The results showed that age HTC and N stage are independent negative prognostic factors for overall survival, and female gender and N stage for the development of metachronous metastases (data not shown). Taking these factors into account, we identified higher FHL2 expression at the tumour invasion front as well as at the tumour centre being an independent negative prognostic factor (both with P<0.001, see Table 2), associated with the development of metachronous metastases. Moreover, FHL2 expression in the tumour invasion front was also associated with overall survival independently of the clinicopathological variables (P<0.05, see Table 2).

Table 2 Multivariate analysis of overall survival and metastasis-free survival By analysing the distribution of the FHL2 LI values in order to stratify the patients in relation to their metastasis-free survival, we found that <20% of the patients with a LI 40% in the tumour invasion front or in the tumour centre had a short metastasis-free survival (in contrast to patients with LI>40%, see Supplementary Figure 1). The Kaplan�CMeier curves shown in Figure 3B and C confirm the significant prognostic value of this FHL2 threshold for the invasion front and the centre, respectively, (both with P<0.001). Figure 3A also demonstrates the significant prognostic value of this threshold for the invasion front with regard to overall survival (P<0.01). A similar result was obtained for FHL2 expression in the tumour centre (data not shown); however, this expression is not an independent prognostic factor (see Table 2). Figure 3 Prognostic value of FHL2 expression.

(A) Overall survival curve of patients dichotomised on the basis of the FHL2 LI evaluated in tumour invasion front (P<0.01). (B and C): Metastasis-free survival curves of patients dichotomised on the basis ... Expression of FHL2 in relation to E-cadherin and ��-catenin In a pilot experiment including 10 cases, foci of intense FHL2 expression with concomitant reduced E-cadherin expression and appearance of nuclear expression of ��-catenin could be demonstrated in consecutive sections. These foci were found in EMT areas, characterised by poor differentiation of the cancer, and presence of small clusters of cancer cells and isolated cancer cells in the extracellular matrix-rich peritumoural stroma (Figure 4). Figure 4 Consecutive sections Drug_discovery with foci of (upper left) intense FHL2 expression with concomitant (upper right) reduced E-cadherin expression and (lower) appearance of nuclear expression of ��-catenin (arrows) in areas of EMT. Note the lower FHL2 expression …