Cancer Stem Cell SNPs Have Opposite Prognostic Outcome in Patients with Gastric Cancer Among Asian and Western Countries

Melissa J. LaBonte1, Takeru Wakatsuki1, Wu Zhang1, Dongyun Yang2, Mizutomo Azuma3, Armin Gerger5, Michael Stotz5, Yan Ning1, Nico Volz1, Sebastian Stintzing1, Joseph E Li1, Rita El-Khoueiry1, Peter M Wilson1, Wasaburo Koizumi3, Masahiko Watanabe4, Martin Maus1, Afsaneh Barzi1, Syma Iqbal1, Anthony El-Khoueiry1, and Heinz-Josef Lenz1,2

1. Department of Medical Oncology, 2. Department of Preventive Medicine; University of Southern California/Norris Comprehensive Cancer Center, Los Angeles, CA3. Department of Gastroenterology, 4 Department of Surgery; Kitasato University East Hospital

5. Division of Clinical Oncology, Medical University of Graz

369 patients with histopathologically-confirmed localized GC were enrolled (stage Ib-IV; TNM 6th), 169 patients from Japan, 137 patients from the USA, and 63 patients from Austria between 2002 and 2010. D2 lymphadenectomy based surgery were conducted in Japan and Austria, while D1 lymphadenectomy based surgery were conducted in USA. Adjuvant fluoropyrimidine based chemotherapy was conducted in ~60% patients. Genomic DNA was extracted from peripheral blood or formalin fixed paraffin embedded tumor tissue using the QIAmp kit. All samples were analyzed by means of PCR-based direct DNA- sequencing. Microdissection and gene expression analysis in the Japanese cohort were conducted in order to assess a correlation between genotypes and relative gene expression levels. The primary endpoint of this study was overall survival (OS). To evaluate prognostic value of these polymorphisms, endpoints were estimated using the Kaplan-Meier method and compared by log-rank test. The level of significance was set to p < 0.05.

The clinical significance of cancer stem cells (CSC) has been well established; however, their prognostic role remains controversial. CD44 is recognized as a CSC marker in gastric cancer (GC) (1) and, recently, the clinical impact of CD166 in GC was reported (2). Our group previously reported SNPs in CD44 rs187116 G>A and CD166 rs1157 G>A are associated with outcome in USA patients with adjuvant GC and colorectal cancer, respectively (3, 4); however these results have not been validated in an independent cohort to date. Since GC has regional differences in epidemiology and clinicopathology, we tested the hypothesis that ethnicity and regional differences in GC could influence the prognostic role of CD44 and CD166 in GC.

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Patients characteristics of 3 cohorts are shown in Table 1. In CD44 rs187116, AA showed a shorter OS than AG/GG (2.0 years vs. not reached, HR: 2.87 [95%CI: 1.61-5.13], p<0.001) in the Japanese cohort, while AA showed a longer OS than AG/GG (7.3 vs. 4.1 years, HR: 2.0 [95%CI: 0.90-4.55], p=0.079) in the US cohort (Figure 1). In CD166 rs1157, AA/AG showed a shorter OS than GG (3.9 years vs. not reached, HR: 1.81 [95%CI: 1.05-3.12], p=0.033) in the Japanese cohort, while AA showed a longer OS that AG/GG (not reached vs. 4.7 years, HR: 5.00 [95%CI: 0.70-35.95], p=0.073) in the USA cohort (Figure 2).

SNP profiles in CSC markers predicted opposite prognostic outcomes in patients with GC among Asian and Western countries. This is the first report suggesting that the prognostic role of CSC markers in GC may differ based on ethnic groups or etiology differences.

In CD44 rs187116, AA was associated with lower gene expression level than AG/GG (p=0.061), while no significant difference was shown between AA/AG and GG in CD166 (Figure 3).

References

Figure 1: CD44 rs187116 Figure 2: CD166 rs1157 Figure 3:Table 1: Patient characteristics Gene expression analysis of CD44 and CD166

AA vs. AG/GG; P=0.061

AA/AG vs. GG; P=0.494

1) Takaishi S, et al; Stem Cells. 27, 2009

2) Ishigami S, et al; J Surgical Oncol. 103, 2011

3) Winder T, et al; Int J Cancer. 129, 2011

4 ) Gerger A, et al; Clin Cancer Res. 17, 2011

Japanese Austrian USCTotal number (%) Total number (%) Total number (%)

169 63   137Ethnicity Ethnicity     Ethnicity

Japanese 169 100 Caucasian 63 100 Caucasian 63 46    Hispanic 45 32.8    others 29 21.2

Median Age, yrs Median Age, yrs     Median AgeAll 68 (31-88) All 66 (37-86) All 64 (26-85)

Gender Gender     GenderMale 109 64.5 Male 32 50.8 Male 83 60.6

Female 60 35.5 Female 31 49.2 Female 54 39.4Stage Stage     Stage

Ib 28 16.6 Ib 12 19.4 Ib 12 8.8II 53 31.4 II 25 40.3 II 36 26.3III 60 35.5 III 25 40.3 III 71 51.8IV 28 16.6 IV 0   IV 18 13.1

T category T category     T category1 12 7.1 1 1 1.6 1 4 2.92 66 39.1 2 37 58.7 2 44 32.13 88 52.1 3 24 38.1 3 79 57.74 3 1.8 4 1 1.6 4 10 7.3

N category N category     N category0 36 21.3 0 19 30.2 0 27 19.71 85 50.3 1 31 49.2 1 64 46.72 33 19.5 2 13 20.6 2 31 22.63 15 8.9 3 0   3 15 10.9

ECOG PS ECOG PS     ECOG PS

0 157 92.9 0 NA NA 0 62 45.3

1 12 7.1 1 NA NA 1 65 47.4

2 0 0 2 NA NA 2 10 7.3

Tumor Site     Tumor Site     Tumor Site    Stomach 166 98.2 Stomach 48 77.4 Stomach 88 69.8GEJ 3 1.8 GEJ 14 22.6 GEJ 38 30.2

Tumor differentiation Tumor differentiation Tumor differentiationwell/mode 68 40.2 moderate 7 11.1 well/mode 37 27

poor 101 59.8 poor 50 79.4 poor 97 70.8      Undifferentiated 6 9.5      Lauren class. Lauren class.     Lauren class.

Diffuse 101 59.8 Diffuse 27 60.0 Diffuse 40 29.2Intestinal 68 40.2 Intestinal 11 24.4 Intestinal 50 36.5

Mixed 0 0 Mixed 7 15.6 Mixed 21 15.3Type of chemo. Type of chemo.     Type of chemo.

S-1 based 87 51.5 5-FU or cape. based 9 14.3 5-FU/LV 70 51.1

UFT 16 9.5 Taxan based 2 3.2 5-FU/Oxali. 19 13.9others 6 3.6 others 3 4.8 5-FU, Cis, CPT-11 25 18.2none 60 35.5 none 49 77.8 none 23 16.8

Radiation Radiation     Radiationyes 0 0 yes 3 4.8 yes 88 64.2no 169 100 no 60 95.2 no 48 35

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B

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B

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B