International Journal of Myeloma 4(1): 1–6, 2014
©日本骨髄腫学会

REVIEW

Clinical and biological significance of surface molecules in myeloma
Yawara KAWANO1,2 and Hiroyuki HATA3
Phenotypic analysis of tumor cells by flow cytometry is essential for various hematological malignancies. Surface
antigen analysis of multiple myeloma cells is also widely used in clinical practice. However, the heterogeneity ofsurface molecule expression for malignant plasma cells often makes it difficult for hematologists and physicians tointerpret the data. In this review, we focus on: 1) surface molecules for detection of myeloma cells; 2) surface molecules
targeted by antibody-based therapy; and 3) surface molecules possibly expressed on myeloma-initiating cells.
Understanding the significance and heterogeneity of surface molecule expression in multiple myeloma cells could
lead to a better understanding of myeloma biology and myeloma treatment strategies.
Key words:

multiple myeloma, surface molecules, anti-body therapy, myeloma-initiating cells

1. Introduction
Phenotypic analysis of tumor cells by flow cytometry
is now essential for various hematological malignancies,
such as acute leukemia and malignant lymphoma. Flowcytometric analysis is also widely used in the clinic forthe diagnosis of multiple myeloma (MM) patients. The
advantages of flow cytometry over other techniques,
such as morphological analysis or immunohistochemistry,
are that it is objective and results can be quantified.
Considering these advantages, phenotypic analysis byflow cytometry is used for the differential diagnosis ofMM, monoclonal gammopathy of undetermined sig­
nificance and other related disorders, prediction of
prognosis, quantification of minimal residual disease,and evaluation of treatment response.
The detection of plasma and MM cells by flow cytometry has previously been conducted using the CD38
gating method. However, although plasma cells are
Received: October 25, 2013, accepted: November 8, 2013
1
Department of Medical Oncology, Dana-Farber Cancer Institute,
Harvard Medical School2
Department of Hematology, Kumamoto University School of Medicine
3
Division of Informative Clinical Science, Kumamoto University Schoolof Medicine
Corresponding author: Yawara KAWANO MD. PhD
Department of Medical Oncology, Dana-Farber Cancer Institute,
Harvard Medical School, Harvard Institute of Medicine Building,Room 237, 4 Blackfan Circle, Boston, MA 02115
E-mail: yawara.kawano@gmail.com

often a bright CD38+ population, the heterogeneity ofsurface molecule expressions on malignant plasma cells
often makes it difficult to interpret the data. It is widely
debated whether antibodies against CD19, CD45, CD56,
and CD138, should be combined with anti-CD38 antibodies to detect and distinguish malignant plasma cells
from their normal counterparts.
Progress in flow cytometry techniques has led to theidentification of cancer-initiating cells (cancer stem cells)
in various tumors. The existence and phenotypes of
myeloma-initiating cells has also been proposed byseveral groups, although these findings remain controversial. In this review, we discuss the current state offlow cytometry analysis related to MM.
2. Surface molecules used to detect myeloma cells
2-1. Molecules expressed in normal and malignant
plasma cells

CD38 is a type II transmembrane glycoprotein that is
involved in cell adhesion, signal transduction, and regulation of intracellular calcium mobilization. Plasma cells
and MM cells have been detected using CD38 as a
primary gating antigen because they exist in CD38++(strongly positive) lesions 1). Although hematopoietic
stem cells, and T and B lymphocytes express CD38,
plasma cells express CD38 at a much higher intensity.
However, MM cells have been reported to express CD38
at lower levels than those in normal plasma cells 2–4).
CD138, also known as syndecan-1, is a heparan sulfate
proteoglycan expressed by plasma cells but not by T or
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B lymphocytes. CD138 mediates the adhesion of MM
cells to extracellular matrix, especially type-1 collagen 5).
CD138 is also considered to be a differentiation antigen;during normal B-cell development, abundant CD138
expression is highly specific for terminally differentiatedplasma cells in the bone marrow 6). Because CD138
expression is also a hallmark of MM cells, it has beenused for MM cell purification from bone marrow samplesof patients 7). CD138 expression should be evaluatedcarefully as it is known to be lost from the cell surface
during apoptosis 8). Heterogeneity of CD138 expression
in bone marrow MM cells, and the existence of CD138−
MM cells have been reported by several groups 9). We
reported a significant decrease in CD138 expression inpatients with relapsed/progressive disease comparedwith that in untreated MM patients. Patients with lowlevels of CD138 had worse overall survival comparedwith those that had high levels of CD138 expression 10).
CS1 is a cell surface glycoprotein that belongs to theimmunoglobulin (Ig) gene superfamily. CS1 is specifically expressed at high levels in normal and malignant
plasma cells, and is expressed at low levels in naturalkiller (NK) cells and CD8+ T cells 11). Because CS1 is a downstream target of interferon regulatory factor 4 (IRF4) 12), a
key transcription factor for plasma cell differentiation, it
is considered a plasma cell differentiation molecule.
2-2. Differential expression of molecules between normal
and malignant plasma cells

CD19, which is a pan-B-cell marker, is preserved innormal plasma cells but is absent in MM cells 1). Expression of CD19 is absent in greater than 95% of MM
patients 2); the reasons for this absence are not clearly
understood. However, alteration of PAX-5, a B-cellspecific transcription factor, which has been seen in
MM cells, could be associated with the absence of CD19
expression 13). CD20 is a transmembrane phosphoprotein
specifically expressed in B cells, but not expressed innormal plasma cells. CD20 is known to be expressed in
15–20% of MM patients 2,14); it is associated with small
mature plasma cell morphology and t(11;14) 15). A higher
frequency of CD20 expression in plasma cell leukemia
has been reported 16).
CD27 is a marker of memory B cells, and is also
expressed in normal plasma cells. Interaction of CD27
with its ligand, CD70, is important for the differentiationof plasma cells. Although CD27 has been detected on
normal plasma cells, its expression is lost as MM prog­
resses. It is also known that CD27 expression is associated with better MM prognosis 17,18). CD45, known as
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vol. 4 no. 1 (2014)

Table 1. Aberrant surface antigen expression in MM cells comparedwith normal plasma cells
AntigenExpression innormal plasma cells

MM cases withaberrant expression (%)

CD19
CD20
CD27
CD45
CD56

Positive
Negative
Positive
Negative or Positive
Negative to Dim

Negative (>95%)
Positive (15–20%)
Negative (40–50%)
Negative to Dim (70–80%)
Positive (70–80%)

leukocyte common antigen, is a protein tyrosine
phosphatase widely expressed in hematopoietic cells.
CD45 is highly expressed in tonsil and peripheral bloodplasma cells, while normal bone marrow plasma cells
are a mixed population of CD45+ and CD45− cells 19). MM
cells in the bone marrow are also a mixture of CD45+
and CD45− cells, however the majority are CD45− 19,20).
Several reports have shown that patients lacking CD45
expression have relatively short survival 20,21). CD45 iscomposed of several isoforms that vary in their extracellular region as a result of alternative splicing 22). The
major isoform expressed in MM cells is CD45RO, while
normal plasma cells express CD45RA 23). In vitro studies
have shown that CD45+ MM cells have a greater proliferative capacity 19) and are responsive to IL-6 stimulation 24).
CD56, also referred to as NCAM-1 (neural cell adhesion
molecule 1), is a cell adhesion protein that is anothermember of the Ig superfamily, and is often consideredan NK cell marker. CD56 is weakly expressed, or absent,
in normal plasma cells; however it is over expressed in
MM cells 25). Lack of CD56 expression in MM cells isreportedly associated with a lambda isotype 25), t(11;14) 26),
plasma cell leukemia 27), and fewer osteolytic lesions 28).
It was reported that CD56− MM patients have a poorer
prognosis compared with CD56+ patients 29). Prognosis
when combination therapies using novel agents are
employed need to be re-evaluated. The aberrant expressions of surface molecules in MM cells are summarizedin Table 1.
2-3. Combinations of surface molecules for gating MM cells

Traditionally, MM cells have been identified as CD38++
CD45− or dim cells; however there are some CD45+ MM
cells 14). Harada et al. targeted three surface antigens(CD38, CD19, and CD56) to distinguish MM cells fromnormal plasma cells 1). They reported that MM cells are
CD38++CD19−CD56+ or CD38++CD19−CD56−, while normal
plasma cells are CD38++CD19+CD56−; as a consequenceof these findings, several groups are now employing sixcolor screening panels. A British group used a combina-

tion of cytoplasm-λ, cytoplasm-κ, CD19, CD38, CD45,
and CD138 to detect minimal residual disease with highefficacy 30). The European Myeloma Network recommends using CD38, CD138, and CD45 for plasma cell
identification, and CD19 and CD56 to detect abnormal
plasma cells 3). This network prefers to include CD20,
CD117, CD28, and CD27 for additional immunophenotyping.
Many groups have used different gating patterns to
identify MM cells. The fewer antibodies used for analysis,
the higher the possibility of contamination by nonplasma cells, however using too many antibodies for the
initial gating could underestimate the proportion of MM
cells. To avoid this, researchers should gate with a combination of antigens that are expressed at equal levels
on MM and normal plasma cells, in conjunction with
one or two antigens differentially expressed between
normal and malignant plasma cells. However, researchers
should be aware that MM cells could be unexpectedly
missed by gating analysis because they are highly heterogeneous with respect to expression of surface antigens.
3. Surface molecules as targets
for antibody-based therapies
Daratumumab is a human CD38 antibody designed tokill MM cells via antibody-dependent cellular cytotoxicity(ADCC) and complement-dependent cytotoxicity (CDC) 31).
Daratumumab stood out from other CD38 antibodies
because of its strong ability to induce CDC in MM cells.
Its cellular toxicity is significantly strengthened with
Lenalidomide, through activation of effector cells showing ADCC activity 32). CD138 monoclonal antibodies havealso been considered for targeted therapy. Ikeda et al.
investigated the antitumor effects of a murine/humanchimeric CD138-specific monoclonal antibody, nBT062,
conjugated with maytansinoid derivatives against MM
cells 33). The immunoconjugates significantly inhibited
growth of MM cell lines and primary tumor cells from
MM patients, and lacked cytotoxicity against peripheral
blood mononuclear cells from healthy controls. They
also inhibited MM tumor growth in vivo and prolonged
the survival of xenograft mouse models of human MM,
and a SCID-hu mouse model. Rousseau et al. reported
preliminary biodistribution and dosimetry results inrefractory MM patients in a phase 1/2 RAIT study using
an iodine-131-labeled anti-CD138 monoclonal antibody 34).
Four patients with progressive disease were enrolled
after three lines of therapy. Grade 3 thrombocytopenia
was observed in two cases, with grade 4 hematologic

toxicity not observed. One patient experienced partial
response and total relief of pain, which lasted for one
year.
Elotuzumab, a humanized anti-CS1 monoclonal antibody, inhibited MM cell binding to bone marrow stromalcells (BMSCs) and induced ADCC against MM cells 11).
Administration of elotuzumab significantly induced
tumor regression in MM xenograft models 11). A combination of elotuzumab with bortezomib significantly
increased the in vivo therapeutic efficacy in the SCID-hu
mouse model 35). In a phase I study of elotuzumab
involving relapsed/refractory MM patients, 26.5% of
patients had stable disease 36). The most common
adverse events were cough, headache, back pain, fever,and chills; these were generally mild to moderate inseverity. Clinical trials of elotuzumab in combination
with novel agents have also been conducted. A phase
I trial of elotuzumab and bortezomib in relapsed/refractory MM patients showed that 48% of patients had an
objective response in terms of a partial response or
better 37). The most frequent grade 3–4 toxicities werelymphopenia (25%) and fatigue (14%). Elotuzumab in
combination with lenalidomide, and a low dose of
dexamethasone, resulted in an objective response in
82% of patients 38). The most frequent grade 3–4
toxicities­were neutropenia (36%) and thrombocyto­
penia (21%). Considering these findings, combining
elotuzumab and novel agents is well tolerated by­­individuals and could be a potential new combination
therapy.
Many other surface molecules are under consideration
for use in antibody therapies against MM 39). Although
antibody therapy has the potential to improve the outcome of MM treatments, they might not be powerfulenough to work as a single agent, and should be combined with other chemotherapeutic agents. It should be
remembered surface molecules on MM cells are heterogeneous between patients, and could change according
to disease progression.
4. Phenotype of myeloma-initiating cells
4-1. CD19+ and myeloma-initiating cells

Cancer stem cells (CSCs) are often considered tocontribute­to relapse and drug resistance in various­cancers 40). Advances in flow cytometry technologies
have led to the identification of CSCs in various tumors,
including hematological malignancies, by searching forthe progenitors of lineage-specific antigens. Previous
reports regarding CSCs, such as acute myeloid leukemia
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Table 2. Reported phenotypes of MM-initiating cells
Reports
Matsui W, et al. Blood.
2004; 103: 2332-6
Matsui W, et al. Cancer Res.
2008; 68: 190-7

Phenotype of MM initiating cells

CD19+/CD20+/CD27+/CD138–

Kim D, et al. Leukemia.
2012; 26: 2530-7

CD19–/CD45 (low or –)/CD38 high/CD138+

Hosen N, et al. Leukemia.
2012; 26: 2135-41

CD38 high/CD19–/CD138+ or –stem cells 41), derived from undifferentiated progenitor
cells have led researchers to focus on the B-cell compartment of MM. Previous reports have shown that
CD19+ B cells in MM patients express the same Ig genesequence as that in MM cells 42,43). These reports indicate
the involvement of a less mature cell fraction comparedwith plasma cells during MM pathogenesis. Pilarski et al.
showed that CD19+ B cells from MM patients can betransplanted into NOD/SCID mice and generate MM 44,45).
Matsui et al. reported that clonogenic MM cells exist
in CD19+CD20+CD27+CD138− lesions and can be treatedwith rituximab, a CD20 monoclonal antibody 46,47).
However, Kim et al. showed that CD19−CD45low or −CD38highCD138+ cells are tumorigenic MM cells 48). Hosenet al. reported that both CD138+ and CD138− cells in
CD38highCD19− lesions can initiate MM 49). These conflicting results (Table 2) are because of multiple issues,
including the use of different xenograft models and thevariation in the heterogenous phenotype of patients.
The determination of antigen phenotypes for myelomainitiating cells is still being widely debated.
Figure. 1
Immature MM cells(MM initiating cells ?)4-2. Characteristics of CD138− MM cells

CD138− cells have been proposed as candidate
myeloma-initiating cells by several groups, althoughwhether CD19+ or CD20+ B cells are MM progenitors is
yet to be fully confirmed. Because CD138 is highly
expressed in mature plasma cells and is considered adifferentiation molecule 6), it has been hypothesized that
CD138− cells have a less mature phenotype. It has beenreported that CD138− MM cells have high levels of B
cell-specific transcription factor (BCL6) and low expression levels of plasma cell-specific transcription factors(IRF4, PRDM1, and XBP1) compared with CD138+ cells inmurine 50) and human MM cell lines 10). Oct-4, SOX2, and
NANOG, which are stem cell-related transcription
factors, are reported to be up-regulated in CD138− MMcells 51,52). We reported that patients with an increased
number of CD138− MM cells had a poor prognosis 10).
Chaidos et al. showed that CD19−CD138− cells are localized at extramedullar lesions, and are more resistant tochemotherapy compared with mature plasma cells 53).
Recently, it was reported that CD138−Xbp1s− MMprogenitors are resistant to proteasome inhibition 54),
suggesting that CD138− cells are a source of drugresistant or tumor-initiating cells.
The mechanisms regulating CD138 expression havealso been studied. It has been reported that cytokines 55),
osteoclasts 56), and bone marrow stromal cells 57) contribute to CD138 down-regulation in MM cells in vitro. We
recently reported that hypoxia reduces CD138 expression levels and induces an immature phenotype in MM
cells 58). These data indicate that the bone marrow
microenvironment has an important role for inducing
an immature phenotype in MM cells (Figure 1). We
Mature MM cells

Bone marrow
microenvironment
CD138↓

Oct‐4 ↑
SOX2 ↑NANOG ↑

Cytokine
Osteoclast
Stromal cell
Hypoxia

IRF4
PRDM1
XBP1
BCL6

4

International Journal of Myeloma

vol. 4 no. 1 (2014)

CD138

Figure 1. Characteristics of immature (CD138−)
MM cells. Immature MM cells are reported
to have lower expression levels of plasma
cell transcription factors (IRF4, PRDM1,
XBP1), and higher expression levels of
B-cell transcription factor 6 (BCL6) andstem cell transcription factors (Oct-4,
SOX2, NANOG) compared with mature
MM cells. The bone marrow microenvironment likely contributes to inducing mature
MM cells toward the immature phenotype. To date, immature cells have notbeen confirmed as MM-initiating cells.

propose that these CD138−CD19− cells are less matureand more resistant to chemotherapy compared withmature plasma cells. A similar model of plasticity
between mature and immature MM cells has also been
proposed 53). However, immature CD138− cells are yet to
be demonstrated as MM-initiating cells. New methods
and further investigation are needed to identify MM
stem cells.
5. Conclusion
Multi-color flow cytometry has enabled us to analyze
the surface molecules of MM cells in a clinical setting.
However, the heterogeneity of surface molecule expression for malignant plasma cells results in complications
when attempting to detect minimal residual disease,and in the use of antibody therapies targeting surface
molecules. Further information regarding the differences and similarities between normal and malignant
plasma cells is essential for solving these problems. The
development of novel agents has improved the survivalof MM patients; however, it is still an incurable disease.
Studying the expression of surface molecules in detailcould lead to a better understanding of MM biology, inparticular the phenotype of myeloma-initiating cells.
A consequence of this, it is hoped, would be bettermolecular-targeting therapies against MM.
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