• CD30;
  • CD44;
  • CD49d;
  • CD70;
  • CD105;
  • CD184;
  • plasma cell myeloma


  1. Top of page
  2. Abstract
  6. Acknowledgments


To investigate the expression profile of potential therapeutic biomarkers in plasma cell myeloma (PCM) by multicolor flow cytometry analysis.


Bone marrow (BM) specimens were collected consecutively and analyzed using a routine PCM panel (CD38/CD138/CD45/CD19/CD20/CD28/CD56/CD117, cyto-kappa/lambda). The specimens were further assessed for CD30, CD44, CD49d, CD70, CD105, and CD184 expression in cases containing a substantial number of neoplastic plasma cells.


Totally, 101 patient BM samples were assessed, including 58 men and 43 women, with a median age of 64 years (34–89). Twenty-nine patients had newly diagnosed/untreated PCM, 40 had persistent/residual disease undergoing various therapies and 32 had relapsed disease. CD49d was expressed brightly and uniformly in all 45 patients tested. Expression of CD44 and CD184 was more variable with a median percentage of 77% (1–100) and 65% (5–100) respectively. Using an arbitrary 20% cutoff, CD44 was positive in 74 (73%) and CD184 in 92 (91%) cases with a mean fluorescence intensity ratio of 42.8 and 21.4. A higher CD44 expression was observed in patients with recurrent/persistent disease (P = 0.028). Additionally, both CD44 (P = 0.002) and CD184 (P = 0.026) showed higher expressions in CD117-positive cases, but there was no correlation with cytogenetic groups. The CD30, CD70, and CD105 were expressed very infrequently in PCM, with a median expression of 0.2%, 0.2%, and 0.4% respectively.


CD49d, CD44, and CD184, are highly expressed in PCM. CD49d expression is bright and uniform, whereas CD44 and CD184 are more heterogeneous. In contrast, surface CD30, CD70, and CD105 are infrequent. These data provide useful preclinical information for the design of potential novel targeted therapies in PCM patients. © 2013 International Clinical Cytometry Society

Despite of recent advances [1, 2], plasma cell myeloma (PCM) remains an incurable disease and new approaches that induce long-term tumor regression and improve disease outcome are needed. Immunotherapy that targets tumor-associated antigens (TAAs) has been shown to be an effective treatment approach in cancer therapy, particularly for diseases re-emerging after therapy, and a number of new agents have been developed. Brentuximab vedotin, a monoclonal antibody (mAb)-monomethyauristatin E conjugate that targets CD30, was recently approved by the Federal Drug Administration for use in patients with refractory Hodgkin lymphoma and anaplastic large-cell lymphoma [3]. SGN-70, a humanized monoclonal antibody that specifically targets CD70, has been shown to exhibit potent anti-myeloma activity in vitro and significantly prolonged the survival of tumor-bearing mice in vivo [4, 5]. Humanized anti-CD70 antibody conjugated with the tubulin inhibitor auristatin induced complete regression of renal cell carcinoma in xenografted mouse model [6]. CXCR4 (CD184), a chemokine receptor, and its ligand, stromal cell-derived factor-1 (SDF-1/CXCL12), serves as the key factor for stem cell and immune cell trafficking and plays a critical role in stem cell mobilization, human immunodeficiency virus infection, autoimmune diseases, cancer, and tissue regeneration [7]. Interruption of the CXCR4-CXCL12 signaling can disrupt the adhesive tumor–stromal interaction that confers survival and drug-resistance signals, and therefore, makes the tumor cells accessible to conventional chemotherapy. CXCR4 antagonists are now in Phase I/II clinical studies for acute leukemia and chronic lymphocytic leukemia [8, 9]. AMD 3100, a specific small-molecule antagonist of CXCL12 binding to CXCR4, has been shown to be effective in inhibiting PCM cell survival and proliferation in vitro [10, 11]. Feng et al. found that myeloma stem cells expressed CD184, and obtained growth support from bone marrow microenvironment, at least partially via the CXCR4 signaling pathway[10]. Kim et al. [11] found that AMD3100 targeting CD184 inhibited the survival and proliferation of myeloma cells. CD44 is a member of cell-surface proteoglycan family, and ligand binding through CD44 induces internalization and intracellular drug release [12]. CD44v6 is found frequently expressed in multiple myeloma and associated with deletion of chromosome 13q. Bivatuzumab mertansine is a novel cytotoxic immunoconjugate specifically targeting the CD44 splice variant CD44v6 [13]. Integrin-a4 (CD49d) is a major molecule serving as an anchor mediating physical interactions of PCM cells with the extracellular matrix (ECM), as well as cellular microenvironmental elements [14]. Natalizumab, a monoclonal antibody against CD49d, is highly effective in multiple sclerosis [15], and has recently been shown to block tumor cell adhesion and chemosensitize PCM cells to bortezomib in vitro [16]. Endoglin (CD105) expressed by endothelial cells, has been speculated to play a role in tumor-related angiogenesis and neovascularization [17]. A phase 1 clinical trial with anti-CD105 (c-SN6j; also known as TRC105) is ongoing in patients with advanced or metastatic solid tumors [18].

In this study, we assessed 101 cases of PCM for expression of CD30, CD44, CD49d, CD70, CD105, and CD184 by multicolor flow cytometry immunophenotypic analysis. The goal of this study is to provide preclinical data that will be helpful for design of possible novel targeted therapies for patients with PCM.


  1. Top of page
  2. Abstract
  6. Acknowledgments


Bone marrow (BM) aspirate samples were collected from patients with a diagnosis of PCM between August 2011 and January 2012. All the samples were submitted to the clinical flow cytometry laboratory as part of the routine clinical work-up. The study was conducted consecutively on BM samples containing a substantial number of neoplastic plasma cells regardless of disease status (first diagnosis, persistent or relapsed disease). This study was conducted in accordance with a protocol approved by the Institutional Review Board at MDACC. Demographic and laboratory data were retrieved from electronic medical record system.

Flow Cytometry Immunophenotyping Analysis

BM aspirate specimens were collected in EDTA-anticoagulant and processed within 24 h (h) of collection, and all experiments were performed in Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. Instrument performance was checked daily by recording fluorescence intensity with calibrating beads (BD Biosciences). Reagent and antibody performance was checked by analyzing control cells (CDChex; Streck Laboratories, Omaha, NE) and peripheral blood from blood bank donors at MD Anderson Cancer Center. After incubation with monoclonal antibodies for 15 min (min) at 4°C, erythrocytes were lysed with ammonium chloride (PharmLyse, BD Biosciences, San Diego, California) at room temperature for 10 min using a standard lyse/wash technique. Samples were acquired on FACSCanto II instruments (BD Biosciences). For the detection of cytoplasmic antigens, cells were fixed and permeabilized using 4% formaldehyde and 0.25% Saponin. CD30-PE was purchased from Beckman Coulter and all other antibodies were purchased from BD Biosciences. All cases included had a substantial number of neoplastic plasma cells detected by Tube 1: CD38-FITC; CD28-PE; CD138-APC; CD117- PE-Cy7; CD19-PerCP-Cy5.5; CD56-V450; CD45-V500; and Tube 2: cytoKappa-FITC; cytoLambda-PE; CD138-APC; CD38-PerCP-Cy5.5; CD20-V450; CD45-V500, similar to an approach described previously [19]. Each case was analyzed with the above markers as part of the routine clinical work-up where at least 100,000 total events or minimal 200 CD38+CD138+ plasma cells were acquired. For the investigated antibodies, the fluorochromes and clones used in this study were: CD30-PE (clone HRS4), CD44-APC (G44-26); CD49d-PE (9F10), CD70-APC (Ki-24), CD105-PE (266) and CD184-APC (12G5). A total of 200,000 events were acquired for assessment of these investigated antibodies.

Data were analyzed using FCS Express software (De Novo Software, Los Angeles, California). Non-viable cells, debris, and aggregates were excluded based on forward scatter-height/forward scatter-area (FSC-H/FSC-A). Plasma cell populations were identified by bright CD38/CD138 expression, and further redefined based on FSC-H/SSC-A properties. Fluorescence minus one (FMO) (CD138/CD38 without investigated antibody) was used consistently as negative controls in all cases. The results were presented as a percentage of neoplastic plasma cells with expression of each respective antigen. Mean fluorescence intensity (MFI) ratio was calculated by comparison with negative control.

Conventional Karyotyping and Fluorescence In Situ Hybridization (FISH)

Conventional chromosomal analysis was performed on G-banded metaphase cells prepared from unstimulated BM aspirate cultures using standard techniques. Twenty metaphases were analyzed and the results reported using the International System for Human Cytogenetic Nomenclature.

Fluorescence in situ hybridization (FISH) was performed with selected probes (Abbott Molecular/Vysis, Des Plaines, IL), including D13S319 locus (13q14.3), CCND1–IGH dual-fusion probe set, TP53 locus (17p13.3), loci D5S32 and D5S721 (5p15.2), D9Z1 (9cen), and D15Z4 (15cen) to detect hyperdiploidy. Laboratory cutoffs have been determined with this probe set to identify IGH rearrangements with other partner genes. A total of 100 nuclei were counted per assay in overnight unstimulated cultures.


  1. Top of page
  2. Abstract
  6. Acknowledgments

Patient Characteristics

Over a five-month period, we performed flow cytometry immunophenotyping analysis consecutively on all patients with a clinical diagnosis of PCM; and 101 of these cases had a well-defined and characterized neoplastic plasma cell population. A median of 1.7% (range, 0.1%–95.4%) of plasma cells was obtained for flow cytometry. Cases with lesser number of plasma cells (<200 events in the CD38/CD138 plasma cell gate) were excluded. All patients had concurrent BM biopsy specimens and aspirate smears, which confirmed the presence of PCM. The percentage of BM plasma cells by morphologic examination and manual counting was 13.5% (range, 0–84) (Table 1). Of these patients, 29 were untreated and/or newly diagnosed; 40 patients had persistent/residual PCM undergoing various treatments including mephalan, Velcade, dexamethasone, and lenalidomide; and 32 patients had relapsed disease. The latter group included 27 patients who had disease recurrence after autologous stem cell transplant. These patients included 58 men and 43 women with a median age of 64 years (range, 34–89).

Table 1. Demographic and Laboratory Data
PatientsN = 101
  1. a

    After autologous Stem cell transplant.

Age (years) (median, range)64 (34–89)
Gender (male/female)58/43
Disease Status
First diagnosis/no treatment29 (29%)
Persistent/ relapsed diseasea72 (71%)
Plasma cells by manual count (median, range)13.5% (0–84.0)
Plasma cells by flow cytometry (median, range)1.7% (0.1–95.4)
Flow cytometry markers (20% cutoff)
CD2826/101 (26%)
CD5676/101 (76%)
CD11734/101 (34%)
Abnormal karyotype30/100 (30%)
Abnormal by fluorescence in situ hybridization (FISH)44/98 (44%)

Conventional chromosomal analysis and FISH (unenriched) were performed in 100 and 98 cases, respectively (Table 1). Thirty (30%) cases had an abnormal karyotype, 26 of which had a complex karyotype. By FISH analysis, 15/94 (16%) had IGH/CCND1/ t(11;14); 27/98 (28%) had del13/13q; 3/94 (3%) cases had IGH/FGFR3 t(4:14) or IGH/MAF t(14;16); 5/94 (5%) had other IGH (14q32) abnormalities, and 4/98 (4%) p53 (17p13.1) deletion, 14/94 (15%) with a hyperdiploidy by 11q13/15q22/9q34 probes.

Flow Cytometry Immunophenotypic Analysis

Routine diagnostic flow cytometry immunophenotyping was performed in all cases. All 101 cases had confirmed cytoplasmic light chain restriction. Immunophenotypic abnormalities were detected variably in all cases, including loss of CD45, CD19 expression, and aberrant CD56, CD117, CD28, or CD20 expression (Table 1). In particular, two markers linked to prognosis, CD28 and CD117, were expressed in 26 (26%) and 34 (34%) cases, respectively.

Owing to a very infrequent CD105 expression and the uniform and bright positive expression of CD49d by PCM cells, these two markers were only assessed in a subset of cases, including 18 cases for CD105, and 45 cases for CD49d. CD30, CD44, CD70, and CD184 were assessed in all cases. The medians and ranges of expression of all markers are shown in Table 2 and Figure 1. Using an arbitrary cutoff of 20% or more positive cells, only two cases (2%) were considered positive for CD30; 74 (73%) cases positive for CD44; 45 (100%) cases positive for CD49d; 3 (3%) cases positive for CD70; 1(6%) case was positive for CD105 and 92 (91%) cases positive for CD184. Internal controls were appropriate for all the markers, especially for those infrequently expressed markers as CD30, CD70, and CD44. In bone marrow, CD30 is expressed in a subset of myeloid precursors [20] and a small subset of lymphocytes; CD70 is expressed in a small subset of lymphocytes; CD105 on early erythroid precursors.


Figure 1. CD30, CD184, CD70, CD44, CD49d, and CD105 expression in cases of plasma cell myeloma. Each symbol represents a case, and is expressed as the percentage (%) of neoplastic plasma cells with expression as compared with negative controls (fluorescence minus one controls).

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Table 2. Expressions of CD30, CD44, CD49d, CD70, CD105, and CD184 in Plasma Cell Myeloma
 Case no.Percentage of positive cells (median, range)Case with ≥20% positive cells
CD301010.2 (0–90)2 (2%)
CD4410176.9 (1.3–100)74 (73%)
CD49d4599.5 (74.7–99.9)45 (100%)
CD701010.2 (0–50.7)3 (3%)
CD105180.4 (0–26.5)1 (6%)
CD18410165 (5.2–100)92 (91%)

The mean fluorescence intensity (MFI) ratio for CD44, CD49d, and CD184 expression (compared with control) in all cases (positive or negative) was 21.4(0.5–844.6), 69.5(9.6–337.5), and 9.0(1.0–343.1), respectively. The MFI ratio for cases with ≥ 20% expression (positive cases) of CD44, CD49d, and CD184 was 42.8(1.1–844.6), 69.5(9.6–337.5), and 10.1(2.1–343.1), respectively. Figure 2 illustrates MFI ratio for CD44, CD49d, and CD184 in a case of PCM.


Figure 2. Flow cytometry analysis with a case illustration. Upper panel: Left-plasma cells identified by CD38/CD138; Middle-plasma cell population refined by CD45/side scatter; Right-control tube (fluorescence minus one-FMO). Middle Panel: Expressions of CD44, CD49d, and CD184; Lower panel: Left- cases strongly positive for CD44 and CD184 are more frequently to be CD117 positive; Right-Mean Fluorescence Intensity (MFI) ratio of CD44, CD49d, and CD184 over FMO control. [Color figure can be viewed in the online issue which is available at]

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CD44 and CD184 were the two markers that were most variably expressed by PCM cells. The heterogeneity of CD184 expression was reflected mainly in the myeloma cells in a given case, whereas, the variability of CD44 expression was among individual cases as either positive or negative (Fig. 1). Because of the presence of heterogeneity, expressions of CD44 and CD184 were correlated with other parameters. A significantly higher expression of CD44 was observed in patients with persistent/recurrent PCM undergoing various therapies compared with patients who were untreated, either newly diagnosed or observation only (P = 0.028); this difference remained significant when the results were compared using a ≥20% cutoff for positivity (P = 0.0130) (Table 3). In contrast, CD184 expression was not significantly different between these two groups. Cases positive for CD117 (Fig. 2) had a significantly higher CD44 expression level, analyzed either by the percentage of positive cells (P = 0.002) or by the number of cases with ≥ 20% positive cells (p < 0.001). CD184 expression was significantly higher in CD117-positive cases when assessed by the percentage of positive cells (P = 0.026), but not when using a 20% cutoff. The latter is likely due to an overall high CD184 expression in PCM cells that only 9 (9%) cases had <20% expression. Neither CD44 nor CD184 expression correlated with CD28 or CD56 expression by PCM cells.

Table 3. CD44 and CD184 Expressions and Their Correlations with Disease Status and CD117 Expression
 CD117-positive N = 34CD117-negative N = 67pNo Treatment N = 29Recurrent/ persistent N = 72p
% positive cells (median, range)97% (13–100)34% (1–100)0.00228% (1–100)86% (3–100)0.028
Cases with ≥20% expressionn = 33 (97%)n = 41 (61%)<0.001n = 16 (55%)n = 58 (81%)0.013
% positive cells (median, range)76% (5–98)55% (9–100)0.02677% (17–100)62% (5–99)0.156
Cases with ≥20% expressionn = 32 (94%)n = 60 (90%)0.714n = 26 (90%)n = 66 (92%)0.714


  1. Top of page
  2. Abstract
  6. Acknowledgments

In this study, we used multicolor flow cytometry immunophenotypic analysis to assess PCM cases for expression of some recently described biomarkers that have exhibited promise in targeted cancer therapy, including CD30, CD44, CD49d, CD70, CD105, and CD184. The advantage of flow cytometry is that the technology allows us to precisely assess surface marker expression in a large number of BM neoplastic plasma cells.

We identified uniform and bright CD49d expression in all cases of PCM analyzed, in keeping with results reported by Kraj et al. [21]. This finding supports the concept that the interaction between CD49d and the BM microenvironment may be a requisite for proliferation of PCM cells., These results also support the idea that targeting CD49d, using agents such as Natalizumab, may inhibit PCM cell growth by disrupting the adhesion of PCM cells to non-cellular and cellular components of the microenvironment [16]. We also show in this study that CD184, another molecule involved in cross-talk between PCM and BM stromal cells, is frequently expressed in PCM cases, in over 90% of cases. CD184 expression, however, is much more heterogeneous in a given case. The variation in CD184 expression did not correlate with disease status, but likely reflect individual case variability in the expressions of myeloma anchor proteins. Interestingly, we did observe a significantly higher CD184 expression in CD117+ PCM cases (see discussion below).

CD44, another adhesion molecule, was found expressed in approximately 75% of PCM cases and significantly higher in patients with persistent/recurrent disease. Similarly, Kraj et al. [21] reported a heterogeneous CD44 expression in PCM in contrast with a higher and more uniform expression of CD44 in plasma cell leukemia. CD44 serves as a cell surface receptor for extracellular matrix components including hyaluronan, and mediates interactions of PCM cells and BM stroma. Ohwada et al. showed that CD44 engagement by an anti-CD44 mAb led to phosphorylation and degradation of IkappaB-alpha, and contributed to myeloma cell resistance to Dexamethasone [22]. Our findings suggest that CD44 expression might be upregulated via therapy or as a manifestation of disease progression or, cases with a higher CD44 expression might be more resistant to dexamethasone treatment. Similar to CD184, we observed a significantly higher expression of CD44 in CD117+ PCM cases. A recent study showed that CD117 expression in PCM might act as an additional anchor molecule, resulting in alterations of both BM myeloid and lymphoid maturation, and contributing to the greater maintenance of the homeostatic role of residual normal plasma cells and a more limited spread of (mono)clonal plasma cells [23]. However, based on current understanding, we cannot explain the underlying biological connection between the higher expressions of CD44 and CD184 and CD117 expression in myeloma cells. We did not observe an association of CD44 and CD184 with CD28 and CD56; nor with conventional cytogenetics or FISH results in PCM. Liebisch et al.[13] observed that CD44v6 expression in myeloma cells correlated with del(13)q cytogenetic abnormalities. In this study, we did not find any correlation with the expression of standard form of CD44 (P = 0.495).

In this study, CD30, CD70, and CD105 were expressed infrequently in PCM cells. In physiologic states, CD30 expression is restricted in subpopulations of activated B- and T-cells [24]. By immunohistochemistry (IHC), CD30 immunostaining is often observed in a variable number of normal plasma cells at a low level; however, CD30 was found much less often expressed in neoplastic plasma cells by IHC [25]. Our results using flow cytometry are in accord with IHC results and suggest that neoplastic plasma cells might lose their capability to express CD30 as one of the normal functions. CD105 (endoglin), is predominantly expressed in angiogenic endothelial cells and is upregulated by hypoxia. Recently, we found that CD105 was highly expressed in myeloblasts in certain subtypes of acute myeloid leukemia (unpublished data); and in normal bone marrow cells, CD105 is expressed in immature erythroid precursors. Our study showed that CD105 expression was nearly absent in PCM cells. Interestingly, Tsirakis et al. reported increased soluble (serum) CD105 (sCD105) in PCM patients and the levels were correlated positively with advanced stage of disease of PCM [26]. Our findings suggest that increased sCD105 in advanced stage of PCM likely reflects an increased tumor angiogenesis, but not directly related to the number of neoplastic plasma cells. CD70 is a member of the Tumor necrosis factor family that is aberrantly expressed by a number of hematological malignancies and some carcinomas. McEarchern et al. performed IHC on PCM tissue microarray slides and reported a variable CD70 expression in 13 of 31 (42%) cases of PCM [5]. They described that the staining pattern for CD70 was both membranous and cytoplasmic. In our study, the median CD70 expression level was 0.2% (range, 0–50.7%) and only 3 of 101 (3%) PCM cases had 20% or more cells that were CD70 positive. It is noteworthy that we used flow cytometry method that allowed us to measure the surface CD70 expression only; and to specifically assess its expression on neoplastic plasma cells. Additionally, we obtained a large number of tumor cells (median, 3400 PCM cells) for assessment. Our results indicate that CD70 may not be a suitable marker to target in majority of the myeloma patients. Other biomarkers currently under investigation for potential target therapy include CD56, CD138, CD200, and CS-1 [27, 28]. Elotuzumab, an anti-CS1 monoclonal antibody, has recently achieved clinically meaningful responses when combined with lenalidomide or bortezomib in patients with relapsed and relapsed/refractory PCM [29].

In summary, we showed that CD49d, CD44, and CD184 are highly expressed in plasma cell myeloma. All three of these antigens are involved in interactions between PCM and the BM microenvironment. CD44 expression is significantly higher in persistent/relapsed PCM than untreated patients. Both CD44 and CD184 are expressed at significantly higher levels in CD117+ PCM cases. In contract, CD30, CD70, and CD105 are very infrequently expressed on PCM. These data provide useful information for the future design of potential novel targeted therapies.


  1. Top of page
  2. Abstract
  6. Acknowledgments

The authors thank our staff in the flow cytometry laboratory at MD Anderson Cancer Center for their technical support.


  1. Top of page
  2. Abstract
  6. Acknowledgments
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