Intrinsic subtypes of gastric cancer, based on gene expression pattern, predict survival and respond differently to chemotherapy

Abstract

Background & aims: Gastric cancer (GC) is a heterogeneous disease comprising multiple subtypes that have distinct biological properties and effects in patients. We sought to identify new, intrinsic subtypes of GC by gene expression analysis of a large panel of GC cell lines. We tested if these subtypes might be associated with differences in patient survival times and responses to various standard-of-care cytotoxic drugs.

Methods: We analyzed gene expression profiles for 37 GC cell lines to identify intrinsic GC subtypes. These subtypes were validated in primary tumors from 521 patients in 4 independent cohorts, where the subtypes were determined by either expression profiling or subtype-specific immunohistochemical markers (LGALS4, CDH17). In vitro sensitivity to 3 chemotherapy drugs (5-fluorouracil, cisplatin, oxaliplatin) was also assessed.

Results: Unsupervised cell line analysis identified 2 major intrinsic genomic subtypes (G-INT and G-DIF) that had distinct patterns of gene expression. The intrinsic subtypes, but not subtypes based on Lauren's histopathologic classification, were prognostic of survival, based on univariate and multivariate analysis in multiple patient cohorts. The G-INT cell lines were significantly more sensitive to 5-fluorouracil and oxaliplatin, but more resistant to cisplatin, than the G-DIF cell lines. In patients, intrinsic subtypes were associated with survival time following adjuvant, 5-fluorouracil-based therapy.

Conclusions: Intrinsic subtypes of GC, based on distinct patterns of expression, are associated with patient survival and response to chemotherapy. Classification of GC based on intrinsic subtypes might be used to determine prognosis and customize therapy.

Figure 1. Unsupervised clustering of GCCLs reveals 2 major intrinsic subtypes

(A) Hierarchical dendrogram depicting clustering of 37 GCCLs 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 of within it s 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).

Figure 1. Unsupervised clustering of GCCLs reveals 2 major intrinsic subtypes

(A) Hierarchical dendrogram depicting clustering of 37 GCCLs 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 of within it s 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).

Figure 1. Unsupervised clustering of GCCLs reveals 2 major intrinsic subtypes

(A) Hierarchical dendrogram depicting clustering of 37 GCCLs 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 of within it s 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).

Figure 2. Associations of intrinsic subtypes with Lauren’s 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). 1st row label: Laurens class; light blue: intestinal, brown: diffuse, white: mixed. 2nd row label: Intrinsic classes: blue: G-INT, red: G-DIF. C. Representative H&E of (C) G-DIF/intestinal cancer and (D) G-INT/Diffuse cancer.

Figure 2. Associations of intrinsic subtypes with Lauren’s 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). 1st row label: Laurens class; light blue: intestinal, brown: diffuse, white: mixed. 2nd row label: Intrinsic classes: blue: G-INT, red: G-DIF. C. Representative H&E of (C) G-DIF/intestinal cancer and (D) G-INT/Diffuse cancer.

Figure 2. Associations of intrinsic subtypes with Lauren’s 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). 1st row label: Laurens class; light blue: intestinal, brown: diffuse, white: mixed. 2nd row label: Intrinsic classes: blue: G-INT, red: G-DIF. C. Representative H&E of (C) G-DIF/intestinal cancer and (D) G-INT/Diffuse cancer.

Figure 2. Associations of intrinsic subtypes with Lauren’s 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). 1st row label: Laurens class; light blue: intestinal, brown: diffuse, white: mixed. 2nd row label: Intrinsic classes: blue: G-INT, red: G-DIF. C. Representative H&E of (C) G-DIF/intestinal cancer and (D) G-INT/Diffuse cancer.

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=0.001) and (B) when the intrinsic classification and Lauren’s classes are discordant (HR 1.83, 95%CI: 1.02–3.30, p=0.04).

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=0.001) and (B) when the intrinsic classification and Lauren’s classes are discordant (HR 1.83, 95%CI: 1.02–3.30, p=0.04).

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-50s refer to the drug concentration at which 50% growth inhibition is achieved. (y-axis: GI-50 enumerated in negative log₁₀). The horizontal grey lines represent the therapeutic concentration patients are exposed to based on pharmacokinetic data^–. Mean GI-50 concentrations for G-INT and G-DIF cell lines respectively: 5FU: 5.20 μM, 23.22 μM; Cisplatin: 38.61 μM, 13.35 μM; Oxaliplatin: 1.33 μM, 5.49 μM.