Summary. Multiple myeloma (MM) is a malignant
disease that results from an excess of monotypic plasma
cells in the bone marrow (BM). This malignancy is
characterised by complex karyotypic aberrancies. In
60% of all MM there are recurrent primary
translocations involving the heavy chain gene locus. The
MM cells strongly interact with the BM micro-
environment, which is composed of endothelial cells,
stromal cells, osteoclasts, osteoblasts, immune cells, fat
cells and the extracellular matrix. This interaction is
responsible for the specific homing in the BM, the
proliferation and survival of the MM cells, the resistance
of MM cells to chemotherapy, the development of
osteolysis, immunodeficiency and anaemia. New
therapeutic agents target both the MM, as well as the
interaction MM cell – BM microenviroment.
Key words: Multiple myeloma, Microenvironment,
Osteoclast, Bone marrow stromal cell
Epidemiology and clinical presentation
Multiple myeloma (MM) is a malignant disease that
results from an excess of monotypic plasma cells (PCs)
which are usually actively secreting antibody (M protein
or paraprotein). The malignant PC cells (MM cells) are
mainly localised in the bone marrow (BM), although
small numbers of MM cells can be encountered in the
peripheral blood circulation. MM comprises about 2.5%
of all malignancies and 10% of the haematological
malignancies and MM has an annual incidence of 40 per
million. The median age at diagnosis is 63 years; fewer
than 2% of MM patients are under 40 years of age at
diagnosis. It has a twofold higher incidence in Afro-
Caribbean ethnic groups than in Caucasians (UK
Myeloma forum, 2001). Men are 1.7 times more affected
than women. The aetiology of the disease is unknown.
Genetic factors (Grosbois et al., 1999), rheumatoid
arthritis, exposure to ionizing radiation, benzene,
dioxins, certain herbicides and insecticides are potential
risk factors (Kyle, 1999). Multiple myeloma (MM) is a
B-cell malignancy which remains largely incurable
despite advances in systemic and supportive therapies.
Signs and symptoms of multiple myeloma include
bone pain, which may be present in three-fourths of
patients. Osteolytic lesions and compression fractures
may be seen in the axial skeleton and proximal long
bones, the most common being the spine. There is an
increased osteoclastic activity in myeloma patients
mediated by osteoclastic stimulating factors. This
increased bone resorption may lead to hypercalcemia.
Renal insufficiency is often multi-factorial but is
predominantly due to the development of "myeloma
kidney" in which the distal convoluted tubules and
collecting tubules become obstructed with casts
consisting mainly of Bence Jones (monoclonal urinary
light chain) protein.
Low levels of endogenous erythropoietin and
inhibition of erythroid lineage synthesis often results in
anemia with manifestations such as weakness, dyspnoea
and pallor. Cellular and humoral immune dysfunction
are also commonly observed and results in an enhanced
susceptibility to bacterial or herpes zoster infections.
Neurological dysfunction is common during the
course of the disease and may be from spinal cord
compression manifested as back pain, sciatica, or muscle
weakness, or from a demyelinating neuropathy (a
paraneoplastic manifestation of the myeloma
paraprotein) (Zaidi, 2001).
The most common type of monoclonal protein
produced is IgG, followed in frequency by IgA, IgD and
extremely rarely IgE. IgA MM is more associated with
extra-skeletal disease, whereas IgD MM is more
commonly associated with plasma cell leukemia (PCL)
and renal damage (Durie et al., 2003).
Part of the MM is de novo and part is preceded by a
monoclonal gammopathy of undetermined significance
(MGUS). In one Mayo Clinical Study, 58 % of the MM
had prior MGUS or plasmocytoma (Kyle et al., 1994).
The role of the bone marrow
microenvironment in multiple myeloma
H.R. De Raeve1and K. Vanderkerken2
1Department of Pathology, University of Antwerp (UA), Antwerp and
2Department of Haematology and Immunology, Vrije Universiteit Brussel (VUB), Brussels, Belgium
Histol Histopathol (2005) 20: 1227-1250
Offprint requests to: Dr. Hendrik de Raeve, University Hospital Antwerp,
Department of Pathology, Wilrijkstraat 10, B-2650 Edegen, Belgium. e-
Cellular and Molecular Biology
By definition, MGUS patients are asymptomatic and
have stable M-protein measurements. Patients with
MGUS usually have less than 10% BM plasmacytosis, a
serum monoclonal protein level for IgG ≤ 3.5 g/dl and
for IgA ≤ 2 g/dl, a urinary Bence-Jones protein ≤ 1 g/24
h. MGUS is much more common than MM occurring in
1 % of the population over age 50 and 3% over age 70
(Greipp, 1995). MGUS patients can be safely observed
without chemotherapy. During long-term follow-up 25
% of patients with MGUS will develop MM or IgM
expressing lymphoma, primary amyloidosis,
macroglobulinaemie, chronic lymphocytic leukaemia or
plasmocytoma, at a rate of 1% per year (Kyle et al.,
2002). The transition of MGUS into MM does not
always pass through a period of smouldering MM.
Smouldering MM (2% of the MM) is characterised
by a stable intramedullary tumour-cell content of ≥ 10%
but ≤ 30%, and none of the other complications of MM
(Rosinol et al., 2003) (Table 1).
Patients with active MM (Table 1) present with a
BM plasmacytosis of ≥ 10%, a serum monoclonal
protein of >3.5 g/dl for IgG and >2 g/dl for IgA, a 24-
hour urine monoclonal protein of >1 g and lytic bone
lesions. Active MM is, during progression, associated
with increasingly severe features of lytic bone disease,
anaemia, immunodeficiency and renal impairment and
also, in a fraction of patients, the occurrence of tumours
in extramedullary sites, such as blood, liver, spleen,
lymph nodes, pleural fluid and skin. Exceptionally,
infiltration of other organs, such as thyroid (personal
observation), the gastro-intestinal tract (Amonkar et al.,
2003), prostate (Yasuda et al., 1994), and testis
(Oppenheim et al., 1991) can be observed in end-stage
disease. Extramedullary MM involving the blood in the
terminal phase of MM is called secondary plasma cell
Variants of MM are primary PCL, non-secretory
MM and osteosclerotic MM. Primary PCL refers to
patients not in the terminal phase of multiple myeloma
in which abnormal PCs comprise at least 20% of the
differential peripheral blood count. Hepato- and
splenomegaly are common. PCL is usually an aggressive
illness which has clinical resemblance to acute
leukaemia. Compared to MM there is a tendency in PCL
for more frequent extramedullary disease,
thrombocytopenia, and high serum lactate
dehydrogenase (Dimopoulos et al., 1994). Non-secretory
MM is defined by the absence of M-protein in the serum
and urine. The MM cells either do not produce or do not
secrete immunoglobulins. Mutations in genes encoding
VL(Dul, 1990) and CL(Coriu et al., 2004) have been
implicated as causal factors in non-secretory MM.
Osteosclerotic MM is a very rare entity associated with
peripheral neuropathy, organomegaly, endocrinopathy,
M-protein and skin changes (POEMS syndrome).
The Durie-Salmon staging system continues to be
Bone marrow microenvironment in multiple myeloma
Table 1. Diagnosis of MM, indolent and smouldering MM.
I. Plasmocytoma on tissue biopsy
II. BM plasmocytosis with > 30 % plasma cells
III. Monoclonal globulin spike on serum electrophoresis: > 3.5 g/dl for IgG, > 2 g/dl for IgA, ≥ 1 g/24 h for k or l Bence Jones proteinuria
a. BM plasmocytosis with 10-30 %
b. Monoclonal globulin spike: 3.5 g/dl for IgG, 2 g/dl for IgA, ≤ 1 g/24 h for Bence Jones proteinuria
c. Lytic bone lesions
d. Uninvolved immunoglobulin levels IgM <50 mg/dl, IgA <100 mg/dl, or IgG <600 mg/dl
Diagnosis of MM
In a symptomatic patient requires a minimum of one major + one minor criterion:
I + b, I + c, I + d
II + b, II + c, II + d
III + a, III + c, III + d
or three minor criteria that must include a + b:
a + b + c or a + b + d
No bone lesions or limited bone lesions ( ≤ 3 lytic lesions)
No compression fractures
M protein levels IgG ≤ 7 g/dl, IgA ≤ 5 g/dl
No symptoms or associated disease features: performace status >70%, hemoglobin >10 g/dl, serum calcium normal, serum creatinine <2 mg/dl,
Same as indolent MM except:
no bone lesions
BM plasmocytosis ≤ 30 %
used worldwide. This system integrates the major
clinical parameters in correlation with measured
myeloma cell mass (the total number of MM cell in the
body) (Durie and Salmon, 1975) (Table 2). Numerous
groups have proposed new systems to more accurately
and simply stage and/or classify MM patients into
prognostic categories. A simple alternative to the Durie-
Salmon staging system was developed by the Southwest
Oncology Group (SWOG) (Jacobson et al., 2003) (Table
3). This SWOG staging system focused on two common
measures with prognostic importance: serum ß2
microglobulin and serum albumin. ß2microglobulin may
be a product of MM cells and can be used as a tumour
marker to predict the course of the disease (Norfolk et
al., 1980). Serum albumin is an indirect indicator of IL-6
levels, liver function and nutritional status of the
patients. Low serum albumin correlates with both rapid
MM growth and the patients overall performance status.
Histopathology of MM
The trephine biopsy is helpful for the diagnosis,
prognosis, evaluation of the haematopoietic reserve, and
finally, for the evaluation of the treatment efficacy, for
the detection of therapy-related and other complications
(aplasia, fibrosis, amyloidosis,
osteosclerosis, secondary myelodysplasia/leukaemia)
and for the study of angiogenesis.
The trephine biopsy allows us, in contrast to the
aspirate, to evaluate the architectural organisation of BM
PCs. The topographic organisation of PCs is useful in
distinguishing reactive plasmocytosis from MM. In the
former the PCs tend to be arranged along the capillaries.
In the latter, the clustering of PC is randomly distributed
between fat cells, without topographic orientation to
capillaries, even if the percentage of MM cells in the
aspirate smears is below 10%. In MM with a nodular
growth pattern of the MM cell, and especially in MM
with BM fibrosis, the percentage of PC in the aspirate
smears is usually underestimated.
Regarding the PC cell type, the most significant
prognostic differences are found when MM cells are
subdivided into two broad categories (Bartl et al., 1987):
the plasmacytic type, with predominantly non-
nucleolated PC (68% of MM, 32 months median
survival) and the plasmablastic type, with predominantly
nucleolated PCs (32%, 8 months median survival).
However, when other characteristics such as cellular
size, cytoplasmic structure and nuclear configuration are
taken into consideration, the spectrum of MM cells
could be divided into six cell types which can be
combined into three prognostic grades: MM, low-grade
malignancy (Marschalko and small cell type, 70% of
MM, 40 months median survival); MM, intermediate-
grade malignancy (cleaved, polymorphous and
asynchronous, 20% of MM, 20 months median survival)
and MM, high-grade malignancy (blastic, 2%, 8 months
Some unusual cytological variations of MM cells
such as the “flaming cells”, “mott, morular or grape
cells” and the “thesaurocytes” are well known in the
haematological literature. Less well known and therefore
not readily recognised, are the MM cells with
Bone marrow microenvironment in multiple myeloma
Table 2. Durie and salmon staging system.
STAGE I (low cell mass: 600 billion MM cells/m2)
All of the following:
Haemoglobin value > 10.5 g/dl
Serum calcium value normal or < 10.5 mg/dl
Bone X-ray, normal bone structure (scale 0) or solitary
Bone plasmocytoma only
Low M-component production rates: IgG value <5.0 g/dl; IgA value <3.0 g/dl; Urine light chain M-component on electrophoresis <4 g/24 h
STAGE II (intermediate cell mass: 600 to 1,200 billion MM cells/m2)
Fitting neither stage I or III.
STAGE III (high cell mass: > 1,200 billion MM cells/m2)
One or more of the following:
Haemoglobin value <8.5 g/dl
Serum calcium value >12 mg/dl
Advanced lytic bone lesions (scale 3)
High M-component production rates: IgG value >7.0 g/dl; IgA value >5.0 g/dl; Urine light chain M-component on electrophoresis >12 g/24 h
SUBCLASSIFICATION (either A or B)
A: relatively normal renal function: serum creatinine value < 2.0 mg/dl
B: abnormal renal function: serum creatinine value ≥ 2.0 mg/dl
Table 3. Swog classification.
ß2M>5.5 mg/l & albumin ≥ 30 g/l
ß2M≥ 5.5 mg/l & albumin <30 g/l
monocytoid features, signet-ring MM cells, histiocytoid
MM cells, clear MM cells, MM cells with spindle cell
morphology, and MM cells with oncocytic changes
(Banerjee et al., 2004). The prognostic significance of
these unusual cytological variants is not well
The growth pattern in MM is of prognostic
significance: the presence of nodules of MM signals a
progressive course, osteolytic lesions and an
unfavourable prognosis (Bartl and Frisch, 1995).
Although not used in daily practise, the plasma cell
burden (tumour load) measured histomorphometrically
as the “percentage of infiltration volume” had been
shown to have prognostic value (Carbone et al., 1987):
- stage I: minimal infiltration (<5 vol%), in 14% of
MM cases at diagnosis, median survival of 86 months.
- stage II: low infiltration (5-19 vol%), in 28% of
MM cases at diagnosis, median survival of 46 months.
- stage III: intermediate infiltration (20-50 vol%), in
40% of MM cases at diagnosis, median survival of 25
- stage IV: high infiltration (>50 vol%), in 18 % of
MM cases at diagnosis, median survival of 15 months.
The PC labeling index (PCLI), a measure of the the
percentage of PC cells in the S-phase, is determined by
measuring the bromodeoxyuridine incorporation using
the anti-bromodeoxyuridine antibody BU-1. A PCLI
higher than 1% is considered high. The PCLI has been
demonstrated to be an independent prognostic factor in
newly diagnosed patients (Greipp et al., 1988, 1993;
Boccadoro et al., 1987). The percentage of abnormal
metaphases in conventional cytogenetic analysis
correlates with the PCLI as well as with the extent of
BM involvement by MM (BM PC percentage)
(Rajkumar et al., 1999). This is not unexpected, since
chromosomal abnormalities may offer a proliferative
advantage to the MM cells, thereby leading to an
unfavorable prognosis. Plasma cell type also correlates
with PCLI (Schambeck et al., 1996). Although
exceptions occur, patients with PC of the Marshalko type
or small cell type have generally a low PCLI. A high
PCLI in MM patients with apparently stable, plateau
phase with minimal numbers of residual monoclonal PC,
is an adverse parameter that may predict a short time to
disease progression and death (Steensma et al., 2001).
High dose chemotherapy has been shown to improve the
overall survival of patients with a high PCLI (>1.2%),
whereas it did not prolong overall survival in those with
a low PCLI (Boccadoro et al., 1999).
Evolving genetic events in MM
MM cells have extensive somatic mutations of
rearranged immunoglobulin (Ig) genes, and in the vast
majority of cases, they express an Ig isotype other than
IgM, which indicates post-follicular B-cell origin.
Further support for the post-follicular origin of MM
comes from the analysis of the mutational status of the
variable region of the IgH gene. MM is characterised by
mutated homogeneous variable gene sequences, which
indicates that the B cell has passed the germinal centre
and is no longer under continuous influence from the
somatic hypermutation mechanism.
The low rate of proliferation of MM cells makes it
difficult to perform conventional cytogenetics.
Interphase FISH and molecular genetic investigations of
MM have provided evidence of a marked karyotypic
instability. Numerical chromosome structural changes
are present in virtually all MM, and in most, if not all
cases of MGUS (Avet-Loiseau et al., 1999; Drach et al.,
1995; Flactif et al., 1995; Zandecki et al., 1996, 1997).
MM can be subdivided into two cytogenetic categories
(Debes-Marun et al., 2003): hypodiploid/pseudodiploid
(which also includes the near-tetraploid karyotypes) and
the hyperdiploid. The latter is defined by the presence of
multiple trisomic chromosomes (mostly chromosomes 3,
5, 7, 9, 11, 15 and 19) associated with a gain of DNA-
observed as aneuploidy with flow cytometry. The
number of structural abnormalities per cell is lower in
the hyperdiploid group (average 5/1) than in the
hypodiploid group (average 9/1) but the types of
abnormalities are similar in both groups (Smadja et al.,
2001). Hypodiploidy is associated with an unfavourable
prognosis (Smadja et al., 2001; Debes-Marun et al.,
Unbalanced chromosomal structural changes are
present in most MM (Cigudosa et al., 1998). Karyotypic
complexity is thought to increase during tumour
progression. Chromosomal gains that recur in more than
30% of MM include 1q, 3q, 9q, 11q and 15q.
Monoallelic loss of 13q sequences is one of the most
frequent abnormalities in MM (50%) and is an
independent predictor of poor prognosis (Avet-Louseau
et al., 2000; Kuehl, 2002).
During the pathogenesis of MM, most primary
translocations are reciprocal translocations that
juxtapose an oncogene and a IgH gene sequence. These
reciprocal translocations result in activation of
oncogenes, because they come under the influence of
enhancer regions at the IgH gene locus. They are
mediated mainly by errors in Ig heavy chain switch
recombinations, but sometimes by errors in somatic
hypermutation during plasma-cell generation in germinal
centres (Bergsagel et al., 2001). These translocations are
almost universal in MM cell lines and are present in 70%
of PCL, in about 60-75% of MM and in 50% of MGUS
and smouldering MM (Fonseca et al., 2002). Despite the
promiscuity of translocation partners, most Ig
translocations involve three groups of genes: the first
group contains the cyclins D1 (on 11q13), D3 (on 6p21),
and possibly D2 (on 12q13) which are translocated in 25
% of tumours. The second group comprises two proteins
that are encoded on 4p16: MMSET – a nuclear SET
DOMAIN protein – and fibroblast growth factor
receptor 3 (FGFR3), an oncogene receptor tyrosine
kinase. These are translocated in 15% of MM. The third
group of proteins in which encoding genes are
translocated in 10% of MM comprises two B-ZIP
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Accepted April 27, 2005
Bone marrow microenvironment in multiple myeloma