Haematologica 2003 - Supplements

005
HETEROGENEOUS PATTERN OF THE
CHROMOSOMAL BREAKPOINTS INVOLVING THE MYC
LOCUS IN MULTIPLE MYELOMA
Sonia Fabris,1 Tiziana Storlazzi,2 Luca Baldini,1 Lucia
Nobili,1 Luigia Lombardi,1 Mariano Rocchi,2 and Antonino
Neri1
1Laboratorio di Ematologia Sperimentale e Genetica Molecolare,
U.O. Ematologia 1, Dipartimento di Scienze Mediche, Università
degli Studi di Milano, Ospedale Maggiore IRCCS, Milan; 2DAPEG
Sezione Genetica, Università di Bari, Bari, Italy
Chromosomal translocations juxtaposing the MYC locus with
one of the immunoglobulin (IG) loci represent an invariant
oncogenetic event in Burkitt’s lymphomas and murine
plasmacitomas. In human multiple myeloma (MM), a malignant
tumor of somatically mutated, isotype-switched plasma cells, the
involvement of the MYC locus has been thought to be a rare
event. Conventional cytogenetic studies have identified 8q24
translocations involving the IG loci in less than 5%, and Southern
blotting has revealed rearrangements of the MYC locus in only a
few cases , generally not involving the IG loci. Over the last few
years, novel molecular approaches such as fluorescence in situ
hybridization (FISH) have demonstrated that IG translocations,
mainly affecting the IGH switch regions, occur in virtually all
MM cell lines and most primary tumors, and may involve a large
number of target loci. Using this approach, recent studies have
shown that MYC locus abnormalities, often characterized by
complex translocations and insertions not involving the IG loci,
are very common in MM cell lines and represent a recurrent
event in MM patients with aggressive disease and a poor
outcome. In our study, we used dual-colour FISH to characterize
the breakpoint locations of chromosomal translocations/
rearrangements involving the MYC locus at 8q24 found in a
panel of 14 MM cell lines and 70 primary tumors (66 MM and 4
PCL). MYC locus alterations were observed in 21 cases:
MYC/IG (mainly IGH) fusions in 11 cell lines and three patients
(2 MM and 1 PCL), and extra signals and/or abnormal MYC
localizations in seven patients (5 MM and 2 PCL). Fourteen of
these cases were investigated by FISH analyses using a panel of
BAC clones covering about 6 Mb encompassing the MYC locus.
The breakpoints were localized in a region 100-250 kb
centromeric to MYC in four cases, a region 500-800 kb telomeric
to the gene in four cases, and regions ≥ 2 Mb centromeric or
telomeric to MYC in five cases. Two different breakpoints were
detected in KMS-18 cell line, while the insertion of a MYC allele
was found in a complex t(16;22) chromosomal translocation in
RPMI8226 cell line. Our data document a relatively high
dispersion of 8q24 breakpoints in MM.
S. Fabris and T. Storlazzi contributed equally to this work
Supported by: the Associazione Italiana Ricerca sul Cancro
(AIRC) and the Italian Ministry of Health.
006
Distinct developmental pathways of the multiple
myeloma identified by mRNA expression analysis of
the chromosomal translocation-associated
protooncogenes
Miyuki Kato, Shinsuke Iida, Hirokazu Komatsu, Takashi
Ishida, Ichiro Hanamura, Yoshihito Hayami, Takae
Kataoka, Takemi Otsuki, Emi Tajima, Hiroshi Miwa,
Masakazu Nitta, Ryuzo Ueda
Department of Internal Medicine & Molecular Science, Nagoya City
University Graduate School of Medical Science; Division of
Hematology/Oncology, Nagoya Memorial Hospital; Department of
Hygiene, Kawasaki Medical School; Department of Internal
Medicine, Aichi Medical University
[Purpose] Accumulation of chromosomal translocations
involving immunoglobulin heavy chain gene locus is responsible
for the development of multiple myeloma (MM). Among them,
deregulation of CCND1 and FGFR3 expression is often found in
MGUS, indicating that t(11;14) and t(4;14) are the primary
translocations, which result in the development of MGUS.
However, t(6;14), t(8;14), t(14;16) and t(14;20), by which
respectively deregulate MUM1, c-MYC, c-MAF and MAFB
genes are rarely found in MGUS, although they are occasionally
encountered in MM cases, suggesting that they belong to the
secondary translocations, which are relevant to the disease
progression of the MM. To pursue the developmental pathways
and to classify MM based on the genetic alterations, we
established RQ/RT-PCR system to quantify their mRNA
expression and applied it to investigate primary MM samples.
[Method] mRNA expression of the abovementioned six genes
was quantified by RQ/RT-PCR with an aid of Light Cycler.
After establishing the optimal conditions, we studied the gene
expression in 19 MM cell lines, 30 MM, 4 MGUS and 5 reactive
plasmacytosis patients. In patients’ specimens, 1ug of total RNA
extracted from purified plasma cells using CD138 bead selection
was reverse transcribed for cDNA, diluted and used for RQ-PCR.
Standard curves of the gene expression were generated using
diluted plasmids. Expression level was corrected using
housekeeping gene expression. [Result] In 19 MM cell lines,
mRNA expression of the CCND1, FGFR3, c-MAF and MAFB
genes was extremely high when chromosomal translocations
involving these gene loci exist. In contrast, expression level of
the MUM1 and c-MYC mRNA was exclusively higher in 19 MM
cell lines than in fresh MM samples irrespective of the status of
the chromosomal rearrangements involving these gene loci,
suggesting that high expression of these two genes are associated
with highly proliferative ability. Of the six genes, 10 out of 19
MM cell lines harbored high expression of more than two genes.
Of the 30 MM samples, eight(26%), six(20%) and five(18%)
showed ectopic expression of the CCND1, FGFR3 and c-MAF
genes. Analogous to the data obtained from cell lines, two and
one CCND1+ cases coexpressed high levels of c-MYC and
MUM1 genes, respectively. Three out of the six FGFR3+ cases
showed ectopic expression of the c-MAF. One c-
MAF+FGFR3+c-MYC+ case and one c-MAF+c-MYC+MUM1+
case were diagnosed as having primary plasma cell leukemia and
highly aggressive MM, respectively. Interestingly, CCND1+
MM cases tended to carry c-MYC or MUM1 overexpression and
never carried c-MAF mRNA expression, and FGFR3+ cases
tended to carry c-MAF overexpression (P