Haematologica 2003 - Supplements

molecules expressed on the surface of T cells. This, in turn, leads
to the rapid activation of T cells and cell division.
Twenty-two expansions have been conducted to date using
samples from multiple myeloma patients who have received a
variety of prior therapies. The number of T cells increased a mean
of 404 fold following an 8-14 day culture process. Purity and
viability of T cells post-Xcellerate averaged 97% and 96%,
respectively. The CD4:CD8 ratio increased from 1.3 to 2.1 during
the course of culture. Significant upregulation of key
surface/effector molecules, including CD25 (IL-2 receptor) and
CD154 (CD40 ligand) occurred.
We have also developed a flow cytometric assay for evaluating
residual myeloma cells in the final Xcellerated T cell product.
Using analysis of surface marker expression, including CD38,
CD138, CD45 and CD56, we have been unable to detect any
myeloma cells in the final product within the limits of detection
of the assay (~0.01%).
Based upon these data, we have initiated a clinical trial to
evaluate the activity of Xcellerated T Cells in patients with
multiple myeloma, who are undergoing an autologous stem cell
transplantation. Following induction chemotherapy, patients
undergo a leukapheresis to collect peripheral blood mononuclear
cells for the Xcellerate Process. Patients then undergo stem cell
mobilization and collection, followed by high dose chemotherapy
with Melphalan (200 mg/m2). Patients then receive a stem cell
infusion, followed three days later by an infusion of 5-10 x 1010
autologous Xcellerated T Cells. Three patients have been treated
to date. In the two evaluable patients, lymphocyte recovery to >
1,000/mm3 occurred within three days following T cell infusion
(Day 6 post transplant). In contrast, lymphocyte recovery usually
does not occur for > 3-4 weeks post transplant in myeloma
patients treated with this regimen.
071
Comparison of single nucleotide cytokine
polymorphisms between patients with myeloma and
their siblings
Merih Kýzýl, Ender Soydan, Esin Serbest, Cemaliye Boylu
Akyerli, Klara Dalva, Meral Beksac
Department of Hematology,Ankara University School of Medicine
,Ankara; Department of Molecular Biology and Genetics,Bilkent
University,Ankara,Turkey.
Aim: Multiple Myeloma(MM) is a monoclonal B cell disorder in
which a network of multiple cytokines is highly active. Tumor
Necrosis Factor-alpha (TNFα, Transforming Growth Factorbeta(TGF-β1),
Interleukin-6 (IL-6), Interleukin-10 (IL-10) and
Interferon-gamma (INF-γ are among those studied the most). The
role of these cytokine genes during the evolution to multiple
myeloma is still under investigation. Polymorphisms of cytokines are
associated with high or low secretion patterns. Although the
frequency of some alleles have been analysed and compared to
healthy controls there is no published study which has evaluated
cytokine polymorphism patterns between patients and their siblings.
Patients and Methods: 35 patients (M/F: 21/14) aged 28-73
(median:51) admitted to Ankara University İbn-i Sina Hospital
were included in the study. Patients were Stage I (n:3), IIA
(n:3),IIB(n:1), IIIA(n:17), IIIB(n:6); presented with IgG (n:19),
IgA(n:4), light chain (n:4), IgM (n:1), PCL (n:1) and
plasmacytoma(n:4). Treatment was radiotherapy (n: 8),MP (n:
18) or VAD (n:23). Ten patients have been transplanted(8 auto
and two tandem). Following a period of 3-81 months (median:25)
from diagnosis seven of the patients responded, 21 relapsed or
became refractory following at least one protocol, six patients
arenot evaluable yet. Patients had a median of two siblings(1-5).
Peripheral blood sample DNA was used for detection of TNFα -
308, TGF-β110,25, Interleukin-10-1082-,819,-592 Interleukin–6-
174 and Interferon γ+874genotyping. Gene polymorphism was
detected using the CYTGEN kit (One-Lambda, USA).
Results: Frequency of all cytokines among patients compared to
the frequency among siblings were similar:
Cytokines Frequency % OS Disparities
Patient Sibling % %
TNF-α high
low
11.8
88.2
8.9
91.1
100
81
13.8
TGF
IL-10
high
interm
low
high
interm
low
76.5
23.5
0
8.6
11.4
28.6
65.5
30.9
3.6
18.7
12.7
23.6
82
55
50
75
100
86
0
100
88
67
53.6
81.8
IL6 high 97.1 85.5
41.4
Low 2.9 14.5
IFN-γ high 20 36.4
55.2
interm 51.4 43.6
low 28.6 20
However invidual comparisons between patients and siblings
revealed similar (TNF) or different expression patterns( TGF, IL-
6, IL-10,INF). The greatest disparity was observed with IL-10.
Khi-Square analysis between the the frequency of disparities in
TNF(13.8%) and IL-10(81.3%) was not significant. Among the
patients with single nucleotide poymorphism disparities,
differences were detected in all(n:1), four(n:4), three(n:5),
two(n:11) or one(n:8) of the cytokines. Patients carrying high
TNFα, low IL-10, high IL-6 high IFN γ. alleles had an
insignificant survival advantage. Age at presentation and cytokine
high/low patterns werenot associated with survival.
Conclusion: Increase in the duration of followup and sample size
are required for better evaluation of prognostic-etiological effects
of these cytokine profiles.
072
Elevated plasma OPN in multiple myeloma patients
Therese Standal, Henrik Hjort-Hansen, Inger Marie Dahl*,
Anne-Tove Brenne, Anders Waage, Magne Børset, Anders
Sundan and Øyvind Hjertner
Institute of Cancer Research and Molecular Biology, Norwegian
University of Science and Technology, Trondheim, Norway *
Department of Medicine, University Hospital of Tromsø, Norway
A characteristic feature of multiple myeloma is the development
of osteolytic bone lesions. Osteopontin (OPN) is a
noncollagenous matrix protein produced by various cell types
including osteoblasts, osteoclasts and several types of tumor cells.
OPN is essential for the migration, attachment, and resorptive
activity of osteoclasts. Furthermore, it has been shown that OPN
inhibits hydroxyapatite formation during bone mineralization. An
association between elevated plasma OPN, increased tumor
burden and decreased survival has been reported in women with
metastatic breast cancer and men with prostate carcinoma.
Here, we report that plasma levels of OPN are elevated in
myeloma patients as compared to healthy individuals. However,
levels of OPN are not associated with survival in the myeloma
patients. We further show that myeloma cell-lines and primary
myeloma cells produce OPN. After inoculating OPN-producing
ANBL-6 human myeloma cells in irradiated SCID-mice we were
not able to detect human OPN in mouse plasma-samples.
Interestingly, levels of circulating rodent OPN in these mice were
elevated as compared to control mice. This suggests that
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