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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
Metaplasia
(Adenoma)
MSI-H (0-6%)
E-cadherin mutation (41-45%)
p53 mutation (0-33%)
CD44Cyclin E overexpression (10 %)
CDC25B overexpression
N-cadherin overexpression (43%)Twist 1 overexpression (39%)
K-sam amplification (33%)c-met amplification (39%)
Reduced nm23 (˂52%)
MSI-H (13-20%)
Telomerase activation/TERT expression
p53 mutation (25-63%)K-ras mutation (10%)Reduced p27 expression
APC mutation (40-60%)Bcl-3 loss (43%)c-met amplification (19%)Cyclin E overexpression (14-20%)18q (DCC) loss (50%)ß-Catenin mutation (27%)
C-erbB2 amplification (20%)CD44E-cadherin reduction (60%)SIP1 overexpression (55%)
Reduced nm23 (52%)
Early cancer
Carcinoma
Metastasis
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 post-translational 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
Extracellular domain TM Cyto 3´UTR
s1 s2 s3 s5s4 s6 s8s7 s10s9
v1
5´ 3´1 2 43 5 16 17 18 19 20
6v2 v3 v4 v5 v6 v7 v8 v9 v10
87 109 11 12 13 14 15
CD44 Receptor
CD44 gene structure
Cell membrane
pY PI3-kinasepY
CD44 - pathways
Akt
PDK1
Gab-1
Hyaluronan,Osteopontin
Hsp90/cdc37 ErbB2
Grb2 Vav2
Ras
Raf-1
MEK
Erk Anti-apoptosis
CD44
PTEN
Gene transcriptionCell-cycle progression Invasion
Drug resistance
Cell survival
Proliferation
Feedback loop
CD44 and cellular adhesion
Cell motilityMigration
Ezrin
Radixin
Hyaluronan, Osteopontin
CD44
Moesin
Ank
yrin
Filamentous actinnetwork
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 2009:27:106-1020Al-Hajj M et al. Proc. Natl Acad. Sci. USA 2003;100:3983-3988
Location of gastric stem cells
Quante M et al. Nat Rev Gastroenterol Hepatol. 2009 Dec;6(12):724-37
Gastric stem cells has been localizedto the isthmus.
Migrate bidirectionally to differentiateinto gastric surface mucus cells thatcoat the
• Gastric pits• Gastric parietal and • Zymogenic cells
CD44 as a gastric cancer stem cell marker
Takaishi S et al. Stem Cells 2009:27:106-1020
CD44 positive gastric cancer cell line in the stomach and skin of SCID mice
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)
Relative risk (95% CI)
P value †
Age 0.42 <60 80 2.2 (1.7, 14.5+) 1 ≥60 57 3.7 (2.1, 12.3+) 0.81 (0.48, 1.36) Sex 0.85 Male 83 2.3 (1.8, 7.0) 1 Female 54 7.0 (1.5, 8.3+) 0.95 (0.56, 1.63) Race 0.085 White 63 1.7 (1.2, 4.4) 1 Black 1 0.5+ — Asian 28 7.0 (2.3, 14.5+) 0.45 (0.23, 0.91) Hispanic 45 3.7 (2.1, 10.7+) 0.63 (0.34, 1.17) T-category 0.013 T1a 4 T2a 44 8.3+ (2.9, 8.3+) 1 T3b 79 1.7 (1.4, 4.4) 2.04 (1.14, 3.67) T4b 10 N-category 0.004 Negative 27 7.0 (1.8, 10.7+) 1 N1 64 4.4 (2.2, 14.5+) 0.99 (0.47, 2.11) N2 31 1.3 (1.1, 2.3) 2.62 (1.15, 5.94) N3 15 1.6 (1.0, 3.8+) 1.96 (0.73, 5.32) Lauren 0.87 Diffuse 40 3.7 (1.8, 8.9+) 1 Intestinal 50 7.0 (2.1, 14.5+) 0.87 (0.45, 1.67) Mixed 21 12.3+ (1.7, 12.3+) 1.04 (0.45, 2.41) Type of chemotherapy 0.003 5-FU/LV 70 7.0 (2.8, 10.6+) 1 5-FU/LV/oxaliplatin 19 1.6 (1.1, 2.9) 2.66 (1.23, 5.76) 5-FU, Cisplatin, CPT-11 25 1.7 (1.2, 14.5+) 1.46 (0.71, 3.01) None 23 2.1 (0.8, 2.5) 2.80 (1.48, 5.27) Radiation 0.92 Yes 88 2.5 (1.8, 14.5+) 1 No 48 3.7 (1.7, 12.3+) 1.03 (0.60, 1.76) + Estimates were not reached. † Based on log-rank test. a,bGrouped together for the estimates of relative risk
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
s1 s2 s3 s5s4 s6 s8s7 s10s9
v1
5´ 3´UTR1 2 43 5 16 17 18 19 20
6
v2 v3 v4 v5 v6 v7 v8 v9 v10
87 109 11 12 13 14 15
CD44 rs187116
CD44 rs7116432
CD44 rs8193
CD44 rs4755392
Transcriptional regulation
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
0 2 4 6 8 10 12 14
CD44 rs187116 predicts tumor recurrence
TTR: 2.1 yrs TTR: 7.0 yrs
Years since Diagnosis of Resectable Gastric Cancer
Est
ima
ted
Rec
urre
nce
-Fre
e P
roba
bilit
y
CD44 A/A (n=30)
CD44 A/G or G/G (n=94)
Log-Rank P value = 0.022
CD44 rs187116 is associated with overall survival
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
0 2 4 6 8 10 12 14
Years since Diagnosis of Resectable Gastric Cancer
Est
ima
ted
Pro
babi
lity
of S
urv
ival
OS: 4.1 yrs OS: 7.0 yrs
CD44 A/G or G/G (n=94)
CD44 A/A (n=30)
Log-Rank P value = 0.079
CD44 rs7116432 predicts tumor recurrence
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
0 2 4 6 8 10 12 14
Years since Diagnosis of Resectable Gastric Cancer
Est
ima
ted
Rec
urre
nce
-Fre
e P
roba
bilit
y
TTR: 2.2 yrs TTR: 7.0 yrs
CD44 G/G (n=36)
CD44 A/G or A/A (n=91)
Log-Rank P value = 0.045
CD44 rs7116432 predicts overall survival
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
0 2 4 6 8 10 12 14
Est
ima
ted
Pro
babi
lity
of S
urv
ival
Years since Diagnosis of Resectable Gastric Cancer
OS: 3.8 yrs OS: 7.3 yrs
CD44 G/G (n=36)
CD44 A/G or A/A (n=91)
Log-Rank P value = 0.018
Combined analysis of risk alleles for time to recurrence
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
0 2 4 6 8 10 12 14
Years since Diagnosis of Resectable Gastric Cancer
Est
ima
ted
Rec
urre
nce
-Fre
e P
roba
bilit
y
TTR: 1.7 yrs TTR: 7.0 yrs
CD44 1-2 Favorable alleles (n=55)
CD44 0 Favorable alleles (n=67)
Adjusted P value = 0.016
Combined analysis of risk alleles for overall survival
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
0 2 4 6 8 10 12 14
Est
ima
ted
Pro
babi
lity
of S
urv
ival
Years since Diagnosis of Resectable Gastric Cancer
OS: 3.6 yrs OS: 7.3 yrs
CD44 1–2 Favorable alleles (n=55)
CD44 0 Favorable alleles (n=67)
Adjusted P value = 0.019
Multivariate Analysis
0.122.31 (0.81, 6.60)0.341.49 (0.66, 3.36)88A/G,A/A
1 (Reference)1 (Reference)34G/G
CD44rs7116432
0.0263.52 (1.16, 10.65)0.0073.81 (1.45, 9.98)92A/G,G/G
1 (Reference)1 (Reference)30A/A
CD44rs187116
P value †Relative risk (95% CI)P value †Relative risk (95% CI)N*
Overall survivalTime to recurrence
0.0192.74 (1.18, 6.38)0.0162.41 (1.18, 4.92)670 Favorable
1 (Reference)1 (Reference)551-2 Favorable
Combined
* Patients with incomplese genotyping were excluded in the multivatiate analysis† adjusted for T category, N category, race and type of therapy
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