Haematologica 2003 - Supplements

032
Hyaluronan Synthase Expression by Circulating B
Cells Correlates With Poor Survival in Multiple
Myeloma (MM)
S. Adamia, M. Crainie, T. Reiman, M.J. Mant, A.R.Belch
and L.M Pilarski
Dept. Oncology, University of Alberta and Cross Cancer Institute,
Edmonton, AB, Canada.
Hyaluronan (HA) regulates MM cell behavior through the
interactions with the HA receptor RHAMM, and may also
mediate intracellular interactions. Both HA and its receptor
RHAMM appear to be clinically important in the biology of MM.
HA is synthesized by hyaluronan synthases (HASs)- HAS1,
HAS2, HAS3. We characterized the expression of HASs in MM
peripheral blood (PB) and BM using rigorously controlled single
stage RT-PCR, capillary electrophoresis and GeneScan analysis
software. We examined 142 BM and 70 PB samples from MM
patients taken at the time of diagnosis. Differential expression of
HAS1 or HAS2 was detected in MM PB and BM samples
respectively. For 82/142 MM patients BM cells expressed
HAS1, while 112/142 patients expressed HAS2, suggesting
preferential expression of HAS2 in MM BM. Contrary, MM PB,
44/70 samples expressed HAS1 and 23/70 expressed HAS2
transcripts, suggesting preferential expression of HAS1 in MM
PB. We identified two novel splice variants of HAS1 (HAS1Va
and HAS1Vb) in MM patients. We find that HAS1Va is
overexpressed in MM PB and BM, as well as in human
thymocytes. HAS1Vb is a result of abnormal intronic splicing,
and is detectable only in malignant cells. Although HAS1Va is
found in both PB and BM, expression of HAS1Vb which is
detectable only in the PB, is found in MM B cells, but is absent
from non-B cells in MM PB. Statistical analysis of 41 PBMC
and 117 BM samples from MM patients showed that expression
of HAS1Vb in PBMC either alone or in combination with
HAS1Va, HAS1 or HAS2, was strongly correlated with poor
survival (P=0.002). Furthermore, all 6 MM patients expressing
HAS1Vb in BM cells had inferior survival when compared to 117
MM patients lacking BM expression of HAS1Vb. HAS1Vb is
detected in PB MM B cells, but is undetectable in purified BM
PC from MM, raising the possibility that BM HAS1Vb
expression may derive from BM B cells rather than PC. The
remarkable association between PB HAS1Vb and poor survival,
together with the relative lack of this variant in the BM, suggests
that HAS1Vb may be preferentially upregulated in circulating
malignant B cells, providing further evidence in support of a
central role for early stage MM cells in malignant progression
and suggesting potential mechanisms through which MM B cells
may impact on disease progression. We speculate that HASs,
particularly the newly identified HAS1Vb, play important roles in
disease progression in MM. Based on the clinical significance of
HAS1Vb expression in early stage components of the MM
hierarchy, HASs may also participate in the initial oncogenic
events giving rise to MM. In addition to a role in promoting
malignant dissemination, we speculate that novel variants of
HAS1 may synthesize intracellular HA, a ligand for intracellular
RHAMM. This may modulate intracellular associations of
RHAMM with the mitotic apparatus and thus promote survival of
malignant cells with altered chromosomal complements,
increasing genetic instability of the MM clone and facilitating the
emergence of aggressive clonal variants. Funded by CIHR and
the National Cancer Institute (USA).
033
Centrosome Amplification in Plasma Cell Proliferative
Disorders (PPD)
Gregory J. Ahmann, Kimberly J. Henderson, Jeffrey L.
Salisbury, Philip R. Greipp, Rafael Fonseca.
Mayo Clinic
Introduction: Multiple myeloma is a PPD that displays overt
karyotypic instability. Aneuploidy has been reported in up to
90% of patients with PPD. In other malignancies centrosome
amplification is associated with chromosomal instability. Thus,
we decided to determine a possible role of centrosomal
amplification as a contributing factor to aneuploidy in PPD. To
identify and quantify the centrosomes we used a centrin-2
antibody to label centrioles: centrin-2 is a crucial protein for the
duplication of centrioles. Normal cells have two (G1) or four
(S/G2/M) centrioles depending on the stage of the cell cycle.
Patients and methods: We prepared cytospin slides from bone
marrow cells from patients with PPD. The slides were fixed then
stained with a mouse anti-human centrin-2 antibody followed by
an anti-mouse FITC. To simultaneously detect clonal plasma
cells we used anti-light chain specific antibodies conjugated with
AMCA. We scored the number of centrin signals in both cIg+
and cIg- cells. Two independent scorers scored a total of 200
plasma cells and 200 non plasma cells per patient. The centrin
signals were enumerated and classified as no signal, normal (1-4
centrioles), abnormal (> 4 centrioles). We used a cut off value of
15% of cells with greater than 4 signals as an indicator of
centrosomal amplification. Samples with 5% to 14% cells were
considered equivocal. We determined the DNA index of the
samples by propidium iodide staining and concurrent cIg
expression using flow cytometry.
Results: In 5 of the 10 cases (50%) we found centrosome
amplification as per our criteria. In patients with abnormal
results the median percentage of plasma cells with centrosomal
amplification was 21.7 (16-28.5). The following table indicates
our results:
Diagnosis
Number of Signals
% cells %
with no normal
signals cells
(1-4
%
abnormal
cells
(>4
signals)
DNA
Index
signals)
Amyloid 13.5 58.0 28.5 1.00
Amyloid 9.0 64.0 27.0 1.00
SMM 39.5 44.5 16.0 1.12
New MM 18.0 65.5 16.5 1.00
New MM 30.0 58.5 11.5 1.29
New MM 67.0 31.5 1.5 1.19
Treated MM 68.0 30.0 2.0 2.00
Treated MM 38.0 61.0 1.0 1.00
Treated MM 30.0 58.0 12.0 1.00
Treated MM 24.5 55.0 20.5 0.95
Conclusion: Centrosome amplification is observed in most
patients with PPD, but it involves only a fraction of the plasma
cells. There was no obvious association between centrosomal
amplification and the DNA index. However, the results of this
study are preliminary and limited in that PI only identifies major
DNA gains/losses. Additionally, we only looked at one protein
of the centrosome complex. We are now conducting further
studies using chromosome enumeration FISH probes and
additional centrosomal protein antibodies.
S102