Determination of follistatin-like protein 1 expression in colorectal cancer and its association with clinical outcomes

Abstract

Background: Follistatin-like protein 1 (FSTL1) has been demonstrated to play a controversial role in cancer. In this study, we aimed to investigate the expression of FSTL1 and its characteristics in patients with colorectal cancer (CRC).

Methods: Gene expression microarray assays in 30 CRC patients and a real-time quantitative polymerase chain reaction (RT-qPCR) of 22 patients were performed to compare the mRNA level of FSTL1 in tumor lesions and paired normal tissues. Also, 332 consecutive patients with pathologically confirmed CRC were selected to detect FSTL1 expression by using immunohistochemistry (IHC). Enzyme-linked immunosorbent assay (ELISA) was also applied to determine the serum level of FSTL1 in 60 CRC patients, as well as 34 healthy donors.

Results: Gene expression microarray assays and RT-qPCR in CRC tissues, as well as ELISA in the serum all, revealed that the expression level of FSTL1 was higher in cancer tissue of CRC patients compared with paired normal tissue or healthy donors. The IHC results suggested that FSTL1 was also higher in tumor tissues than in its normal counterparts, however interestingly, a narrow scan focusing on the stromal region indicated that FSTL1 was significantly higher in normal tissues than in cancerous tissues. Besides, higher FSTL1 expression in cancer tissue, as well as lower FSTL1 expression in cancer stroma, both correlated with a worse prognosis, and the latter was an independent prognostic factor.

Conclusions: Our results provide novel insight into the role of FSTL1 in CRC, and it might be an essential factor in CRC development.

Keywords: Colorectal cancer (CRC); follistatin-like protein 1 (FSTL1); prognosis; stroma.

Figure 1

Flow diagram summarizing the patients involved in our study. Clinical samples of tissues and serum collected from Sun Yat-sen University Cancer Center (SYSUCC) were classified into 4 groups for determination of FSTL1 expression via transcriptome array, RT-qPCR, IHC and ELISA. The included CRC patients had received no previous local or systemic treatment prior to surgery. FSTL1, follistatin-like protein 1; RT-qPCR, real-time quantitative polymerase chain reaction; IHC, immunohistochemistry; ELISA, enzyme-linked immunosorbent assay; CRC, colorectal cancer.

Figure 2

The example of image analysis of IHC staining in constructed TMAs. The area inside the dotted line belong to the colorectal epithelium (indicated by the red arrow). Each slide experienced evaluations twice, one for epithelial as well as stroma area (the full scan, i.e., within and outside the dashed line), and the other for only the stroma area (the narrow scan, i.e., outside the dotted line). IHC, immunohistochemistry; TMA, tissue microarray.

Figure 3

FSTL1 is frequently overexpressed in CRC samples both for tumor tissues and serum. (A) Expression of FSTL1 in 30 pairs of CRC and corresponding nontumor tissues [18 patients with stage I to III CRC (6 patients per stage), and 12 patients with stage IV CRC (6 patients with liver-only metastasis and 6 patients with metastasis in organs other than the liver)] examined by transcriptome array (mean ± SD: 9.12±1.32 vs. 7.94±0.91, P<0.001). (B) Expression of FSTL1 in 22 pairs of tumors and nontumor counterparts examined via RT-qPCR (mean ± SD: 12.37±1.95 vs. 10.46±1.58, P=0.002). (C) Box plot showing the ELISA results of the expression of FSTL1 in sera collected from 34 healthy normal individuals and 60 CRC patients (mean ± SD: 1.91±1.40 vs. 1.10±1.25, P<0.001). FSTL1, follistatin-like protein 1; RT-qPCR, real-time quantitative polymerase chain reaction; IHC, immunohistochemistry; ELISA, enzyme-linked immunosorbent assay; CRC, colorectal cancer.

Figure 4

Representative IHC for FSTL1 in full scans and narrow scans of the stroma in tumor tissues and tumor-adjacent normal tissues. Scale bar, 200 and 100 µm. (A,B) CRC tissue with weak FSTL1 expression in both the full scan and the stromal region; (C,D) CRC tissue with strong FSTL1 expression (especially in the stroma); (E,F) weak FSTL1 expression in tumor-adjacent normal tissue in both the full scan and in the stroma; (G,H) strong FSTL1 expression in tumor-adjacent normal tissue (especially in the stroma). FSTL1, follistatin-like protein 1; IHC, immunohistochemistry; CRC, colorectal cancer.

Figure 5

Bar graph summary of the distribution of FSTL1 expression levels in nontumor (n=247) vs. CRC (n=310) tissues examined via IHC of TMA (n=332 total cases). (A) Comparison of FSTL1 expression levels between fully scanned CRC tissues and tumor-adjacent normal tissues (mean ± SD: 7.39±3.18 vs. 3.09±2.38, P<0.001); (B) comparison of FSTL1 expression levels shown on a narrow scan of the stromal region between tumor and tumor-adjacent normal tissues (mean ± SD: 3.71±2.18 vs. 6.34±2.60, P<0.001). FSTL1, follistatin-like protein 1; IHC, immunohistochemistry; CRC, colorectal cancer; TMA, tissue microarray.

Figure 6

Optimal cutoff levels for FSTL1 expression were applied with ROC curves for overall survival. (A) ROC curve for optimal cutoff levels for FSTL1 expression in fully scanned CRC tissues according to overall survival. The optimal IHC score cutoff value was 6.5; (B) ROC curve for optimal cutoff levels for FSTL1 expression in a narrow scan of the CRC stromal region according to overall survival. The optimal IHC score cutoff value was 3.17. ROC, receiver operating characteristic; FSTL1, follistatin-like protein 1; IHC, immunohistochemistry; CRC, colorectal cancer; TMA, tissue microarray.

Figure 7

Kaplan-Meier survival analysis comparing the survival rate of patients with different FSTL1 expression levels. (A) Survival analysis of patients with high FSTL1 expression and low FSTL1 expression in fully scanned CRC tissues (3-year OS rate: 72.7% vs. 62.0%, P=0.041); (B) survival analysis of patients with high FSTL1 expression and low FSTL1 expression in a narrow scan of the CRC stromal region (3-year OS rate: 57.8% vs. 76.5%, P=0.002). FSTL1, follistatin-like protein 1; CRC, colorectal cancer; OS, overall survival.