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