Haematologica 2003 - Supplements

patient samples. In addition, the patient was found to harbor an
inactivating mutation of p53 rendering the remaining p53 allele
inactive. Of interest, the number of t(4;14)(p16.3;q32) fusion
signals detected per cell increased with time indicating, in a
model of ongoing genomic instability, a positive selection of the
derivative chromosomes of the translocation. This effect was
more pronounced in the cell line than in the serial patient
samples. In addition, the cell line duplicated the total
chromosome number becoming near-tetraploid. Further analysis
of these and other primary myeloma samples may reveal a pattern
of progression reflective of events occurring in vivo, facilitating
further investigation into the pathogenesis and treatment options
for this fatal disease.
012
Exclusion of V4-34 expressing B-cells from
transformation to multiple myeloma: no inherent
maturation block to plasma cells in normal and
myeloma circulating CD19+ lymphocytes
Surinder S. Sahota, Karin Tarte*, Niklas Zojer, C. Ian
Mockridge, Gavin Babbage, Richard Oreffo, Bernard Klein*
& Freda K. Stevenson
Molecular Immunology Group, Tenovus Laboratory, Southampton
University Hospitals UK; * Inserm Unite 475, Montpellier, France.
One of the striking features that emerged from immunoglobulin
variable (V) region analysis in multiple myeloma (MM) is that a
specific population of B-cells expressing the IgH V4-34 gene is
excluded from the myelomagenic pathway. Other features have
established a post-follicular stage of neoplastic arrest. This
asymmetry in VH gene use in MM contrasts markedly with usage
in the normal B-cell repertoire of ~7%, and derivation of a range
of other B-cell tumors from such cells. Recently, it was reported
that there may be a developmental censoring of normal B-cells to
end-differentiated plasma cells, using a MoAb (9G4) specific for
V4-34. Such censoring could also explain the fact that V4-34 is
not found in malignant plasma cells. To address this further, we
have examined V4-34 at the gene level in purified normal plasma
cells from bone marrow, and in plasma cells generated in-vitro
from normal circulating CD19+ B cells. We also examined invitro
matured plasma cells from circulating B-cells from a MM
patient. In both the normal and tumor setting, we detected
isotype-switched somatically mutated V4-34 functional
sequences, indicating no block in maturation. Interestingly, in
>50% of cases, somatic mutation had occurred in sequences
involved in binding-site-associated idiotope expression. Our data
suggest that normal B cells expressing V4-34-encoded Ig can
mature to plasma cells in-vivo and in-vitro, but emerging cells
may lose reactivity to 9G4 by somatic mutation. Failure to detect
V4-34 in plasma cell tumors is therefore not due to a maturational
defect, but is likely to be a tumor-specific feature.
013
In multiple myeloma clonotypic memory B-cells
recirculate through bone marrow, peripheral blood and
lymph nodes
Thomas Rasmussen, Marianne Lodahl and Hans Erik
Johnsen
Department of Hematology L, Herlev Hospital, University of
Copenhagen, 2730 Herlev, Denmark
Introduction. It is believed that myeloma cells are derived from a
germinal center (GC) or post GC B- cell. The GC B-cell can
differentiate into both a memory B-cell and a PC. In this study,
we investigated the recirculating potential of memory B-cells
clonally related to the myeloma plasma cell (termed clonotypic).
Materials and methods. From 10 multiple myeloma (MM)
patients bone marrow (BM) aspirates obtained at time of
diagnosis and peripheral blood (PB) samples were collected.
From 7 out of the 10 MM patients a single peripheral lymph node
(PLN) was aspirated. The VHDJH immunoglobulin gene
rearrangement that represents the MM clone was identified for
the 10 MM patients and allele-specific oligonucleotides (ASO)
IgH RT-PCR assays were designed for each patient. BM
mononuclear cells (BMMNC) and PBMNC were stained with the
monoclonal antibodies CD19, CD27, CD38, CD62L, CCR6,
CXCR4, CXCR5, CCR7 and different memory B-cell subsets
were flow-sorted as single cells directly to PCR tubes followed
by ASO RT-PCR analysis.
Results. Clonotypic memory B-cells were identified in 7/10
patients and both CD62L positive and negative clonotypic
memory B- cells were identified suggesting the presence of
clonotypic memory B-cells with different migration/homing
potential. Further, clonotypic memory and later stage B-cells
(CD38+) were identified in CXCR4+/- subsets, whereas all
clonotypic memory and later stage B-cells were CXCR5 positive.
Comparable frequencies of clonotypic cells were found in the
CCR6+/- memory B-cell subsets, but only few clonotypic CCR7+
memory B-cells were observed in a single patient. Different
clonotypic memory B-cell subsets were identified in both PB and
BM.
To extend these studies we investigated whether clonotypic cells
were present in PLNs obtained from 7 myeloma patients. In 2 out
of 7 patients we were able to identify clonotypic cells in the PLN
illustrating that a subset of clonotypic cells enters the PLNs.
Discussion. We identified a CD19+/CD27+/CD38- subset of
clonotypic cells in the majority of MM patients and as all
clonotypic cells have accumulated somatic mutations, these cells
meet all the characteristics of memory B- cells. The
heterogeneous expression of the CD62L, CXCR4, CXCR5 and
CCR6 molecules on clonotypic memory B-cells probably reflects
their diverse homing/recirculating possibilities including a
potential to extravasate secondary lymphoid organs. In
accordance with their immunophenotype, clonotypic memory B-
cells were identified in PLNs. Clonotypic memory B-cells seem
to have the same diverse recirculating/homing capacity as normal
memory B-cells. Although the clonotypic memory B-cells
showed a normal immunophenotype and recirculating potential,
these cells comprised up to 4% of the memory B-cell pool. The
malignant potential of clonotypic memory B-cells is currently
under investigation.
014
Identification of a new human plasma cell subset from
which myeloma and ‘progressive’ MGUS are derived
Andy C Rawstron, James AL Fenton, David Gonzalez de
Castro, Anne Dring, Faith E Davies, Roger G Owen,
Stephen J Richards, Andrew S Jack, Anthony Child, Gareth
J Morgan.
HMDS, Academic Unit of Haematology and Oncology, Algernon
Firth Building, University of Leeds, Leeds LS1 3EX, United
Kingdom.
Plasma cells in MGUS patients are heterogeneous with respect to
phenotype, karyotype, and degree of intraclonal variation. A
correlation between plasma cell characteristics and clinical
outcome has not yet been demonstrated, partly because
progression to myeloma is a rare event. However, many patients
do show progression, with gradually increasing paraprotein,
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