Formalin-fixed paraffin-embedded tissue samples were obtained from the Institute of Pathology, University Hospital Duesseldorf. All tissue samples were newly reviewed prior to this study and the diagnosis of FTC histologically confirmed. FTCs were defined as thyroid carcinomas with follicular differentiation in the absence of papillary nuclear features, with either capsular or vascular invasion. The construction of the tissue microarray, immunohistochemistry and protein expression analyses were performed as described previously21 (link),22 (link). The expression levels of CXCR4 and CXCR7 were scored by two independent investigators (TW, CF) using the immunoreactivity score (IRS) reported by Remmele23 (link) in a blinded manner.
The immunohistochemistry was carried out using mouse monoclonal anti-CXCR4 (1:100 dilution; Abcam, Cambridge, UK) and rabbit polyclonal anti-CXCR7 (1:200 dilution; GeneTex, Irvine, CA, USA) as primary antibodies. Isotype control was performed using mouse IgG1k (MOPC-21; 1:50 dilution; Abcam, Cambridge, UK) and rabbit immunoglobulin fraction (Code X0903; 1:1000 dilution; Dako, Glostrup, Denmark). CXCR4 expressing tonsil tissue and CXCR7 expressing pancreatic adenocarcinoma served as positive controls. The prognostic power of CXCR4 and CXCR7 was assessed in accordance with the REporting recommendations for tumour MARKer prognostic studies (REMARK)24 .