Cell Senescence-Related Pathways Are Enriched in Breast Cancer Patients With Late Toxicity After Radiotherapy and Low Radiation-Induced Lymphocyte Apoptosis

Abstract

Background: Radiation-induced late effects are a common cause of morbidity among cancer survivors. The biomarker with the best evidence as a predictive test of late reactions is the radiation-induced lymphocyte apoptosis (RILA) assay. We aimed to investigate the molecular basis underlying the distinctive RILA levels by using gene expression analysis in patients with and without late effects and in whom we had also first identified differences in RILA levels.

Patients and methods: Peripheral blood mononuclear cells of 10 patients with late severe skin complications and 10 patients without symptoms, selected from those receiving radiotherapy from 1993 to 2007, were mock-irradiated or irradiated with 8 Gy. The 48-h response was analyzed in parallel by RILA assay and gene expression profiling with Affymetrix microarrays. Irradiated and non-irradiated gene expression profiles were compared between both groups. Gene set enrichment analysis was performed to identify differentially expressed biological processes.

Results: Although differentially expressed mRNAs did not reach a significant adjusted p-value between patients suffering and not suffering clinical toxicity, the enriched pathways indicated significant differences between the two groups, either in irradiated or non-irradiated cells. In basal conditions, the main differentially expressed pathways between the toxicity and non-toxicity groups were the transport of small molecules, interferon signaling, and transcription. After 8 Gy, the differences lay in pathways highly related to cell senescence like cell cycle/NF-κB, G-protein-coupled receptors, and interferon signaling.

Conclusion: Patients at risk of developing late toxicity have a distinctive pathway signature driven by deregulation of immune and cell cycle pathways related to senescence, which in turn may underlie their low RILA phenotype.

Keywords: breast cancer; gene set enrichment analysis (GSEA); late skin toxicity; radiation-induced lymphocyte apoptosis (RILA); radiotherapy—adverse effects.

Figure 1

Heatmap and GSEA of basal non-irradiated cells comparing patients with late skin toxicity to normal responders. (A) A different expression pattern between patients with toxicity (dark blue) and without toxicity (green) of baseline expressed genes with non-adjusted p-value < 0.05 is suggested by hierarchical clustering classification of patients (columns). (B) The enrichment map allows the visualization of the general expression landscape of the toxicity group compared with the non-toxicity group in basal conditions. The key is shown in the bottom right of the figure. The map is constructed with an FDR cutoff of <0.25. Each node corresponds to a gene set from the GSEA; its size represents the number of genes in the gene set and its color the p-value. The thickness of the lines connecting the nodes is proportional to the number of genes that overlap in the gene set. Clustering among the various gene sets is visualized with boxes over the nodes. Those sets that are upregulated in patients with toxicity are shown in red and those downregulated are shown in blue. CT, control non-irradiated sample in toxicity patients; CNT, control non-irradiated sample in non-toxicity patients; GSEA, gene set enrichment analysis.

Figure 2

Heatmap and GSEA of 8 Gy-induced expression comparing patients with late skin toxicity to normal responders. (A) A different expression pattern between patients without toxicity (orange) from those with toxicity (purple) of 8 Gy-induced expressed genes with non-adjusted p-value <0.05 is suggested by hierarchical clustering classification of patients (columns). (B) The enrichment map allows the visualization of the general expression landscape induced by 8 Gy of the toxicity group compared with the non-toxicity group. The key is shown in the bottom right of the figure. The map is constructed with an FDR cutoff of <0.05. Each node corresponds to a gene set from the GSEA; its size represents the number of genes in the gene set and its color the p-value. The thickness of the lines connecting the nodes is proportional to the number of genes that overlap in the gene set. Clustering among the various gene sets is visualized with boxes over the nodes. Those sets that are upregulated in patients with toxicity are shown in red and those downregulated are shown in blue. NetT, 8 Gy-induced expression in toxicity patients; NetNT, 8 Gy-induced expression in non-toxicity patients; GSEA, gene set enrichment analysis.

Figure 3

Heatmap and GSEA of overall in-vitro radiation response comparing irradiated to non-irradiated samples in all patients. (A) A clear different expression pattern is observed between non-irradiated (non-toxicity in green and toxicity in dark blue) and 8 Gy-irradiated (non-toxicity in yellow and toxicity in pink) PBMC samples. Hierarchical clustering of the differentially expressed genes with non-adjusted p <0.05 and absolute logFC >0.5 almost perfectly separates the two experimental conditions. (B) The enrichment map allows the visualization of the general expression landscape of the irradiated compared with the non-irradiated PBMCs. The key is shown in the bottom right of the figure. The map is constructed with an FDR cutoff of <0.05. Each node corresponds to a gene set from the GSEA; its size represents the number of genes in the gene set and its color the p-value. The thickness of the lines connecting the nodes is proportional to the number of genes that overlap in the gene set. Clustering among the various gene sets is visualized with boxes over the nodes. Those sets that are upregulated in irradiated samples are shown in red and those downregulated are shown in blue. Abs, absolute; CNT, control non-irradiated sample in non-toxicity patients; 8 Gy NT, 8 Gy-irradiated culture in non-toxicity patients; CT, control non-irradiated sample in toxicity patients; 8 Gy T, 8 Gy-irradiated culture in toxicity patients; PBMCs, peripheral blood mononuclear cells; GSEA, gene set enrichment analysis.

Figure 4

Involved pathways in cell response to 8 Gy comparing irradiated to non-irradiated samples in the toxicity and non-toxicity patients. (A) Gene sets exclusively downregulated (blue) and upregulated (red) in the non-toxicity group after 8 Gy. (B) Gene sets exclusively downregulated (blue) and upregulated (red) in the toxicity group after 8 Gy. NES, normalized enrichment score.