Haematologica 2003 - Supplements

evidence from two t(11;14) cases where DNA from 11q13 was
joined to recombined hybrid (S/S) switch region sequences.
In the first case S sequence and S1 sequence, on der(11), had
apparently recombined before a 3’ join to 11q13. However in the
second case 11q13 sequence was joined to sequence 5’ of a
S/S1 hybrid switch region on der(14). There was also
evidence of another non-CSR-mediated rearrangement having
occurred at the t(11;14) recombination site on der(14), with 5’S
sequence inverted from germline orientation for a run of 40 bases
before being aligned in the conventional orientation running for
approximately 1kb, until the recombination point with S1
sequence. For this case the reciprocal der(11) breakpoint was also
isolated and sequenced, comparison between the two breakpoints
showed that 21 bases of 11q13 sequence was common to both, i.e
had been apparently duplicated in the recombination process
associated with the translocation event. Both this data and the fact
the breakpoint on 14q32 involved the same region 5’ of S and
not another switch region suggests that the translocation did not
occur by an aberrant CSR process. The four cases described
above suggest that primary IgH translocations in MM can occur
via different mechanisms. We speculate that legitimate CSR
rearrangements can occur before a later translocation event
between 4p16 or 11q13 and 14q32, which implies that primary
IgH translocations can occur at different time points. Therefore
the crucial neoplastic event which juxtaposes the IgH enhancers
to the critical oncogene(s) associated with immortalisation of
plasma cells is independent of translocation partner and may not
occur via a CSR mechanism, but rather a later undefined event.
026
t(4;14)(p16;q32) as a Model for Unbalanced
Immunoglobulin Heavy Chain Translocations in
Multiple Myeloma
Jonathan J. Keats, Tony Reiman, Christopher A. Maxwell,
Michael J. Mant, Andrew R. Belch, Linda M. Pilarski
Department of Oncology and Medicine, University of Alberta and
Cross Cancer Institute, Edmonton, AB Canada
Translocations involving the immunoglobulin heavy chain (IgH)
locus are common and recurrent events in multiple myeloma
(MM). The most common IgH translocations identified to date
are t(11;14)(q13;q32), t(4;14)(p16;q32), and t(14;16)(q32;q23).
The frequency of each event in patient populations has become
apparent in the last few years with t(11;14) occurring in 15-20%,
t(4;14) 10-15%, and t(14;16) in approximately 5% of patients.
Moreover, multiple independent research groups have now
reported similar results regarding the clinical impact of IgH
translocations in MM. We and others have found that t(4;14)
predicts for a poor outcome while other groups have shown a
similar prognosis for t(14;16) patients, however, t(11;14) seems
to predict for an improved outcome. Interestingly, as shown by
our group, the clinical outcome associated with t(4;14) appears to
be independent of FGFR3 expression, the proposed target gene of
t(4;14), as a subset of patients lack FGFR3 expression. These
observations lead us to hypothesize that in some patients t(4;14)
may be unbalanced resulting in the selective loss of der(14),
which has recently been validated by Santra et al. Moreover, this
phenomenon does not seem to be localized to t(4;14) as Fonseca
and colleges have recently shown that unbalanced translocations
also occur in both t(11;14) and t(14;16) MM. Therefore, we are
beginning to investigate the importance of genes that flank either
side of the t(4;14) breakpoints to determine their expression
levels and potential contribution to MM.
To access the importance of flanking genes we have updated our
cohort, which now includes 272 MM and 77 MGUS patients.
Using an RT-PCR screening assay we have found 38 (14.0%)
t(4;14) positive MM patients and 1 (1.3%) positive MGUS
patient. This strategy can identify three major breakpoint
clusters, termed MB4-1, 2, and 3. The 38 MM patients segregate
into 26 MB4-1, and 6 each of MB4-2 and MB4-3, while the
MGUS patient is an MB4-1. FGFR3 is expressed in 27/37
(73.0%) positive MM patients and in the one MGUS patient. All
FGFR3 non-expressers lacked a detectable der(14) RT-PCR
product. Furthermore, for 54 negative and 11 t(4;14) positive
patients we have tested multiple BM samples, primarily diagnosis
and relapse, and have yet to find any differences in t(4;14) status
or FGFR3 expression. To define the level of gene expression we
have chosen to use quantitative RT-PCR. We are particularly
interested in genes that are not interrupted by any of the
breakpoints or genes that may promote the oncogenic process by
either overexpression or haplo-insufficiency. One gene that falls
into the latter category is the Response Element-II Binding
Protein (RE-IIBP). Transcription of RE-IIBP initiates
downstream of all known t(4;14) breakpoints and thus, unlike
MMSET type I & II, RE-IIBP could be overexpressed in its
native form by the mu enhancer within all t(4;14) patients. To
date we have observed an 8-15 fold increase in the expression of
RE-IIBP in t(4;14) positive cell lines compared to other human
myeloma cell lines. Work is ongoing to identify other genes
located on chromosome 4 that are deregulated by t(4;14).
027
The recurrent translocation t(14;20)(q32;q12) in
multiple myeloma results in aberrant expression of
MafB and PPP1R16B (TIMAP); a molecular and
genetical analysis of the chromosomal breakpoint
Gienke R Boersma-Vreugdenhil, Jeroen Kuipers, Ton
Peeters, Esther van Straalen, Anne Hagemeijer, Peter L
Pearson, Hans C. Clevers, Bert J.E.G. Bast
Depts Immunology & Medical Genetics, UMC UtrechtCenter for
Human Genetics, KU Leuven
Chromosomal translocations of the Immunoglobulin Heavy gene
region at 14q32 are regularly involved in B lymphoid
malignancies. They are thought to be important in initiating
transformation either by deregulation of existing (proto)
oncogenes or creation of new hybrid genes with transforming
properties. Previously, we reported a novel translocation,
t(14;20)(q32;q12), found in the myeloma cell line UM3 by means
of double color FISH analysis. In this cell line, the t(14;20) is the
only translocation involving the IgH locus. Using a recently
developed sensitive double color Immuno FISH, we found the
t(14;20) in the diagnostic bone marrow sample as well, excluding
a possible in vitro artifact.
We cloned the regions containing the breakpoints in the der(14)
and der(20) chromosomes from the UM3 cell line, and analyzed
ectopic mRNA expression of genes and expressed sequence tags
(EST’s) in the breakpoint regions of both derivative
chromosomes. Ectopic gene expression was observed for the
transcription factor MafB in der(14), and aberrant expression
levels of PPPIR16B, also known as TIMAP (putatively involved
in the regulation of apoptosis), was detected in der(20). In
addition, we characterized the breakpoints in a similar if not
identical t(14;20) in 3 other cell lines; the breakpoint scatter is
comprised within a region of 0,8 Mb, explaining the aberrant
expression in all situations. We also have found such a t(14;20) in
additional patient material.
S99