Haematologica 2003 - Supplements

differentiate to antibody-secreting plasma cells and are eliminated
by apoptosis.
Thus, p18INK4c is specifically required for cell cycle arrest and
differentiation of functional plasma cells, and it modulates
plasmacytoid cell survival. Given that MM cells may represent
inappropriate intermediates of plasma cell differentiation that
survive in the bone microenvironment, work is in progress to
address the expression of CDKI in MM cells. A model for
coordinated cell cycle and apoptosis control in MM pathogenesis
will be discussed.
Supported by NIH grants (CA 80204, AR49436) and a
Specialized Center of Research for Myeloma grant by the
Leukemia and Lymphoma Society of America.
1. Morse, et al., (1997) Immunity 6: 47-56.
2. Franklin et al., (1998) Genes Dev 12: 2899-2911.
3. Tourigny, et al., (2002) Immunity 17: 179-189.
010
Molecular Analysis of the Mitotic Checkpoint Genes
BUB1, BUBR1 MAD1L1, MAD2, and MAD2B in Multiple
Myeloma
Tammy L Price-Troska, Scott A Van Wier, Philip R Greipp
MD, and Rafael Fonseca MD.
Mayo Clinic, Division of Hematology and Department of Internal
Medicine, Rochester, MN, USA
Introduction: Chromosomal instability (CIN) occurs in the
context of defective mitotic checkpoints, as in colorectal cancer,
with the end result being aneuploidy. Multiple myeloma (MM) is
characterized by ubiquitous aneuploidy, which is an early event
detectable in MGUS. Hyperdiploidy, characterized by gains of
chromosomes 3,5,7,9,11, and 15, is seen in 50% of cases,
predominantly those without IgH translocations. Key mitotic
checkpoint genes include BUB1, BUBR1, MAD1L1, MAD2 and
MAD2B. We therefore assayed for abnormalities of these genes
in MM.
Samples and Methods: Our analysis included FISH, Southernblot,
Northern-blot, molecular screening and sequencing of these
genes. We studied 5 human MM cell lines; JJN3, OCI-MY5,
MM1, KAS 6/1, ANBL-6 (all harbor IgH translocations) and 10
patients with MM and no IgH translocations (by FISH). Northern
and Southern blot analysis were done on the cell lines. Both
patient samples and MM cell lines were screened for mutations
using conformation sensitive gel electrophoresis followed by
manual sequencing in abnormal cases. BAC clones including the
genomic loci of these genes were used as FISH probes.
Interphase FISH combined with the cytoplasmic light-chain and
cytomorphology were used to analyze the MM patients.
Metaphase and interphase FISH was used for the analysis of the
cell lines.
Results: No abnormal qualitative RNA production was detected
by Northern blot analysis of the cell lines. The genomic loci
appeared intact as Southern blot, using EcoRI digested fragments,
did not reveal large deletions, insertions or inversions with the
exception of a point mutation in MAD2B IVS(4) which appeared
to be polymorphic. RNA sequencing of the BUBR1 gene
revealed three single base alterations causing the following
transitions: 161C>T (T40M), 1088A>G(Q349R) and
1895T>C(V619A). Population studies from 100 normal
individuals revealed these transitions are polymorphic. Analysis
of MAD1L1 showed a missense mutation at codon
695(2262G>A) in 2 of the cell lines and 1 MM patient. Analysis
of MAD2 revealed three missense mutations (E164G, R181C,
and R183C) in one patient sample, plus R181C also detected in a
second patient sample. A third patient sample showed a mutation
of 442A>T causing an amino acid change of S111C. FISH
analysis on the 10 MM patients revealed no predominant deletion
pattern for any of the 5 checkpoint genes. FISH analysis on the
cell lines displayed abnormal deletion patterns in 3 of the
checkpoint genes. KAS 6/1 showed deletion of BUB1 in 40% of
metaphases while OCI-MY5 showed deletions of MAD2 and
MADIL1 in each metaphase observed. .
Conclusion: Our preliminary findings indicate that mutational
inactivation of BUBR1, MAD1L1 and MAD2 could result in
defective checkpoint allowing the generation of aneuploidy in
some cases (30%) of MM. However none of these mutations
were constant. Further investigations into the role of mitotic
checkpoint genes and their relationship to aneuploidy is
warranted.
011
Establishment of the JMW Myeloma Cell Line: In vitro
Analysis of Multiple Myeloma Clonal Evolution
R.C. Tschumper*, R. Fonseca+, S.A. Van Weir+, T.L.
Price-Troska+, and D.F. Jelinek*
Depts. of Immunology* and Internal Medicine+, Mayo Graduate
and Medical Schools, Mayo Clinic, Rochester, MN 55905.
Human multiple myeloma (MM) cell lines have proven to be
useful tools in understanding this progressive disease. However,
establishment of human myeloma cell lines is a difficult task and
a further complication is the uncertain relationship between in
vivo tumor cells and tumor cells that survive in vitro giving rise
to cell lines. Thus, the selection pressures in vitro may differ
markedly from in vivo selection pressures. We wished to study
this more closely and have done so by genomic profiling and
interphase fluorescent in situ hybridization (FISH) analysis from
the initial stage of procurement of patient tumor cells to
permanence as a cell line (designated as JMW).
The IgA-λ expressing JMW cell line was derived from the blood
of a 67-year-old female presenting with aggressive MM and
many circulating plasma cells. Purified myeloma cells were
cultured in RPMI 1640 media with FCS, IL-6, and IGF-1. The
JMW cell line is CD2-, CD5-, CD19-, CD10-, CD38+, CD40+,
CD44+, CD28+, and EBV negative. The IgVH sequence of the
cell line is identical to the sequence of the primary tumor cells
(VH 4-39 with 6.8% somatic mutations). This cell line is IL-6
dependent, but also displays a smaller proliferative response to
IGF-I. Interferons alpha and gamma inhibited proliferation of the
cell line whereas IL-1, IL-2, IL-3, IL-4, IL-10, IL-11, IL-12, GM-
CSF and TGF-beta were without effect on proliferation.
RNA from both the initial cell population and the established line
was used for cDNA array analysis using the Affymetrix U95Av2
biochip. A significant number of genes were differentially
expressed between the two time points and these results will be
presented. Genes of interest include HSP 70, IAP-1, IGFBP-4
and several human ribosomal proteins.
Concurrent with the genomic profiling, cells were analyzed by
FISH for genetic abnormalities. The t(4;14)(p16.3;q32) was
detected since the time of diagnosis in nearly all cells and was
conserved throughout disease evolution, and in the stable cell
line.. As expected monosomy 13 (94-97% of the cells) was
present in all samples. Patient tumor cells did have a complex
karyotype that was shown both by karyotype analysis and by
FISH to include an unbalanced complex translocation resulting in
LOH of 13q and 17p (der13 t(13;17)(q?;p?)), in the context of a
hypodiploid karyotype. FISH analysis shows divergence in the
chromosome complexity between the cell line and subsequent
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