A gene expression signature of acquired chemoresistance to cisplatin and fluorouracil combination chemotherapy in gastric cancer patients

Abstract

Background: We initiated a prospective trial to identify transcriptional alterations associated with acquired chemotherapy resistance from pre- and post-biopsy samples from the same patient and uncover potential molecular pathways involved in treatment failure to help guide therapeutic alternatives.

Methodology/principal findings: A prospective, high-throughput transcriptional profiling study was performed using endoscopic biopsy samples from 123 metastatic gastric cancer patients prior to cisplatin and fluorouracil (CF) combination chemotherapy. 22 patients who initially responded to CF were re-biopsied after they developed resistance to CF. An acquired chemotherapy resistance signature was identified by analyzing the gene expression profiles from the matched pre- and post-CF treated samples. The acquired resistance signature was able to segregate a separate cohort of 101 newly-diagnosed gastric cancer patients according to the time to progression after CF. Hierarchical clustering using a 633-gene acquired resistance signature (feature selection at P<0.01) separated the 101 pretreatment patient samples into two groups with significantly different times to progression (2.5 vs. 4.7 months). This 633-gene signature included the upregulation of AKT1, EIF4B, and RPS6 (mTOR pathway), DNA repair and drug metabolism genes, and was enriched for genes overexpressed in embryonic stem cell signatures. A 72-gene acquired resistance signature (a subset of the 633 gene signature also identified in ES cell-related gene sets) was an independent predictor for time to progression (adjusted P = 0.011) and survival (adjusted P = 0.034) of these 101 patients.

Conclusion/significance: This signature may offer new insights into identifying new targets and therapies required to overcome the acquired resistance of gastric cancer to CF.

Figure 1. Hieraching clustering analyses of pretreatment samples using acquired resistance signatures.

Hierarchical clustering dendrograms of pretreatment samples from a separate set of 101 gastric cancer patients, using genes differentially expressed between the pretreatment- and chemoresistant-states of 22 rebiopsied responders at various P cutoffs for feature selection. Kaplan-Meier plots for the time to progression (TTP) calculated for each of the two major clusters generated by each dendrogram are shown below. (A) Hierarchical clustering of 101 pretreatment samples using the 2,446–gene acquired resistance signature (P for feature selection<0.05). Heatmap generated using a log₂-pseudocolor image with gene centering. Kaplan-Meier plots for TTP calculated for each of the two major clusters generated are shown below. Patients in high risk cluster (n = 60, high expression of the genes upregulated at chemoresistant state (upper) had a significantly shorter TTP than patients in low risk cluster (n = 41, low expression) (3.0 vs. 5.0 months; P = 0.033). (B) Hierarchical clustering of the same 101 gastric cancer samples using the 633–gene acquired resistance signature (P for feature selection<0.01). Patients in high risk cluster (n = 38, high expression of genes upregulated at chemoresistant state (upper) had a significantly shorter TTP than patients in low risk cluster (n = 63, low expression) (2.5 vs. 4.7 months; P = 0.012).

Figure 2. Acquired resistance signature and stem cell signature.

(A) Hierarchical clustering of 101 pretreatment patient samples using the “ES set without proliferation genes signature”. Kaplan-Meier plots for time to progression (TTP) of patients in each cluster generated are shown on the right side. Patients in high risk cluster (I) (n = 44, high expression of “ES set without proliferation genes”) had a significantly shorter TTP than patients in the low risk cluster (II) (n = 57, low expression) (2.7 vs. 4.7 months; Log-rank P value = 0.014). (B) Principal component analysis plot using a published U133A microarray meta-analysis dataset containing 24 human ES cell samples (shown in red) and 193 various fetal and adult differentiated tissue samples (shown in green) using the 633-gene acquired resistance signature (feature selection P<0.01). Each sphere represents a single sample. Samples whose expression profiles of 633 genes are similar are shown close together. (C) a. Expression of the 633-gene acquired resistance signature using the same published meta-analysis microarray data¹⁴ as in (B). Heatmap generated using a log₂-pseudocolor image with gene centering. Red and green colors represent high and low gene expression levels, respectively. Genes upregulated at the chemoresistant state of our study patients (post/pre>1, I) show coordinated overexpression in ES cells (left), while genes downregulated at the chemoresistant state (post/pre<1, II) show coordinated overexpression in differentiated tissue samples (right). b. Expression of the same 633-gene acquired resistance signature in 101 pretreatment samples collected from a separate set of gastric cancer patients. Each row represents each patient, sorted according to the increasing order of TTP from left to right, as matched with Kaplan-Meier curves for TTP of 101 patients (top right). Genes upregulated at the chemoresistant state of our study patients (I) show the concordant overexpression in patients with shorter TTP (left), while genes downregulated at the chemoresistant state (II) show the concordant overexpression in patients with longer TTP (right).

Figure 3. Hierarchical clustering analyses of pretreatment samples using the 72 genes.

(A) Hierarchical clustering of the 101 gastric cancer samples using the 72 genes that are upregulated at chemoresistant state (P<0.01) and belong to “ES cell-related gene sets”. (B) Patients in high risk cluster according to (A) (n = 51, high expression of 72 genes) had a significantly shorter time to progression (TTP) than patients in low risk cluster (n = 50, low expression) (2.7 vs. 4.0 months; P = 0.025). (C) Patients in high risk cluster according to (A) (n = 51, high expression of 72 genes) had a significantly shorter survival than patients in low risk cluster (n = 50, low expression) (6.8 vs. 9.2 months; P = 0.028).