Haematologica 2003 - Supplements

may be related to pesticide exposure. The higher incidence of
computer use and lower educational background of the cases is
difficult to explain although electromagnetic radiation could be
implicated in the former.
019
COEXISTENCE OF PRODUCTIVE AND NON-
PRODUCTIVE IGH GENE REARRANGEMENTS IN
MULTIPLE MYELOMA
E. Novella, E. Albiero, D. Madeo, F. Elice , I. Giaretta and
F. Rodeghiero
Department of Cell Therapy and Hematology, San Bortolo
Hospital, Vicenza
Multiple Myeloma (MM) is a B-cell neoplastic disease
characterized by clonal expansion of plasma cells in the bone
marrow. Analysis of the VDJ rearrangement of the
immunoglobulin heavy chain (IgH) gene provides a unique
clonotypic marker to monitor the residual tumor cells and to
verify the efficacy of chemotherapy. The VDJ rearrangement is
considered to remain constant in the patient thorough the course
of the disease; furthermore, the presence of independent clones in
the same MM patient is rarely reported, mostly in association
with a second hematological malignancy.
The aim of this study was to assess the recurrence of different
clones in a panel of consecutive MM patients included in a highdose
treatment program enrolled at our Department from January
1999 to December 2002.
Ninety consecutive MM patients were first screened at the
genomic DNA level; mRNA analysis was subsequently
performed in those patients showing multiple rearrangements to
verify the real coexistence of multiple productive clones evolved
independently or the presence of rearrangements involving both
alleles of a single cell. VDJ rearrangement was determined using
different set of VH family-specific primers for FR1. PCR
products were subjected to capillary electrophoresis and analyzed
by gene scanning in an automated DNA sequencer ABI Prism
310. A monoclonal population was detected in 64/90 (71%); three
patients (3%) showed double rearrangements. These
rearrangements used different VH, D and JH genes, as confirmed
by sequencing. Total RNA from samples collected prior to highdose
therapy was reverse transcribed using random primers and
cDNA was separately amplified with the same VH familyspecific
primers which gave positivity at the DNA level. PCR
products were analyzed by gene scanning. The absence of
contaminating DNA was assessed using primers for the beta-actin
gene, designed to discriminate between DNA and RNA.
In two of the three patients a monoclonal peak was detected on
cDNA only with one VH-family. This finding confirms the
hypothesis of rearrangements involving both alleles, one of
which was a non-productive rearrangement. In the third patient,
two monoclonal population were identified, for family VH.1 and
VH.3 respectively. In this case two monoclonal population are
really detectable, although the presence of an associated,
clinically silent disease, can not be excluded.
Biclonality or oligoclonality are widely documented for ALL and
lymphomas; this study strongly suggests that multiple
rearrangements could be identified even in a small number of
MM patients (∼3%), although the presence of more than one
functional clone remains a rare event (∼1%). In these cases
mRNA analysis is mandatory to distinguish between real
biclonality and productive or non-productive rearrangements
involving the same cell and to monitor residual tumor plasma
cells. Clinical and molecular follow-up using tumor-specific
primers of the patients with double clonality is currently under
way.
020
Isolation and culture of human multiple myeloma
medullary plasmocytes
L. Garderet, C. Mazurier, M. Lagrange, S. Bouchet, J. Frick,
NC. Gorin, M. Lopez
Department of Hematology, Inserm U76, CHU and Hospital Saint
Antoine, Paris, France.
Aim: Studies of freshly isolated multiple myeloma patient bone
marrow samples are often limited because of massive and rapid
cell death. So far, no human myeloma cell line has been obtained
from patients with medullary involvement alone. We are
developing a plasma cell culture, which keeps some of them alive
after a week. Materials and methods: Bone marrow from five
multiple myeloma patients (stage III) at diagnosis were collected
into heparin with the patient’s informed consent. Mononuclear
cells were isolated by Ficoll Hypaque sedimentation, then plasma
cells were purified using the anti-CD138 plasma cell isolation
system (Miltenyi-Biotec). Purity of isolated plasma cells was
determined by incubation with PE anti-CD138 monoclonal
antibody and analysed on a FACScalibur flow cytometry system
(Becton Dickinson). Purified plasma cells were used immediately
or frozen in fetal calf serum containing 10 % DMSO and stored
in liquid nitrogen before use. Fresh or thawed plasma cells were
maintained in liquid culture in ISCOVE medium with IL-6, GM-
CSF, IL-3 and SCF for 8 days. On day 0, 4 and 8, the plasmocyte
culture was examined for viability (using trypan blue dye
exclusion), morphology (air-dried cytospin stained with Wright-
Giemsa) and cell surface phenotype (CD38 and CD138
monoclonal antibodies). Results: After immunomagnetic bead
separation, purity was 76% (55%-98%) and yield was 54% (11%-
100%). On day 0, the culture was started with a cell range from
0.6 to 4.6 X 106 plasmocytes. After a 4-day culture, 41% of
myeloma cells (15%-69%) were viable as assayed by trypan blue
and checked by morphological examination. On day 8, the mean
plasmocyte number decreased to 15% (4%-24%). In terms of
viability, we didn’t observe any difference starting the culture
with fresh plasma cells or after thawing. FACS analysis was very
heterogeneous, CD138 decreasing steadily during the cell culture
as cells were dying and failed to express Syndecan-1. CD38 had a
higher expression compared to CD138 because of a more nonspecific
staining.
Conclusion: In this preliminary study, we showed that the
combined use of IL-6, GM-CSF, IL-3 and SCF enabled some
viability of medullary plasma cells during at least an 8-day
culture and as such avoided the common early death of freshly
uprooted myeloma cells. This culture method could be used for
short-term functional or cytogenetic studies.
S96