Download CD44

Germline polymorphisms in the
CD44 gene are associated with
clinical outcome in localized gastric
adenocarcinoma.
Thomas Winder, M.D.
University of Southern California
Norris Comprehensive Cancer Center
USC Keck School of Medicine
Sharon A. Carpenter Laboratory
Los Angeles, CA
Background
• Gastric Cancer is the 4th most common
cancer type worldwide
– 21.130 newly diagnosed patients in the US in 2009
• 2nd cause of cancer death worldwide
– 10.620 deaths in the US in 2009
• Prognosis depends on:
– Stage
– Pathological differentiation
National Cancer institute http://www.cancer.gov/cancertopics/types/stomach
Genetic alterations in gastric
cancer
Diffuse type
Intestinal type
Normal gastric mucosa
MSI-H (0-6%)
E-cadherin mutation (41-45%)
MSI-H (13-20%)
Metaplasia
Telomerase activation/
TERT expression
p53 mutation (25-63%)
p53 mutation (0-33%)
(Adenoma)
APC mutation (40-60%)
Bcl-3 loss (43%)
CD44
Cyclin E overexpression (10 %)
CDC25B overexpression
N-cadherin overexpression (43%)
Twist 1 overexpression (39%)
c-met amplification (19%)
Early cancer
Carcinoma
K-sam amplification (33%)
Cyclin E overexpression (14-20%)
18q (DCC) loss (50%)
ß-Catenin mutation (27%)
C-erbB2 amplification (20%)
CD44
E-cadherin reduction (60%)
SIP1 overexpression (55%)
c-met amplification (39%)
Reduced nm23 (˂52%)
K-ras mutation (10%)
Reduced p27 expression
Metastasis
Reduced nm23 (52%)
Adapted from Keller et al. 2005 Expert Rev in Mol Medicine 7;17
CD44 - Background
• CD44 is a glycoprotein encoded on the short
arm of chromosome 11.
• CD44 was first isolated in haemopoietic cells
and has since been found on a wide range of
tissues (e.g. gastric, lung, liver, pancreas)
• The main ligands of CD44 are hyaluronan and
osteopontin.
• The protein isoforms are encoded by a single
gene by alternative splicing and posttranslational modification.
CD44 and its function
• Cellular adhesion (transmembrane link between
extracellular matrix and cytoskeleton)
• CD44 positive cells are tumor initiating cells in
gastric cancer
• Immune System (e.g. lymphocyte homeing, T
cell activation)
• High CD44 protein expression has been
associated with poor prognosis in gastric
adenocarcinoma*
*Ghaffarzadehgan K et al. World J Gastroenterol 2008;14(41):6376-6381
CD44 gene structure
Cell membrane
CD44 Receptor
TM Cyto 3´UTR
Extracellular domain
1 2 3 4 5
5´
s1
s2
s3
s4
s5
16 17 18 19 20
s6
s7
s8
CD44 gene structure
6
7 8 9 10 11 12 13 14 15
v1
v2 v3 v4 v5 v6 v7 v8 v9 v10
s9 s10
3´
CD44 - pathways
Hyaluronan,
Osteopontin
Feedback loop
CD44
Hsp90/
pY
cdc37 ErbB2
Grb2 Vav2
pY PI3-kinase
Gab-1
PTEN
Ras
PDK1
Raf-1
Akt
MEK
Erk
Cell survival
Anti-apoptosis
Gene transcription
Cell-cycle progression
Proliferation
Invasion
Drug resistance
CD44 and cellular adhesion
Hyaluronan,
Osteopontin
CD44
Ezrin
Radixin
Moesin
Filamentous actin
network
Migration
Cell motility
CD44 gastric stem cell marker
• Property of self-renewal, longevity and
multipotency.
• High CD44 protein-expression correlates
with the presence of dysplasia in murine
and human gastric cancer.
• CD44 overexpression is associated with
chemo- and radio-resistance
Takaishi S et al. Stem Cells 2009:27:106-1020
Al-Hajj M et al. Proc. Natl Acad. Sci. USA 2003;100:3983-3988
Location of gastric stem cells
Gastric stem cells has been localized
to the isthmus.
Migrate bidirectionally to differentiate
into gastric surface mucus cells that
coat the
• Gastric pits
• Gastric parietal and
• Zymogenic cells
Quante M et al. Nat Rev Gastroenterol Hepatol. 2009 Dec;6(12):724-37
CD44 as a gastric cancer stem
cell marker
CD44 positive gastric cancer cell line in the stomach and skin of SCID mice
Takaishi S et al. Stem Cells 2009:27:106-1020
Objectives
• Identifying germline polymorphisms
within the CD44 gene for clinical
outcome in patients with localized
gastric adenocarcinoma.
Patient Characteristics
N=137
Median time to recurrence (TTR)
yrs (95% CI)
Age
<60
80
2.2 (1.7, 14.5+)
≥60
57
3.7 (2.1, 12.3+)
Sex
Male
83
2.3 (1.8, 7.0)
Female
54
7.0 (1.5, 8.3+)
Race
White
63
1.7 (1.2, 4.4)
Black
1
0.5+
Asian
28
7.0 (2.3, 14.5+)
Hispanic
45
3.7 (2.1, 10.7+)
T-category
T1a
4
T2a
44
8.3+ (2.9, 8.3+)
T3b
79
1.7 (1.4, 4.4)
T4b
10
N-category
Negative
27
7.0 (1.8, 10.7+)
N1
64
4.4 (2.2, 14.5+)
N2
31
1.3 (1.1, 2.3)
N3
15
1.6 (1.0, 3.8+)
Lauren
Diffuse
40
3.7 (1.8, 8.9+)
Intestinal
50
7.0 (2.1, 14.5+)
Mixed
21
12.3+ (1.7, 12.3+)
Type of chemotherapy
5-FU/LV
70
7.0 (2.8, 10.6+)
5-FU/LV/oxaliplatin
19
1.6 (1.1, 2.9)
5-FU, Cisplatin, CPT-11
25
1.7 (1.2, 14.5+)
None
23
2.1 (0.8, 2.5)
Radiation
Yes
88
2.5 (1.8, 14.5+)
No
48
3.7 (1.7, 12.3+)
+ Estimates were not reached.
† Based on log-rank test.
a,b
Grouped together for the estimates of relative risk
Relative risk
(95% CI)
P value
†
0.42
1
0.81 (0.48, 1.36)
0.85
1
0.95 (0.56, 1.63)
0.085
1
—
0.45 (0.23, 0.91)
0.63 (0.34, 1.17)
0.013
1
2.04 (1.14, 3.67)
0.004
1
0.99 (0.47, 2.11)
2.62 (1.15, 5.94)
1.96 (0.73, 5.32)
0.87
1
0.87 (0.45, 1.67)
1.04 (0.45, 2.41)
0.003
1
2.66 (1.23, 5.76)
1.46 (0.71, 3.01)
2.80 (1.48, 5.27)
0.92
1
1.03 (0.60, 1.76)
Methods
• gDNA was isolated either from blood or
from formalin-fixed paraffin-embedded
tissue samples.
• PCR-RFLP was used to determine the
polymorphisms
Selected germline
polymorphisms
CD44 rs4755392
CD44 rs187116
5´
1
2
3
4
5
s1
s2
s3
s4
s5
8
16 17 18 19
s6
s7
s8
6
7
9 10 11 12 13 14 15
v1
v2 v3 v4 v5 v6 v7 v8 v9 v10
s9
20
s10
3´UTR
CD44 rs8193
CD44 rs7116432
Transcriptional
regulation
CD44 rs187116 predicts tumor
recurrence
1,0
Log-Rank P value = 0.022
Estimated Recurrence-Free Probability
0,9
0,8
0,7
0,6
0,5
0,4
CD44 A/A (n=30)
0,3
CD44 A/G or G/G (n=94)
0,2
0,1
0,0
TTR: 2.1 yrs
0
2
TTR: 7.0 yrs
4
6
8
10
Years since Diagnosis of Resectable Gastric Cancer
12
14
CD44 rs187116 is associated
with overall survival
1,0
Log-Rank P value = 0.079
Estimated Probability of Survival
0,9
0,8
0,7
0,6
0,5
0,4
CD44 A/A (n=30)
0,3
0,2
CD44 A/G or G/G (n=94)
0,1
OS: 4.1 yrs
0,0
0
2
OS: 7.0 yrs
4
6
8
10
12
Years since Diagnosis of Resectable Gastric Cancer
14
CD44 rs7116432 predicts tumor
recurrence
1,0
Estimated Recurrence-Free Probability
Log-Rank P value = 0.045
0,9
0,8
0,7
0,6
0,5
CD44 G/G (n=36)
0,4
0,3
CD44 A/G or A/A (n=91)
0,2
0,1
0,0
TTR: 2.2 yrs
0
2
TTR: 7.0 yrs
4
6
8
10
Years since Diagnosis of Resectable Gastric Cancer
12
14
CD44 rs7116432 predicts
overall survival
1,0
Log-Rank P value = 0.018
Estimated Probability of Survival
0,9
0,8
0,7
0,6
0,5
CD44 G/G (n=36)
0,4
0,3
0,2
CD44 A/G or A/A (n=91)
0,1
OS: 3.8 yrs
0,0
0
2
OS: 7.3 yrs
4
6
8
10
12
Years since Diagnosis of Resectable Gastric Cancer
14
Combined analysis of risk
alleles for time to recurrence
1,0
Estimated Recurrence-Free Probability
Adjusted P value = 0.016
0,9
0,8
0,7
0,6
0,5
CD44 1-2 Favorable alleles (n=55)
0,4
0,3
CD44 0 Favorable alleles (n=67)
0,2
0,1
0,0
TTR: 7.0 yrs
TTR: 1.7 yrs
0
2
4
6
8
10
12
Years since Diagnosis of Resectable Gastric Cancer
14
Combined analysis of risk
alleles for overall survival
1,0
Adjusted P value = 0.019
Estimated Probability of Survival
0,9
0,8
0,7
0,6
0,5
CD44 1–2 Favorable alleles (n=55)
0,4
0,3
CD44 0 Favorable alleles (n=67)
0,2
0,1
OS: 3.6 yrs
0,0
0
2
OS: 7.3 yrs
4
6
8
10
Years since Diagnosis of Resectable Gastric Cancer
12
14
Multivariate Analysis
Time to recurrence
N*
Relative risk (95% CI)
A/A
30
1 (Reference)
A/G,G/G
92
3.81 (1.45, 9.98)
G/G
34
1 (Reference)
A/G,A/A
88
1.49 (0.66, 3.36)
1-2 Favorable
55
1 (Reference)
0 Favorable
67
2.41 (1.18, 4.92)
P value †
Overall survival
Relative risk (95% CI)
P value †
CD44rs187116
1 (Reference)
0.007
3.52 (1.16, 10.65)
0.026
CD44rs7116432
1 (Reference)
0.34
2.31 (0.81, 6.60)
0.12
Combined
1 (Reference)
0.016
2.74 (1.18, 6.38)
* Patients with incomplese genotyping were excluded in the multivatiate analysis
† adjusted for T category, N category, race and type of therapy
0.019
Conclusions
• CD44 gene polymorphisms are
associated with TTR and OS in the
multivariate analysis
• CD44 polymorphisms may identify
patients at high risk for tumor recurrence
• CD44 might be a promising target for
drug development
Future directions
• These results need to be validated in
large, prospective biomarker embedded
clinical trials.
• Mechanistic studies need to explore the
function of these polymorphisms.
• The pharmacogenetic analysis should be
expanded to the CD44 pathway.
Acknowledgements
Medical Oncology:
Heinz-Josef Lenz, Syma Iqbal
Anthony El-Khoueiry,
Statistics:
Dongyun Yang, Susan Groshen
Dr. Lenz´ Lab:
Georg Lurje, Wu Zhang, Yan Ning,
Pierre Bohanes, Siwen Hu, Rita El-Khoueiry
Memorial Sloan-Kettering Cancer Center:
Derek G. Power, Laura H. Tang, Manish Shah
Grants:
Dhont Foundation
Austrian Society of Hematology and Oncology