MULT
MUL IPLE MYELOMA
Redefining risk
Cytogenetics
il
Proteomic analyses
Johannes Drach, MD
Professor of Medicine, Hematology & Oncology
Program Director
Multiple Myeloma and Malignant Lymphoma
Medical University of Vienna
Vienna, Austria
MULTIPLE MYELOMA
... not just one disease !
3 decades
- Risk stratification
- Individuali a
z tion
ation of treatment
tr
RISK CLASSIFICATION OF ACTIVE MM (mSMART)
High-ri
High sk
risk (25%)
(25%)
Standard-ri
Standard sk
risk (75%)
(75%)
FISH: Del 17p
All others including
t(4 14)
;
Hd
Hyper i
di l
p oid
idy
t(14;16)
t(11;14) by FISH
t(6;14)
() by FISH
Cytogenetics:
Del13q
Hypodiploidy
(beta
(
-2
- -
2 M<55m
-M < 5.5
g/l;
mg/l;
LDH < 250)
PCLI > 3%
Mayo Clinic, Leukemia 2007; 21: 529-534
Bortezomib in MM Patients with Cytogenetic Abnormalities
Bortezomib + MP (VMP)
No impact f
o cytogeneti
tic (FISH)
(FISH) abnormalities
FISH: any unfavorable [t(4;14), t(14;16), del(17p)] vs.
None
Best M-protein
Total
High Risk
Std Risk
Response, n (%)
(N=165)
(N=26)
(N=139)
CR (IF )
-
32%
33%
32%
TTP
OS
VMP standard risk
VMP standard risk
VMP high risk
VMP high risk
VMP standard risk (N=142): 23.1 months (34 events)
VMP standard risk (N=142): not reached (16 events)
VMP high risk (N=26): 19.8 months (7 events)
VMP high risk (N=26): not reached (3 events)
HR = 1.297 (95% CI: 0 5
. 5
55, 3 06)
.
HR = 1.009 (95% CI: 0.278, 3 663)
.
San Miguel J et al., NEJM 2008
Bortezomib in relapsed MM with gain of 1q21
N = 109
Medi
ed an
a number
ube of
o prior
po th
t er
e api
ap es: 3 (r
3( ange
ag ,
e 1 9)
100%
96%
63%
80%
60%
60%
40%
40%
20%
Median time from MM diagnosis to treatment with bortezomib:
4.8 years (range, 0.3 15.1)
RESPONSE TO SINGLE AGENT BORTEZOMIB
1q21
q
GAIN vs. 1q21
q
NORMAL
58%
60
8%
50
g
MR
n
40
27%
30%
respondi
30
PR
5%
20
patients
15%
%
23%
10
CR, nearCR
P = .06
10%
MM, 1q21 normal
MM, 1q21 gain
N = 28
N = 21
SURVIVAL AFTER SINGLE AGENT BORTEZOMIB
1q21 GAIN
qG
vs. 1q21 NORMAL
qO
Time to Treatment Failure
Overall Survival
1,0
P = .02
1,0
0,9
P = .002
0,9
0,
0,8
0,8
median, 32.4 months
0,7
0,7
n = 28
ts
0,6
n 0,6
median,
eti
0,5
a 0,5
Patients
6.6 months
P
04
% 0,4
% 0,4
0,3
0,3
0,2
0,2
median,
median, 4.4 months
0,1
n = 28
0,1
2.5 months
n = 21
n = 21
0,0
0,0
, 0
5
10
15
20
25
30
35
40
45
50
0
5
10
15
20
25
30
35
40
45
50
Months
Months
Months from start of bortezomib
MM with 1q21 gain
MM with normal 1q21
RESPONSE TO A BORTEZOMIB COMBINATION
1q21
q
GAIN vs. 1q21
q
NORMAL
70
63%
57%
60
12%
3%
50
g
MR
n
40
24%
27%
respondi
30
PR
30
20
patients
%
29%
CR, nearCR
27%
10
P = .67
MM, 1q21 normal
MM, 1q21 gain
N = 33
N = 27
SURVIVAL AFTER A BORTEZOMIB COMBINATION
1q21 GAIN
qG
vs. 1q21 NORMAL
qO
Time to Treatment Failure
Overall Survival
1,0
0,9
0,
P = .43
P = .50
0,8
median, not reached
median, 12.2 months
n = 27
n = 27
median, 23.3 months
n = 32
median, 8.9 months
n = 32
Months from start of bortezomib
MM with 1q21 gain
MM with normal 1q21
PROGNOSTIC FACTORS FOR SURVIVAL
single-a
g
gent
g
BORTEZOMIB
1q21 GAIN AND SERUM ALBUMIN 3.5
1,0
P = .001
09
0,9
08
median, 42.1 months
n = 18
0 factor
1 factor
atientsp
2 factors
%
median, 22.4 months
n= 20
median, 2.9 months
n = 9
Months from start of bortezomib
PROGNOSTIC FACTORS FOR SURVIVAL
BORTEZOMIB combination
1q21 GAIN AND SERUM ALBUMIN 3.5
1,0
median, 24.0 months
0,9
n=
n = 21
0,8
median, 26.8 months
atients
0 factor
a
n=
n = 30
p
1 factor
%
median, 22 months
2 factors
n = 8
P = .32
Months from start of bortezomib
Summary
· Bortezomib appears to be active in patients with poor-
risk cytogenetics
· Regarding lenalidomide, more data are required to
define ist role in such patient populations
· Any risk factor can only be evaluated in the context of a
specific ther
e apeuti
apeu c interventi
ee
on
· It is our aim to evaluate biomarkers and risk-factors
bd
beyond
tt
cytogene i
tics
BM stromal cell/IL6 dependence
Increased DNA index
Inmortalization
Malignant
g
Aggressive
gg
growth
g
In vitro
non malignant accumulation transformation
stromal independent
proliferation
Plasma cell
Normal
MGUS
MM
Cell line
leukemia
Primary IgH
IgH
Secondary IgH translocations:
translocations: C-MYC
translocations
Karyotypic instability
11q13
q
Tr
T isomies
del 13/p16
6p21
16q23
Mutations of N, K-RAS, FGFR3
20q11
0q
4p16
p53 mutations
Adapted from Hallek et al. Blood 1998;91:3-21
Role of bone marrow
microenvironment
· Secretion of bioactive
mol
o ecul
ecu es (cyt
(cy okin
o
es,
chemokines)
· Direct
ll
ce
ll
-ce i
i
nteract on
· Supports survival of tumour
cells
Protein secretion
secretion is fulfilling a biological function rather than
maintenance of basic metabolism - secretome analysis
Methodology: shotgun
shotgun proteomics
Methodology II: Shotgun Analysis
Separation by nano-liquid
chromatography
Identification by peptide
Protein extraction
fragmentation analysis
and digestion
digestion
(MS/MS)
Clinical proteomics database
Cl
Complex
i
pept d
ide
i
m xture
More sensitive; identification of more than 1000 different proteins in a sample, but
rather semi-quantitative.
Methodology: 2D-PAGE
·
Comparison of
state A (,,control") to
tt
state B(
B (,,di
")
sease
·
List proteins such as
A
p
Upregulated
Downregulated
Modified
B
Current database status
Lymphocytes
Monocytes
·
>1.800.000 independent peptide identifications
Neutrophils
·
>63.000 different peptides
Dendritic cells
·>4.900 different human proteins (SwissProt only)
Platelets
Composed from:
Erythrocytes
Plasma
primary
py cells
Fibroblasts
cultured cells
Epithelial cells
tissues
Keratinocytes
· Subcellular fractions:
Hepatocytes
Cytoplasmic proteins
Stellate cells
Mitochondrial/ribosomal proteins
Kupffer cells
Endothelial cells
Nuclear proteins
etc....
Secreted proteins
Easy access to data:
www.meduniwien.ac.at/proteomics/database
Search for MM-specific proteins
A
B
A: MM
MM celllls (1
(1 076
.
diff
dif
t
eren prot i
e
)
ns
B: MGUS in addition to all other primary cells analyzed so far (4.266
different proteins)
only A:
A: cell
cell-type specific proteins (5 hits)
Five MM Specific Proteins Identified
·
Q8N573 Oxidation resistance protein 1
·
Q8NEZ4 Myeloid/lymphoid or mixed-lineage leukemia protein 3
homolog
·
P05771
Pi
Protein ki
kinase C beta type
·
Q9NTK5
Obg-like ATPase 1
·
Q86X76
Nitrilase homolog 1
Proteins upregulated in MM
Cell adhesion molecules
·
CD9/tetraspanin-29, LYRIC/metastatic adhesion protein,
·
Intercellular adhesion
adhesion molecule
molecule 2 precursor (ICAM
(ICAM-2) (CD102
antigen)
St
Secret d
e proteins modulati
ting cellll cell it
interacti
tions
·
P-selectin precursor (granule membrane protein 140) (PADGEM)
·
Sts-1 (Suppressor of T-cell receptor signaling 1)
·
Cystatin F, basigin/collagenase stimulatory factor, small inducible
cytokine B7, PD-ECGF/Gliostatin
Secretion profile of bone marrow fibroblasts from
hl
hea th
lthy bk
back
d
groun
·
ECM: Collagens, laminins, fibronectin, biglycan, lumican, decorin,
perlecan, SPARC etc
·
Proteases: MMP-2, MMP-3, secernin-3
·
Protease inhibitors: TIMP-1, TIMP-2
·
Insulin like growth factor binding proteins 2, 4, 7,
·
Growth factors: connective tissue growth
growth factor,
factor, periostin
periosti
·
Cytokines/Chemokines: CXCL5, MIF, stanniocalcin-2, follistatin-
related
eated prote
ot in
e
1, PEDF, PTX3
·
Alpha-fetoprotein
Secretion profile of bone marrow fibroblasts from
MGUS b k
ac
d
groun is lt
a ered
Specific expression of (not detected in
in fibroblasts from healthy background):
·
CSF-1: inflammatory, inducable in vitro by IL1-beta in endothelial cells and by
LPS in dendritic cells
·
Chitinase-3-like protein 1 (CHI3L1): defense, plays a role in tissue
remodelling and defense against pathogens, otherwise expressed by
neutrophils
·
Insulin-like growth factor II (IGF-II): survival, strongly up-regulated upon co-
culture with MM
MM celllls
·
Insulin-like growth factor binding protein 6 (IGFBP-6): potential early
biomarker, expressed by
by a variety of
of tumour
tumour cells and cancer-related
fibroblasts, but not normal cells
Secretion profile of bone marrow fibroblasts from
MM background is altered as
as well
well
All proteins
proteins specific
specific for MGUS-
MGUS background were
were identified
identified as
as in
in
fibroblasts from MM patients as well
In addition to those, we found the specific expression of:
·
Stem cell growth factor
·
Hamartin
·
MMP-28
·
Stanniocalcin-1
·
Higher expression of alpha-fetoprotein
Stem cell growth factor
·
Stimulates the proliferation and differentiation of hematopoietic
precursor cells from various lineages. Acts synergistically with
other
ki
cyto nes, il
incl d
u i
ding IL-3G
3, G-CSF, GM
GM-CSF
d
an FLT3
ligand.
·
Suppresses SCF-stimulated erythroid cell proliferation.
·
Otherwise identified by us in inflammatory stimulated endothelial
cells and inflammatory stimulated dendritic cells
Stanniocalcin-1
· Involved in the regulation
regulation of
of calcium
calcium and phosphate
homeostasis in humans: Stimulates renal phosphate
reabt
bsorp i
tion,
dl
and cou d
ld th
th
f
ere ore prevent hl
hypercalcemia.
· Not expressed in normal blood and bone marrow cells
· Otherwise identified by us in cancer-related fibroblasts
(melanoma,
(, glioblastoma
g
).
)
Isolation of Human Bone Marrow-Derived
Endothelial Cells
Dispersion with
proteolytic enzymes
(e.g. Collagenase IV)
Primary Culture
>100mm3 sample
Magnetic
Field
Magnetic particles
CD31 positive cells (Culture
p(
d5)
coated with antib
tibody
against CD31
(Dynabeads)
Proteins identified from cell supernatant
Among about 150 different proteins genuinly secreted by bone-marrow-derived
endothelial cells we identified:
·
Cytokines
IL-6
IL-25
Growth factors:
CSF-1
Connective tissue growth
growth factor
factor
CXCL1
Placenta growth factor
CXCL-10
ECM-proteins:
Proepithelin
Biglycan
·
Proteases:
Fib
i
ronect n
ADAM 30
Laminins alpha-2, gamma-1
MMP-1
Multimerin-1
MMP-2
VE-cadherin
·
Protease inhibitors:
Vascular cell adhesion protein 1
MIF
PAI-1
TIMP-1
TIMP-2
CD31
11q13 (cyclinD1)
14q32 (IgH)
IgH
Bone marrow biopsy of MM with t(11;14)
Summary
· Our results show that complex protein mixtures
derived from human MM cells as well as BM stromal
ll
ce s can be
l
ana yzed by h
s
t
o gun
t
pro eomics
· Ii
In comparison to
l
norma
l
ce llls, a b
d
roa
i
var t
e y f
o
aberrantly expressed proteins was identified in MM
cells (including
(g 5 proteins
p
found exclusively in MM
cells)
Summary
· BM fibroblasts from MGUS and MM background display a
specifically altered secretion profile, and BM fibroblasts
from MGUS background already show tumor promoting
-
activities as indicated by the expression of IGF-II
·
We suggest that our approach will contribute to an
understanding of tumor progression in monoclonal
gammopathies by including the analysis of the
microenvironment.
ACKNOWLEDGMENTS
Jutta Ackermann
Medical University of Vienna
HK
Hannes K
f
au mann
Department of Medicine I
Verena Sagaster
Institute of Cancer Research
Sonja Holzer
Johannes Drach
Christopher Gerner
Heinz Ludwig (Wilhelminenspital)
Astrid Slany
Thomas Mohr
Roman Hajek (Brno)
Johannes Griess