IGFBP7 is associated with poor prognosis in oesophageal adenocarcinoma and is regulated by promoter DNA methylation

Abstract

Background: We examined whether silencing of IGFBP7 was associated with survival in patients with oesophageal adenocarcinoma.

Methods: Protein expression of IGFBP7 was determined using immunohistochemistry in a tissue microarray representing tumours from 65 patients with oesophageal adenocarcinoma who had not had neoadjuvant therapy. DNA methylation of the IGFBP7 promoter was determined with the melt curve analysis in cell lines and patient tissues.

Results: Expression of IGFBP7 was observed in the oesophageal adenocarcinoma of 34 out of 65 (52%) patients and was associated with significantly reduced median (11 vs 92 months) and 5-year survival (25% vs 52%). Multivariate analysis identified expression as an independent prognostic indicator for survival (hazard ratio=3.24, 95% confidence interval=1.58-6.67, P-value=0.0014). Hypermethylation of IGFBP7 was associated with silencing of gene expression in cell lines and patient tissues (P-value=0.0225). Methylation was observed in the squamous mucosa of 2 out of 15 (13%) patients with Barrett's oesophagus and 3 out of 17 (18%) with oesophageal adenocarcinoma. Methylation was observed in 14 out of 18 (78%) of biopsies of Barrett's mucosa and 23 out of 34 (68%) patients with oesophageal adenocarcinoma.

Conclusion: Reduced IGFBP7 protein expression was associated with longer survival in patients with oesophageal adenocarcinoma. Methylation of the IGFBP7 promoter was associated with silencing of gene expression and was frequent in Barrett's oesophagus and oesophageal adenocarcinoma.

Figure 1

Immunohistochemistry of IGFBP7 in human oesophageal adenocarcinoma. Representative images of IGFBP7 immunohistochemistry displaying (A) negative expression in tumour cells and stroma; (B) negative expression in tumour cells, and positive in stroma; (C) positive expression in tumour cells, but negative in stroma; (D) positive expression in tumour cells and stroma.

Figure 2

Kaplan–Meier analysis of overall survival of patients with oesophageal adenocarcinoma in relation to IGFBP7 expression. Expression of IGFBP7 in tumour cells, determined by immunohistochemistry, scored as negative (n=31) or positive (n=34). Log-rank (Mantel–Cox) test P-value=0.0140.

Figure 3

Expression of IGFBP7 in oesophageal tissues. Expression of IGFBP7 in histologically normal squamous mucosa (Normal), Barrett's oesophagus (Barrett's), and oesophageal adenocarcinoma (OAC) tissues was determined using transcriptional microarrays in four different data sets as named and published on Oncomine.

Figure 4

Expression and DNA methylation of IGFBP7 in oesophageal cell lines. The Barrett's associated oesophageal adenocarcinoma (OE33) and oesophageal squamous cell carcinoma (OE21 and TE7) cell lines were treated with either vehicle (veh) or 1 μmol l⁻¹ 5-aza-2'-deoxycytidine (aza) and analysed for (A) expression of IGFBP7, measured by QPCR (mean±s.e.m. of three replicates), and (B) DNA methylation, measured with the melt curve analysis (mean±s.e.m. of two to three replicates). (C) Methylation of individual CpGs in the IGFBP7 promoter was measured in OE33 using an Infinium HumanMethylation450 BeadChip Kit. The position of individual CpGs was determined relative to the transcription start site (TSS) of IGFBP7 (assembly GRCh37/hg19, chr4:57898404-57976551). The black horizontal bar denotes the region analysed with the melt curve analysis. (D) Methylation, measured with the melt curve analysis, in oesophageal adenocarcinoma (OE19 and JH-EsoAd1), Barrett's oesophagus (CP-A, CP-B, and CP-D) and squamous epithelial cell lines (mean±s.e.m. of two to three replicates).

Figure 5

Kaplan–Meier analysis of overall survival of patients with oesophageal adenocarcinoma in relation to IGFBP7 methylation. Methylation of the IGFBP7 promoter, determined with the melt curve analysis, was recorded as unmethylated (n=11) or methylated (n=23). Log-rank (Mantel–Cox) test P-value=0.0707.