The expression of CXCL13 and its relation to unfavorable clinical characteristics in young breast cancer

Abstract

Background: Young breast cancer occupies a higher and higher proportion of breast cancer, especially in Asia, and is associated with a more unfavorable prognosis compared with the disease arising in older women. However, the poor prognosis of young breast cancer cannot be fully explained by the clinical and molecular factors.

Methods: This study investigated 1125 Chinese breast cancer patients diagnosed from 2009 to 2013. A data mining of gene expression profiles was performed for the young and older breast cancer patients, identifying significantly differentially expressed genes. Quantitative RT-PCR, Western blotting and immunohistochemistry assay were carried out for the clinical sample validations.

Results: The investigation firstly displayed that young patients (≤45 years) accounted for 47.6 % (535/1125) of breast cancer, and clinically associated with some unfavorable factors related to poor prognosis, such as invasive pathological type, high tumor grade, lymph node positive, ER negative and triple-negative subtype. Subsequently, 553 significantly differentially expressed genes were identified by the data mining. Of them, a set of genes related to immune function were observed to be up-regulated in young patients with breast cancer. Impressively, the CXCL13 (C-X-C motif chemokine 13) expression level showed the most significant difference (FC = 2.64, P = 8.2 × 10(-4)). Furthermore, the validations with clinical samples and correlation analysis demonstrated that CXCL13 was indeed highly expressed in young breast cancer and closely associated with some prognostic factors including lymph node positive and ER negative.

Conclusion: This is the first to indicate the clinical relevance of CXCL13 to young breast cancer and represents a potential therapeutic target for young breast cancer.

Fig. 1

Data mining of Gene expression profiles. a Significance analysis of microarray (SAM) was performed to identify differentially expressed genes between young and older breast cancer tissues. Delta was set to 0.6, and the threshold of FDR was set to 0.182. b Supervised hierarchical clustering of 553 differentially expressed genes. The heat map revealed the gene expression patterns between young patients and older patients. All samples were denoted in columns and genes were denoted in rows (gene symbols for a cluster of genes were listed on the right and the details of all differentially expressed genes could be found in Additional file 2: Table S2). The mapped expression levels for all genes were depicted using a color scale; highly expressed genes were indicated in red and lowly expressed in blue. c GO analysis of a cluster of differentially expressed genes was performed with GenCLiP software (http://ci.smu.edu.cn)

Fig. 2

Real-time PCR validation of mRNA expression in patients with breast cancer. a, c, e The ratios of CXCL13, GABRP and ESR1 mRNA expression in cancer tissues to their corresponding adjacent normal tissues were calculated after real-time PCR detection and normalization to GAPDH expression. X-axis indicates the ratio of mRNA expression in the cancer tissues to their corresponding adjacent normal tissues and Y-axis indicates the number of the specimens. b, d, f Expression levels of CXCL13, GABRP and ESR1 in young women group (n = 130) and older women group (n = 22) were analyzed by real-time PCR analysis and normalized to GAPDH expression. *P < 0.05, **P < 0.01

Fig. 3

Western blotting detection of CXCL13 protein in patients with breast cancer. a Expression levels of CXCL13 protein were assessed by Western blotting analysis and normalized to GAPDH. T_Y1–6 and T_O1–6 represented randomly selected cancer tissues from young women group and older women group, respectively, and N_Y1–6 and N_O1–6 represented the randomly selected corresponding adjacent normal tissues from young women group and older women group, respectively. b A semi-quantitative analysis of the Western blotting of CXCL13 protein was performed between cancer tissues (n = 12) and their corresponding adjacent normal tissues (n = 12). c A semi-quantitative analysis of the Western blotting of CXCL13 protein in cancer tissues between young patients (n = 6) and their older counterparts (n = 6). *P < 0.05

Fig. 4

Immunohistochemistry detection of CXCL13 protein in patients with breast cancer. a Representative IHC of breast cancer samples, showing the negative, weak, moderate and high expression level of CXCL13, respectively. b The percentage of patients with the negative, weak, moderate and high expression of CXCL13 protein in young and older women group. *P < 0.05

Fig. 5

Analysis of the correlation of CXCL13 expression with clinicopathological features. a The CXCL13 mRNA expression in 152 clinical tissue specimens was compared according to clinicopathological features. b, c, d 138 microarrays from GSE45255 and GSE15852 were divided into two groups according to their relative expression levels of CXCL13 (low expression group and high expression group). The CXCL13 expression and clinicopathological features were compared between these two groups. *P < 0.05, **P < 0.01, ***P < 0.001