The extracellular matrix protein ITIH5 is a novel prognostic marker in invasive node-negative breast cancer and its aberrant expression is caused by promoter hypermethylation

Inter- α -trypsin inhibitors (ITIs) are protease inhibitors stabilizing the extracellular matrix. ITIs consist of one light (bikunin) and two heavy chains (ITIHs). We have recently characterized ITIH5 , a novel member of the ITIH gene family, and showed that its messenger RNA is lost in a high proportion of breast tumours. In the present study, an ITIH5-specific polyclonal antibody was generated, validated with western blot and used for immunohistochemical analysis on a tissue microarray; ITIH5 was strongly expressed in epithelial cells of normal breast ( n =11/15), while it was lost or strongly reduced in 42% (92/217) of invasive breast cancers. ITIH5 expression in invasive carcinomas was associated with positive expression of oestrogen receptor ( P =0.008) and histological grade ( P =0.024). Correlation of ITIH5 expression with clinical outcome revealed that patients with primary tumours retaining abundant ITIH5 expression had longer recurrence-free survival (RFS; P =0.037) and overall survival (OS; P =0.044), compared to those with reduced expression (mean RFS: 102 vs 78 months; mean OS: 120 vs 105 months). Methylation-specific PCR analysis frequently showed strong methylation of the ITIH5 promoter in primary breast tumours (41%, n =109) and breast cancer cell lines ( n =6). Methylation was significantly associated with mRNA loss ( P