Haematologica 2003 - Supplements

P2.3
DEVELOPMENT OF A MULTIPLE MYELOMA SPECIFIC
ARRAY
A. Keith Stewart
Medical Oncology, Princess Margaret Hospital, 610 University
Avenue, Room 5-126, Toronto, Ontario, M5G 2M9, Canada.
The initiating molecular event in multiple myeloma is defined by
several nonrandom chromosomal translocations leading to
immortalization of malignant plasma cells. In order to understand
the heterogeneity of multiple myeloma and perhaps identify key
genes that are altered in the diseased state, we have developed a
myeloma enriched 4.3K array from a cDNA library of pooled
patient samples 1 . Using our custom-made myeloma array and a
19K generic cDNA array produced by the University Health
Networks microarray facility we are examining the global gene
expression of normal and malignant plasma cells. Using these
resources, we have profiled the differences in expression of 18
myeloma and 8 other hematopoietic malignancy cell lines, 45
myeloma patient samples (at various stages of disease
progression) 6 healthy donor plasma cell controls and 7 CD19 + B
cell controls. Supervised cluster analysis of cell line data from
4.3K and 19K array identified 52 and 13 genes respectively, that
were differentially expressed between myeloma and nonmyeloma
cell lines. This data set includes genes known to be
involved in plasma cell biology, such as the multiple myeloma
oncogene 1 (MUM/IRF4), BlyS/BAFF receptor BCMA, plasma
cell marker CD138 (syndecan-1), serine threonine-protein kinase
oncogene (PIM2), plasma cell transcription factor XBP1, and a
gene involved in osteoclast formation and bone resorption
(ANXA2). In addition, novel genes such as putative disulfide
isomerase (MGC 3128), hypothetical proteins (MGC 5395,
FLJ22167), ESTs and unannotated genes from uncharacterized
chromosomal regions were also identified as differentiating these
two groups. Of the differentially expressed genes, three genes
PIM2, XBP1 and TRA1 were identified as MM specific on both
the 4.3K and the 19K array thus further validating the integrity of
these results. Further analysis of the cell line data attempted to
define a gene signature specific for FGFR3 and c-maf
translocations but revealed no genes significantly predictive of
either group suggesting that the primary oncogenic role of these
proteins has been overwhelmed by other factors in late stage
disease. CD138 + and CD19 + magnetic bead sorted cells from 45
MM patients and 13 healthy donors were examined. Supervised
cluster analysis of data from the 4.3K array detected 118 genes
that were altered between myeloma and non-malignant plasma
cells. Further scrutiny of these genes revealed upregulation of
endoplasmic reticulum stress response genes that include the
plasma cell marker x-box binding protein XBP-1,
immunoglobulin binding protein BiP (GRP78/HSPA5) and tumor
rejection antigen TRA1(GRP96). These genes are key members
of an adaptive pathway termed the unfolded protein response
(UPR), a phenomenon suggested to be critical for B celldifferentiation
into plasma cells 2,3 . Unsupervised cluster analysis
of all the 58 samples identified significant changes in expression
of 435 genes that distinguished the myeloma patients into two
major groups, a B cell-like (BL) and a normal plasma cell-like
(PL). Although the BL group has characteristic expression similar
to CD19 + B cells, the BL group are consistently differentiated by
upregulation of UPR genes. Further analysis of the BL signature
revealed the presence of several genes that are involved in
proliferation such as NF-kB subunit p65 (RELA), MAP kinase
kinase MKK2/SSP33 (PBK1), phosphoprotein regulated by
mitogenic pathways (C8FW), core promoter element binding
protein (CPBP) and cyclin A2 (CCN1). Our results demonstrate
two subclasses of Myeloma one of which appears proliferative
and is characterized by activation of the unfolded protein
response pathway which differentiates this class form normal B
cells. A better understanding of the stress response pathway in
plasma cell development may provide avenues for developing
novel diagnostic and therapeutic strategies.
References
1. Claudio JO, Masih-Khan E, Tang H, et al: A molecular
compendium of genes expressed in multiple myeloma. Blood
100:2175-2186., 2002
2. Reimold AM, Iwakoshi NN, Manis J, et al: Plasma cell
differentiation requires the transcription factor XBP-1. Nature
412:300-7., 2001
3. Gass JN, Gifford NM, Brewer JW: Activation of an
unfolded protein response during differentiation of antibodysecreting
B cells. J Biol Chem 8:8, 2002
P2.4
MOLECULAR CYTOGENETIC CLASSIFICATION OF
MYELOMA: CYTOGENETICS AND MICRO-ARRAYS
Prof Hervé Avet-Loiseau, MD, PhD
Laboratoire d’Hématologie, Centre Hospitalier Universitaire,
Nantes, France.
So far, no constant recurrent genetic abnormality has been
identified in multiple myeloma (MM). Several intrinsic MM
characteristics may explain this apparent difference with other B-
cell malignancies. First of all, myeloma PCs are not proliferative,
in agreement with the fact that they correspond to terminally
differentiated cells. This low proliferative index is a major pitfall
for cytogenetic studies, in which, by essence, cells have to pass
through the mitosis phase to be analyzable. The second reason
could be that, when informative (i.e., abnormal), karyotypes are
very complex, with multiple numerical and/or structural changes.
Finally, the absence of a so far unrecognized unique recurrent
abnormality may reflect the heterogeneity of MM, which could
represents several different entities.
Extended analyses of published cytogenetic reports have revealed
several recurrent abnormalities and/or profiles. The most frequent
profile is hyperdiploidy, observed in 50% to 60% of the patients
with an informative karyotype. Interestingly, the gained
chromosomes are not random, and involve especially odd
chromosomes. Of note, this is in marked contrast with another B-
cell malignancy, i.e., acute lymphoblastic leukemia, in which
hyperdiploidy is related to the gain of even chromosomes. The
second most frequent abnormality is the loss of chromosome 13,
observed in half of the patients. Finally, abnormalities of the
14q32 region are found in about 30% of the patients, most
frequently through t(11;14). This incidence is markedly different
from that observed in B-cell lymphomas, in which 100% of the
cases present a translocation involving an Ig gene. However,
more recent data obtained on human myeloma cell lines (HMCL)
have enhanced the interest to the IgH gene in MM. Cytogenetic
and molecular analyses of HMCL have shown that almost all the
HMCL present an illegitimate IgH rearrangement. However, in
contrast to B-cell lymphomas, in which a unique partner is
involved, several partners have been identified in HMCL. Three
partners are more frequently involved: FGFR3/MMSET at 4p16,
CCND1 at 11q13, and c-maf at 16q23, observed in about 25% of
the HMCL each. In the other quarter of HMCL, several
chromosomal bands are translocated to 14q32. Do these results
reflect the situation observed in patients? Actually, analyses using
techniques that do not require metaphases (i.e., independent of
the proliferative index), such as interphase fluorescence in situ
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