDO for our studies. CD19+ CD138+ cells from bone marrow biopsy specimens obtained from WM patients were isolated by positive selection and were treated with varying concentrations of CDDO-Im (62.5 nM to 750 nM ) and cell viability was determined after 24 hours (n=3). Compared to the nil control 47% of the malignant cells remained viable at a CDDO-Im concentration of 62.5 nM and only 11% remained viable at 125 nM CDDO-Im. To determine if CDDO-Im had specific toxic effects on non-malignant cells, we cultured CD19- CD138- cells from WM patient bone marrows with CDDO-Im and found that non-malignant cells were less sensitive to the drug, 80% being viable at 62.5 nM and 65% being viable at 125 nM. Similarly, we found that normal peripheral blood B cells and CD19+ CD138+ bone marrow B cells from healthy donors were less sensitive to CDDO-Im. Compared to the nil control 93% of the CD19+ CD138+ bone marrow B cells and 70% of the peripheral blood B cells remained viable at a CDDO-Im concentration of 62.5 nM and 95% and 68% remained viable at 125 nM CDDO-Im respectively. We next examined the effect of CDDO-Im on WM cell proliferation and found that CDDO-Im inhibited cell proliferation in a dose-dependent manner. Similar to the viability assays, there was a differential effect of CDDO-Im on malignant and non-malignant cells. Compared to the nil control, at 125 nM, there was a complete inhibition of malignant cell growth, while approximately 40% of the non-malignant cells remained proliferative. To determine the mechanism of cell death, CD19+ CD138+ cells were cultured in the presence or absence of various doses of CDDO-Im for 6 hours and cell lysates were examined for cleavage of PARP. There was evidence of PARP cleavage in a dose-dependent manner, suggesting that CDDO-Im induced malignant cell death occurs through a caspase-dependent mechanism. In summary, the synthetic triterpenoid CDDO-Im decreased the viability of WM B cells in a dose-dependent manner, and CDDO-Im had a greater effect on the viability of the malignant cells compared to non-malignant cells from the same WM patients. CDDO-Im also inhibited malignant cell growth in a dose-dependent manner and the mechanism of CDDO-Im mediated cell death appears to be a caspase-mediated event. Overall, our data indicate that CDDO-Im may have potential efficacy in WM patients.

Download Full-text