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 4 th most common cancer type worldwide – newly diagnosed patients in the US in nd cause of cancer death worldwide – deaths in the US in 2009 Prognosis depends on: –Stage –Pathological differentiation National Cancer institute

Genetic alterations in gastric cancer Diffuse typeIntestinal type Normal gastric mucosa Metaplasia (Adenoma) MSI-H (0-6%) E-cadherin mutation (41-45%) p53 mutation (0-33%) CD44 Cyclin 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%) CD44 E-cadherin reduction (60%) SIP1 overexpression (55%) Reduced nm23 (52%) Early cancer Carcinoma Metastasis Adapted from Keller et al 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):

CD44 gene structure Extracellular domain TMCyto3´UTR s1s2s3s5s4s6s8s7s10s9 v1 5´3´ v2v3v4v5v6v7v8v9v CD44 Receptor CD44 gene structure Cell membrane

pY PI3-kinase pY CD44 - pathways Akt PDK1 Gab-1 Hyaluronan, Osteopontin Hsp90/ cdc37 ErbB2 Grb2Vav2 Ras Raf-1 MEK ErkAnti-apoptosis CD44 PTEN Gene transcription Cell-cycle progression Invasion Drug resistance Cell survival Proliferation Feedback loop

CD44 and cellular adhesion Cell motility Migration Ezrin Radixin Hyaluronan, Osteopontin CD44 Moesin Ankyrin Filamentous actin network

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: Al-Hajj M et al. Proc. Natl Acad. Sci. USA 2003;100:

Location of gastric stem cells Quante M et al. Nat Rev Gastroenterol Hepatol Dec;6(12): 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

CD44 as a gastric cancer stem cell marker Takaishi S et al. Stem Cells 2009:27: 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

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 s1s2s3s5s4 s6s8s7s10s9 v1 5´ 3´UTR v2v3v4v5v6v7v8v9v CD44 rs CD44 rs CD44 rs8193 CD44 rs Transcriptional regulation

CD44 rs predicts tumor recurrence TTR: 2.1 yrsTTR: 7.0 yrs Years since Diagnosis of Resectable Gastric Cancer Estimated Recurrence-Free Probability CD44 A/A (n=30) CD44 A/G or G/G (n=94) Log-Rank P value = 0.022

CD44 rs is associated with overall survival Years since Diagnosis of Resectable Gastric Cancer Estimated Probability of Survival OS: 4.1 yrsOS: 7.0 yrs CD44 A/G or G/G (n=94) CD44 A/A (n=30) Log-Rank P value = 0.079

CD44 rs predicts tumor recurrence Years since Diagnosis of Resectable Gastric Cancer Estimated Recurrence-Free Probability TTR: 2.2 yrsTTR: 7.0 yrs CD44 G/G (n=36) CD44 A/G or A/A (n=91) Log-Rank P value = 0.045

CD44 rs predicts overall survival Estimated Probability of Survival Years since Diagnosis of Resectable Gastric Cancer OS: 3.8 yrsOS: 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 Years since Diagnosis of Resectable Gastric Cancer Estimated Recurrence-Free Probability TTR: 1.7 yrs TTR: 7.0 yrs CD Favorable alleles (n=55) CD44 0 Favorable alleles (n=67) Adjusted P value = 0.016

Combined analysis of risk alleles for overall survival Estimated Probability of Survival Years since Diagnosis of Resectable Gastric Cancer OS: 3.6 yrsOS: 7.3 yrs CD44 1–2 Favorable alleles (n=55) CD44 0 Favorable alleles (n=67) Adjusted P value = 0.019

Multivariate Analysis (0.81, 6.60) (0.66, 3.36)88A/G,A/A 1 (Reference) 34G/G CD44rs (1.16, 10.65) (1.45, 9.98)92A/G,G/G 1 (Reference) 30A/A CD44rs P value †Relative risk (95% CI)P value †Relative risk (95% CI)N* Overall survivalTime to recurrence (1.18, 6.38) (1.18, 4.92)670 Favorable 1 (Reference) 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