Haematologica 2003 - Supplements

hypothesis that (1) CCND1 and FGFR3 contribute to the early
development of MM, followed by (2) c-MYC/MUM1 and c-
MAF play crucial roles in the progression of CCND1+ and
FGFR3+ MM, respectively. Our diagnostic system will provide a
new insight into understanding genetics-based MM classification
and into its application for the appropriate molecular targeting
therapies.
007
IGH REARRANGEMENTS PATTERNS IN MULTIPLE
MYELOMA.
González D, García-Sanz R, Balanzategui A, Gutiérrez NC,
López-Pérez R, Chillón C, Alonso ME, González M, San
Miguel JF.
Hematology Department, University Hospital of Salamanca, SPAIN
An increased rate of complete responses in myeloma multiple
(MM) patients through high dose chemotherapy and
transplantation of cell progenitors, has lead to the design of
strategies for the analysis of minimal residual disease (MRD).
One example is the use of PCR for amplification of
immunoglobulin (Ig) gene rearrangements that are specific for
each malignancy. Recent studies have shown that “real time
quantitative PCR” (RQ-PCR) analysis of the Ig heavy chain gene
(IgH) rearrangements can be used for MRD detection in acute
lymphoblastic leukaemias. This approach is more complicated
when applied to B-cell malignancies with somatically mutated
IgH genes like MM. We analyzed the presence of incomplete
DH-JH and complete VH-JH rearrangements in a series of 84
MM with DH and VH family primers in combination with a
consensus JH primer. Genescanning was used to evaluate the
clonality of the rearrangements. After purification in
polyacrylamide gels, clonal VH-JH and DH-JH rearrangements
were sequenced to identify the VH, DH and JH gene segments.
Germline VH, DH and JH segments were identified by
comparison to the V, IGMT and BLAST databases. These
analyses also allowed the assessment of somatic mutations in
both VH-JH and DH-JH rearrangements. The overall detection
rate of clonality was 94% (79/84). In particular, VH-JH
rearrangements were detected in 73/84 patients (84%). Moreover,
more than 50% of patients displayed incomplete DH-JH
rearrangements (50 out of the 84 patients, 60%). Of the 11
patients in whom no VH-JH product could be obtained, the DH-
JH PCR showed a monoclonal band in 6 of them. All VH
segments carried >2% deviation from the germline sequence
compared to molecular databases (median 7,2%; range: 2-22). By
contrast, 88% of the incomplete DH-JH rearrangements possesses
>98% homology with its closest germline gene segments. We
also observed a biased VH, DH and JH gene segment usage. In
VH-JH rearrangements we found an over-representation of VH3-
30, VH1-69, and VH3-9, DH3-22 and DH2-21 as well as JH4. In
DH-JH rearrangements we found an over-representation of DH1-
7 and DH4-4, and JH4 gene segments. In conclusion, incomplete
DH-JH rearrangements are present in 60% of MM patients with
the special feature that the vast majority of them are unmutated
which make them the preferential target for MRD studies in MM
patients by RQ-PCR using consensus JH probes. Furthermore,
these findings offer a new insight into the regulatory development
model of IgH rearrangements.
008
DNA mismatch repair defects in the pathogenesis and
evolution of myeloma
Mark Velangi, Graham Jackson, Gareth Morgan, Penny
Taylor, Andy Hall, Julie Irving
Royal Victoria Infirmary, Newcastle Upon Tyne, UK Northern
Institute for Cancer Research, University of Newcastle, UK, Dept
Haematology, Leeds General Infirmary
Genetic instability is a prominent feature in mutliple myeloma
(MM) and progression of this disease from MGUS and
smouldering MM is associated with increasing molecular and
chromosomal abnormalities. The mismatch repair (MMR)
pathway is a post replicational DNA repair system that maintains
genetic stability by repairing mismatched bases and insertion
deletion loops mistakenly incorporated during DNA replication.
Deficiencies in proteins pivotal to this pathway result in a higher
mutation rate, particularly at microsatellites. We have
investigated the proficiency of the MMR pathway in clinical
samples and myeloma cell lines. Microsatellite analyses showed
instability at one or more loci of 9 examined in 15/92 patients:
7.7% of MGUS/SMM, 19.3% of MM/plasma cell leukaemia and
25% relapsed MM/PCL. An in vitro heteroduplex G/T repair
assay found reduced repair in 2 cell lines, JIM1 and JIM3 and in
2 from 4 PCL samples. Thus we show that microsatellite
instability occurs in plasma cell dyscrasias and the increased
frequency during more active stages of disease may suggest
defects in the MMR pathway have a contributory role in disease
progression.
009
Cell Cycle Control of Plasma Cell Differentiation and
Tumorigenesis
Selina Chen-Kiang
Department of Pathology, Weill Medical College of Cornell
University, New York, NY 10021, USA.
sckiang@mail.med.cornell.edu
Cell cycle control and apoptosis are major determinants of
homeostasis during B cell differentiation and tumorigensis.
Although multiple myeloma cells rarely cycle and normal plasma
cells are cell cycle arrested, virtually nothing is known about cell
cycle control of B cell differentiation. Frequent translocation of D
type cyclins in MM cells implies that cell cycle dysregulation
may contribute to MM pathogenesis. Cell cycle progression,
however, is controlled not by the absolute levels of D type
cyclins, but by the balance between positive regulators, cylins
together with cyclin-dependent kinases (CDK), and negative
regulators, the CDK inhibitors. (CDKI) This suggests a critical
role for CDKIs in plasma cell generation and tumorigenesis.
To test this hypothesis, we have dissected the roles of CDKIs in
normal plasma cell differentiation. We demonstrate that one
specific CDKI, p18INK4c, is elevated by IL- 6 to cause G1 cell
cycle arrest through inhibition of Rb phosphorylation by CDK6
during differentiation of human lymphoblastoid cells to plasma
cells (1). Targeted disruption of p18INK4c in mouse leads to B
and T cell hyperproliferation (2). Most significantly, plasma cell
differentiation in vivo and in vitro is defective in the absence of
p18INK4c, but not other CDKIs (3). The requirement for
p18INK4c is temporal specific, because there is no impairment in
the formation of germinal centers and memory cells, class switch
recombination or bone homing in a T-cell dependent antibody
response. Syndecan-1- positive plasmacytoid cells containing
high levels of secreted-form of immunoglobulin are generated in
the absence of p18INK4c. However, they fail to efficiently
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