Volume 141, Issue 2, Pages 476-485.e11 (August 2011) Intrinsic Subtypes of Gastric Cancer, Based on Gene Expression Pattern, Predict Survival and Respond Differently to Chemotherapy  Iain Beehuat Tan, Tatiana Ivanova, Kiat Hon Lim, Chee Wee Ong, Niantao Deng, Julian Lee, Sze Huey Tan, Jeanie Wu, Ming Hui Lee, Chia Huey Ooi, Sun Young Rha, Wai Keong Wong, Alex Boussioutas, Khay Guan Yeoh, Jimmy So, Wei Peng Yong, Akira Tsuburaya, Heike Grabsch, Han Chong Toh, Steven Rozen, Jae Ho Cheong, Sung Hoon Noh, Wei Kiat Wan, Jaffer A. Ajani, Ju–Seog Lee, Manuel Salto–Tellez, Patrick Tan  Gastroenterology  Volume 141, Issue 2, Pages 476-485.e11 (August 2011) DOI: 10.1053/j.gastro.2011.04.042 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Unsupervised clustering of GC cell lines reveals 2 major intrinsic subtypes. (A) Hierarchical dendrogram depicting clustering of 37 GC cell lines into G-INT (blue) and G-DIF (red); height: squared Euclidean distances between cluster means. (B) Silhouette widths of individual cell lines when classified in 2 clusters. Silhouette width: a measure for each sample of membership within its own class against that of another class. (C) Heat map of expression of 171 genes arranged by hierarchal clustering of cell lines (columns) and expression difference for each gene between G-INT and G-DIF as measured by the t test statistic (rows). Gastroenterology 2011 141, 476-485.e11DOI: (10.1053/j.gastro.2011.04.042) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Associations of intrinsic subtypes with Lauren's histopathologic classification in primary GCs. Heat map of gene expression in (A) SG and (B) AU cohorts arranged by strength of association (columns) and expression difference for each gene between G-INT and G-DIF as measured by the t test statistic (rows). First row label (Lauren's class): light blue, intestinal; brown, diffuse; white, mixed. Second row label (intrinsic classes): blue, G-INT; red, G-DIF. (C and D) Representative H&E stain of (C) G-INT/diffuse cancer and (D) G-DIF/intestinal cancer. Gastroenterology 2011 141, 476-485.e11DOI: (10.1053/j.gastro.2011.04.042) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Intrinsic genomic subclasses are prognostic. Kaplan–Meier plots of survival in (A) all patients (HR, 1.79; 95% CI, 1.28–2.51; P = .001) and (B) when the intrinsic classification and Lauren's classes are discordant (HR, 1.83; 95% CI, 1.02–3.30; P = .04). Gastroenterology 2011 141, 476-485.e11DOI: (10.1053/j.gastro.2011.04.042) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 In vitro chemosensitivity of G-INT and G-DIF cell lines. GI-50 values of 11 G-INT and 17 G-DIF cell lines upon treatment with 5-FU, oxaliplatin, and cisplatin. GI-50 refers to the drug concentration at which 50% growth inhibition is achieved (y-axis: GI-50 enumerated in negative log10). The horizontal gray lines represent the therapeutic concentration patients are exposed to based on pharmacokinetic data.25–27 Mean GI-50 concentrations for G-INT and G-DIF cell lines were as follows, respectively: 5-FU, 5.20 μmol/L and 23.22 μmol/L; cisplatin, 38.61 μmol/L and 13.35 μmol/L; oxaliplatin, 1.33 μmol/L and 5.49 μmol/L. Gastroenterology 2011 141, 476-485.e11DOI: (10.1053/j.gastro.2011.04.042) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 PCA and NMF plots of 37 GC cell lines. (A) Principal components analysis of 37 GC cell lines. G-INT and G-DIF cell lines are distinguished by the first principal component. (B) Reordered consensus matrices. An average of 1000 connectivity matrices were computed at k = 2–5 for the 37 GC cell lines using the selected genes. Samples were hierarchically clustered using the consensus clustering matrix, colored from 0 (blue, samples are never in the same cluster) to 1 (red, samples are always in the same cluster). The y-axis lists the cell line names. (C) Cophenetic correlation coefficient plot corresponding to k = 2–7. A 2-class decomposition is suggested. Gastroenterology 2011 141, 476-485.e11DOI: (10.1053/j.gastro.2011.04.042) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 2 G-INT/G-DIF is prognostic in the SG cohort and AU cohorts. Kaplan–Meier plots of survival in the (A) SG cohort (HR, 1.78; 95% CI, 1.19–2.64; P = .004) and (B) AU cohort (HR, 1.73; 95% CI, 0.92–3.26; P = .09). G-INT and G-DIF are prognostic. Gastroenterology 2011 141, 476-485.e11DOI: (10.1053/j.gastro.2011.04.042) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 3 G-INT/G-DIF is prognostic in a third independent cohort. (A) Heat map of gene expression arranged by strength of association (columns) and expression difference for each gene between G-INT and G-DIF as measured by the t test statistic (rows). Row label: G-INT, blue; G-DIF, red. (B) Kaplan–Meier plots of survival (HR, 3.3; 95% CI, 1.03–10.53; P = .04). Gastroenterology 2011 141, 476-485.e11DOI: (10.1053/j.gastro.2011.04.042) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 4 TMA data set. (A) Representative immunostaining expression of CDH17 and LGALS4 in GC. (1 and 4) Positive membranous CDH17 expression. (2 and 5) Negative CDH17 expression. (3 and 6) Positive cytoplasmic LGALS4 expression. (B) Kaplan–Meier plots of survival of tumors positive for both LGALS4 and CDH17 (2-marker positive) compared with tumors negative for both markers (2-marker negative) (HR, 1.95; 95% CI, 1.13–3.38; P = .02, adjusted for stage). In this study, we specifically compared the 2-marker–positive group with the 2-marker–negative group (see main text). We also noted that compared with the 2-marker–negative group, the one-third of patients who are 1-marker positive also exhibit a trend for improved survival (CDH17, P = .08 adjusted for stage; LGALS4, P = .07 adjusted for stage). This may suggest that some 1-marker–positive cancers may also be G-INT cancers. Gastroenterology 2011 141, 476-485.e11DOI: (10.1053/j.gastro.2011.04.042) Copyright © 2011 AGA Institute Terms and Conditions