High S
gSerum Sclerostin Correlates with
Advanced Stage, Increased Bone
Resorption, Reduced Osteoblast Function
and Poor Survival in Newly-Diagnosed
P t
a i
tients with Multiple Myeloma
Evangelos Terpos1, Dimitrios Christoulas1, Eirini Katodritou1,
Cornelia Bratengeier2, Brigitte Lindner2, Silvia Harmelin2, Gerhard
Hawa2
Hawa , Georgios Boutsikas1
Boutsikas , Magdalini Migkou1
Migkou , Maria
Gavriatopoulou1, Eurydiki Michalis1, Anastasia Pouli1, Efstathios
Kastritis1, Konstantinos Zervas1 & Meletios A. Dimopoulos1
Kastritis , Konstantinos Zervas & Meletios A. Dimopoulos
1Greek Myeloma Study
Study Group;
2Biomarker Design Forschungs GmbH, Vienna, Austria
Di
l
sc osures for Evangelos Terpos, MD
MD
In
l
comp i
liance with
ith ACCME
l
po i
licy, th
the ASH
ASH
i
requ res th
the f ll
o owing di
disclosures to th
the
activity audience:
Research Support/P.I.
Support/P
(N/A)
Employee
(N/A)
Consultant
(N/A)
Mj
Ma
M jor
j
St
S
Stockh
khold
llder
ld
(N/A)
Speakers' Bureau
(N/A)
Scientific Advisory Board
(N/A)
(N/A)
N/A = Not Applicable (no conflicts listed)
Osteocyte: Key-cell
yy
for Bone Remodeling
Seeman E, et al. N Engl J Med. 2006;354(21):2250-2261.
van Bezooijen RL, et al. Cytokine Growth Factor Rev. 2005;16(3):319-327.
Regulation of Osteoblast Function
Marie
Peters E, et PJ.
al. Arch
Arch Biochem
Biochem B
B iophys
iophys. . 2008;473(2):98-105.
2008;473(2):112-116.
Osteonectin-
Osteonectin SPARC
-
· SPARC/osteonectin/BM-
40 is the most abundant
non-collagenous
extracellular matrix
protein in bone.
· It regult
lates osteobl
blasti
tic
lineage progression at
multiple levels.
· SPARC may have a
positive role in p
pprogenitor
cell expansion.
· It positively
py influences
osteoblast differentiation.
Delany AM & Hankenson KD. J Cell Commun Signal 2009; in press
Microenvironment & Myeloma
Bone Disease
Osteoblast
OPG
bALP,
Ml
Myeloma
l
cells
tl
osteocalci
(-)
n
RANKL
CD138
IL-6
Dkk-1, sFRP-1 & -2
41 integrin
RANKL
RANKL
VCAM-1
VCAM-1
BMSCs
RANKL
IL-11, IL-1, bFGF
(-)
TNF, M-CSF
OPG
IL-6
OPG
IL
RANK
MIP-
MIP 1
1 ,M
, IP-
MIP 1
1 ,
SDF-1, IL-3,
HGF, OPN
Osteoclast
Activated osteoclasts
precursor
TRACP-5b
Collagen type-1
Terpos & Dimopoulos.
degradation products
Bone resorption
Ann Oncol 2005;16:1223-31
Bone
Aim of
of the
the Study
The aim of
of this
this study
study contacted by
by the
the Greek
Myeloma Study
yy Group in collaboration with
Biomarker Design Forschungs GmbH (BDF),
Vienna, Austria was to evaluate the serum levels
of sclerostin in
in patients
patients with
with MM
MM and
and explore
possible correlations with clinical and laboratory
py
data, including SPARC levels, ISS stage and
survival.
Patient Characteristics
MM
MGUS
Ct
Cont
l
ro s
at diagnosis
No
157
21
21
Gender (M/F)
87-70
12-9
12-9
Median age (range)
68 (36-94)
67 (54-80)
68 (50-79)
Ig-
Ig subtype: gG

/
gG IgA
/
/BJ/IgD
/BJ/
/NS
94-38-21-3-
3 1
13-7-
7 1
1: 56 (35%)
ISS stage
-
2: 51
5 (32%)
(32%)
3: 50 (32%)
Hb 10
<
/dL
g
52 (33
(3 %)
3
-
-
creatinine >UNL
37 (23%)
1 (2.2%)
-
creatinine 2 mg/dl
21 (13%)
-
-
albumin <3.5 g/dL
76 (48%)
-
-
-M >3 mg/L
98 (62%)
-
-
2
-M >6 mg/L
g
48 (30%
(
)
-
-
2
CRP >10 mg/L
31 (20%)
-
-
LDH > 240 U/L
47 (29%)
-
-
Methodology (1)
Sclerostin ELISA
· Microtiter plates were coated with anti-human SOST antibody,
incubated over night at 4°C, and blocked at room temperature for 1
hours.
· Serial dilutions of rhSOST were used to establish a standard curve,
ranging from 0 to 3 ng/ml. A secondary biotinylated anti-human
SOST antibody is used for detection.
· 50 l
µ
f
o sample, t
s
d
an ard or cont
l
ro are i
b
ncu ated togeth
ther with
ith
200 µl of detection antibody over night at room temperature.
· After a washing step,
gp, 200 µl of horseradish peroxidase-con
µp
jugated
jg
streptavidin were added and the plates incubated for one hour at
room temperature in the dark. After a last washing step 3,3',5,5'-
tetramethyl benzidine
benzidine is used as substrate and
and after addition
addition of 50
µl stop solution the optical density is measured at 450 nm.
· The detection limit was 0.18 ng/ml.
· The standard range was set from 0.3-3 ng/ml.
· CV for intra- & inter- assay was 2-6%.
Methodology (2)
SPARC ELISA
· Microtiter plates were coated with anti-human SPARC antibody,
incubated over night at 4°C, and blocked at room temperature for 1
hour.
· Serial dilutions of human SPARC were used to establish a standard
curve, ranging from 0 to 133ng/ml. A secondary biotinylated anti-
human SPARC antibody is used for detection.
· Ft
For h
the assay serum or EDTA
EDTA
l
samp es are dil t
u ed 1 50
:
in assay b ff
u er.
50 µl of diluted sample, standard or control are incubated together with
200 µl of detection antibody for 3 hours at room temperature.
·
After a washing step, 200 µl of horseradish peroxidase-conjugated
streptavidin were added and the plates incubated for one hour at room
temperature in
in the
the dark. After
After a last washing step
step 3,3'
3,3 ,5,5
,
'-
5,5 tetramethyl
benzidine is used as substrate and after addition of 50 µl stop solution
the optical density is measured at 450 nm.
· D t
e
ti
ec on li it
m : 1.95
95x102
10
/l
ng/ml
· CV for intra- & inter- assay was 4-8%
Methods (3)
· In all patients we also measured in their sera
· Cys-C: immunonephelometry (Dade Behring, Liederbach,
Germany).
· CTX: ELISA (Serum CrossLaps, Nordic BD, Herlev, Denmark)
· bALP: ELISA (Metra BAP, Quidel Co., San Diego, CA, USA)
· Statistics:
­ Mann-Whitney test (patients vs. controls)
­ Spearman Rank correlation test (correlations among clinical
characteristics and laboratory values)
­ Kaplan-Meier method (survival)
­ log-rank test (prognostic variables
variables)
­ Cox regression: multivariate models
Results: Sclerostin in Patients and Controls
mean value: 0.31±0.20
0.26±0.29
0.48±0.46
±SD
p=0.01
p=0.004
SPARC: Patients vs. Controls
mean value:
52.8±50.2
27.2±18.0
26.3±16.2
±SD (x102)
p<0.001
p<0.001
Sclerostin: Correlations with Markers of
Myeloma Activity
Activity
Sclerostin: Correlation with Bone Resorption
Sclerostin: Negative Correlation with Bone
Formation (bALP)
Sclerostin and Lytic Lesions
Sclerostin and ISS
Treatment of Patients
· The median number of lines of received therapies was 3
(range: 1-7).
· Only 27 patients had been diagnosed before 2000 and of
those 15
15 had
had never been exposed to novel agents (i.e.
thalidomide, bortezomib or lenalidomide).
· 137/157 (87%)
() had received conventional chemotherapy
py
(either MP or VAD) as first line anti-myeloma therapy, while
of the other 20 patients, 7 had received PAD, 8 TD and 5
MDT
t
par i
ticipating in respecti
tive cli
linical trials.
· 34 patients (21%) had received both bortezomib and an
IMiD (thalidomide or
or lenalidomide)
lenalidomide) during the
the course of
their disease.
· 33/67 (49%) patients
patients who
who aged <65 years had
had been given
high dose melphalan with ASCT.
Sclerostin and Survival
The median survival of all patients was 48 months.
The median f l
ollow-up oft
f h
the
t
pa itients was 20
20
t
mon h
ths
(range: 7-
7 113 months).
months).
Effect of Bortezomib on Sclerostin
L
l
eve s in P ti
a ents with
ith Rel
d
apse MM
MM
· Aim
· 25 patients with relapsed MM
· Treated with bortezomib 1.3 mg/m2 days 1, 4, 8, 11 of 3-
week
cycl4
le x 4
· Responders could receive 4 more cycles
· Non-responders after 4 cycles could have dex added
· Results
· Response data
· 10% CR, 54% PR
Changes of sclerostin
post-
post bortezomib
-
in relapsed MM
p<0.01
n=16
9
N=
Post-VD
Conclusions
· Our study provides evidence that sclerostin is
increased in the serum of patients with MM and
correlates with advanced ISS stage, increased bone
resorption, reduced osteoblast function and
poor survival.
· SPARC is reduced in MM possibly confirming the
reduced osteobl
blast functi
tion observed in these patients.
· Sclerostin is reduced post-bortezomib therapy and this
is another possible mechanism
mechanism of
of action of
of bortezomib
in enhancing bone formation in MM.
· Sclerostin seems to
to participate
participate in the biology
biology of MM
and thus it may be a possible target for the
development of novel therap
ppies that can both increase
osteoblast function and target myeloma cells.