Haematologica 2003 - Supplements

of genes, one of them being the gene for transmembrane protein
carbonic anhydrase isoenzyme IX (CA IX) which is involved in
maintaining the acid-base balance of the cell. Since the degree of
intratumoral hypoxia is positively correlated with HIF 1, the
expression of CA IX has been proven an indirect parameter for
the degree of tissue hypoxia.
Aim of the study: Determining the expression of CA IX, the
endothelial cell proliferation fraction (ECP) and mean vessel density
(MVD) of bone marrow (BM) biopsies in non-neoplastic diseases,
haematological malignancies and metastatic carcinomas.
Materials and methods: BM biopsies were included at the time of
diagnosis before any treatment was administered from 12 patients
without malignancies (NON-NEO), 17 patients with MGUS, 23
myeloma patients (MM), 44 patients with a chronic
myeloproliferative syndrome (CMPS), 13 patients with a CD34-
negative AML, 15 patients with a CLL, 25 patients with a bone
marrow metastasis of an epithelial tumour (META).
The BM biopsies were immunostained with anti-CA IX. The
endothelial cell proliferation was determined by a double staining
for CD34 and Ki-67. The mean vessel density (MVD), the
number of vessels per 0.22 mm2 was determined on sections
immunostained for CD34.
Results: 7 of 25 META had at least a focal membranous
expression of CA IX whereas no expression was observed in all
haematological neoplasms nor in MGUS and bone marrows of
NON-NEO patients. There was a significantly (Kruskal Wallis: p
< 0,0001) increased MVD in MM (19.1(11.3), META (24.0(7.7),
CMPS (19,2(7,6), AML (20,8(6,8) compared to the NON-NEO
(8.6(6.5) cases. There was no significant difference for MGUS
(7.3(3.5) and CLL (9,2(4,6) compared to NON-NEO (p>0,05).
The MM (1.9(2.0), CMPS (0,9+0,8), AML(1,2(1,1) and the
META (2.5(1.4) had a significantly increased ECP compared to
the the NON-NEO(0.1(0.3) cases. For the CLL (0,1(1,3) and the
MGUS (0.0(0.0) no significant difference could be demonstrated.
A significant difference in ECP was observed between MM and
MGUS. The intensity of CD34 staining was graded: grade 1
corresponded to a weak to partially negative staining of the
majority of the (sinusoidal) vessels. Grade 2 was attributed to the
cases in which part of the vessels had a weak to partially negative
staining and part of the vessels had a strong CD34 staining and
grade 3 if the majority were strongly staining for CD34 (small
sprouting vessels). MM with a diffuse growth pattern (7 cases)
had grade 3 staining vessels; in the interstitial-nodular growth
pattern (5 cases), 2 were grade 1, 2 were grade 2 and 1 was grade
1 and in the pure interstitial pattern (9), eight had grade 1 and 1
had grade 3 (chi-squared p-value= 0.0003).
In MM there was a significant correlation between the tumorload
and the MVD (r=0.72, =0.0001), between the tumorload and the
endothelial cell proliferation (r=0.60, p=0.03) and between the
MVD and the endothelial cell proliferation (r=0.60, p=0.009)
6.3 Bone disease
148
Role of myeloma-induced osteoclastogenesis in the
disease.
Shmuel Yaccoby, John Shaughnessy, Fenghuang Zhan,
Qing Yi, Bart Barlogie, and Joshua Epstein.
Myeloma Institute for Research and Therapy, Arkansas Cancer
Research Center, University of Arkansas for Medical Sciences,
Little Rock, Arkansas, USA.
We have studied the role of myeloma-induced osteoclastogenesis
in the disease process. Initially, we determined whether myeloma
plasma cells (MM PC) could induce osteoclastogenesis in a
stromal cell-free environment. Next, we examined the effect of
osteoclasts (OC) on the growth and survival of myeloma and
healthy donor PC, and the molecular consequences of PC
interactions with OC.
Cultures of osteoclast precursors (pOC) and mature active
multinucleated OC were prepared from PBSCs and MNC from 5
healthy donors and PBSCs from 30 myeloma patients. CD138-
enriched PC were purified from BM aspirates from 50 patients
and from 6 healthy donors (NPC).
MM PC conditioned media from 9 patients increased migration
of pOC across a 5 µM pore size membrane 1.6-4.2 fold from
127±37 (mean±SEM) in controls (p=0.01). In co-cultures with
pOC, MM PC from 22 patients induced formation of
multinucleated, bone-resorbing OC by 6 fold from 50±8 in
controls (p