Haematologica 2003 - Supplements

immunofluorescence staining for DKK1 and cytoplasmic
immunoglobulin light chain (cIg) on cytospin preparations from
MM bone marrow aspirates confirmed the heterogeneous
expression pattern in clonotypic PC and also revealed DKK1 in a
subset of cells with neutrophil nuclear morphology. A similar
analysis on normal healthy donors revealed cytoplasmic
DKK1expression unique to 1/3 to 1/2 of cIg-positive plasma
cells. A quantitative ELISA for DKK1 in sera from bone marrow
and peripheral blood showed that normal bone marrow aspirates
contained a mean of 10ng/ml with no case showing greater than
15ng/ml. One the other hand, MM cases could have as much as
125 ng/ml in both the bone marrow and peripheral blood. There
was a strong correlation between plasma levels of DKK1 protein
and DKK1 gene expression.
Since lytic bone lesions develop at sites of MRI-defined
medullary plasmacytoma (MPCT), MRI represents a highly
sensitive surrogate for present and future osteolytic lesions. As
MRI-defined MPCT can be observed in the absence of x-ray
detectable lytic lesions, we hypothesized those cases lacking lytic
lesions, yet having high DKK1 and/or FRZB gene expression
levels may have underlying MRI detectable MPCT. To address
this issue we combined x-ray and MRI data and applied the same
analyses used above to analyze differences between those cases
with ≥ 3 lytic lesions and MPCT (n = 65) and MM with no lytic
lesions or MPCT (n = 45). A total of 107 genes differentiating
the two groups (P < .001) were identified. Here many of the same
genes, e.g. cell cycle genes, identified above remained significant
and, importantly, the degree of significance increased. For
example, whereas the ratio (≥3 lytic lesions/no lytic lesions) of
the mean expression level for DKK1 in the first comparison was
2.45, the mean value increased to 6.25 in the latter comparison.
This reflected the fact that virtually all cases with no lytic lesions
and moderate to high DKK1, had MRI-defined focal lesions. The
mean expression level of DKK1 in the no lytic lesion group was
1674 (range 40 to 10828) whereas the mean DKK1 level in the no
lytic lesion & no MPCT group dropped to 625 (range 57 to
4183). It is important to note that DKK1 and FRZB expression,
as determined from bone marrow aspirates of the iliac crest,
although very powerful, can not account for the presence of bone
lesions in all patients, as 10 of 83 (12%) of cases with >3 x-ray
lesions did not express appreciable levels of DKK1 or FRZB. A
quantitative trait locus (QTL) for low bone mass in the general
population has been identified 5 and may enhance bone loss, even
in the presence of low levels of DKK1 and FRZB. Alternatively,
Wnt signaling antagonism-independent mechanisms also
certainly play a role in bone disease.
We have recently shown that expression of the cell cycle control
and DNA metabolism genes TYMS, UBE2C, CCNB1, PCNA,
TK1, BUB1, BUB1B, EZH2, and TOP2A is significantly higher
in MM with metaphase cytogenetic abnormalities and that these
features are linked to poor survival 6 . These same genes were also
over-expressed in MM with lytic lesions and MPCT and
enhanced in cases with both, suggesting this type of MM is also
likely to have a high proliferation index, thus providing a
molecular explanation for the classification of MM with >3 lytic
lesions as stage III disease in the Durie-Salmon system.
Although exhibiting highly variable and sometimes very high
expression in MM PC, MIP1 (CCL3/SCYA3), a chemokine
implicated in OCL development and MM bone disease 1 , was not
significantly differentially expressed in this analysis. In addition,
RANKL, a known osteoclast differentiation factor with
conflicting data over its expression on MM PC 7-9 was not
detected in any MM PC or normal bone marrow PC sample tested
with our microarray system.
The relevance of elevated DKK1 and FRZB expression in MM
bone disease is derived from several recent studies that have
shown that functional Wnt signaling is critical for osteoblast
differentiation and function 10-14 . Patients with loss-of-function
mutations in the low-density lipoprotein receptor-related protein
5 (LRP5), a co-receptor for the Wnt ligand, have a condition
known as osteoporosis-pseudoglioma (OPPG) 10 . Remarkably,
separate and distinct mutations in LRP5 result in a high bone
mass (HBM) phenotype 11,12 . In contrast to the OPPG mutations,
the HBM defects represent gain-of-function mutations that
effectively block binding of the inhibitory protein DKK1 13 .
Elevated expression of FRZB results in a block in chondrocyte
maturation and function 14 . Thus, data presented here suggests that
MM PC exert a powerful negative effect on bone growth through
the secretion of two independent WNT signaling antagonists that
likely interfere with osteoblast function.
Finally, serial gene expression profiling after short-term in-vivo
drug treatment has revealed that DKK1 is hyperactivated in
nearly half of MM cases receiving dexamethasone and over
three-quarters of MM treated with thalidomide and IMiD, but
rarely by PS-341. Thus, we recommend that all patients receiving
drugs linked to DKK1 activation also receive bisphosphonates.
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