Low FHL2 expression is related to better survival in patients with breast cancer (Gabriel et al, 2006). Four-and-a-half LIM domains protein 2 is highly expressed in human colon cancer, and cell line experiments showed that sellckchem FHL2 is critical for migration and invasion (Zhang et al, 2010). Moreover, suppression of FHL2 in colon cancer cell lines induces cell differentiation and inhibits tumorigenesis (Wang et al, 2007). In the present study, we aimed to investigate a possible prognostic role of FHL2 expression in a large series of CRC cases, without evidence of distant metastasis at the time of presentation; development of metastases and mortality are the outcome parameters. We thereby focus on expression of FHL2 in neoplastic epithelial cells.

Materials and methods Patients We retrospectively analysed tumour samples from consecutive patients who underwent surgical resection of a primary CRC, without evidence of distant metastasis at the time of surgery. All patients underwent surgical resection at the Erasme University Hospital (Brussels, Belgium) between May 1990 and December 2000. Sex, age and pTNM status were retrieved from medical reports. Follow-up was available until August 2009. The study was approved by the local ethics committee. Basic patient demographical data are summarised in Table 1. Table 1 Clinical and histopathological patient data Tissue microarray construction Tissue microarray (TMA) blocks were constructed as described previously (Decaestecker et al, 2009), with a manual microarrayer (Beecher Instruments, Sun Prairie, WI, USA) to include twelve cores (600-��m diameter) from each CRC case.

Six cores were obtained from the central part of the tumour and six cores from the invasion front. Immunohistochemistry for mismatch repair proteins and its evaluation Standard immunohistochemistry was applied to 5-��m thick sections to display MLH1, MSH2, MSH6 and PMS2 expression, using antibodies against MLH1 (Menarini, Zaventem, Belgium; clone ES05, dilution 1:100), MSH2 (Menarini; clone 25D12, dilution 1:200), MSH6 (Menarini; clone PU29, dilution 1:400) and PMS2 (Menarini; clone M0R4G, dilution 1/100), and was performed on the BOND-MAX (Leica, Wetzlar, Germany). Briefly, the immunohistochemical expression was visualised using the Bond Polymer Refine Detection kit (Menarini; kit DS9800). The sections were counterstained with haematoxylin.

Mismatch repair (MMR) protein expression was divided into ��intact’ and Carfilzomib ��deficient’ groups. Intact MMR protein expression was defined by nuclear expression of MLH1, MSH2, MSH6 and PMS2 in neoplastic epithelial cells (Resnick et al, 2010). If stromal cells were negative for at least one of these markers, the case was considered non-evaluable for MMR protein expression by lack of positive internal control.