Reduced expression of ezrin in urothelial bladder cancer signifies more advanced tumours and an impaired survival: validatory study of two independent patient cohorts

In 2008 there were 386 000 estimated new cases of bladder cancer in the world, and approximately 150 000 individuals died from their disease [1]. Bladder cancer is the fourth most common cancer among men in the USA [2].

Standard treatment for non-muscle-invasive carcinoma is transurethral resection of the bladder (TURB), with or without intravesical instillation of bacillus Calmette-Guérin (BCG), to prevent recurrence and progression. In contrast, muscle-invasive carcinoma is treated more aggressively with neoadjuvant chemotherapy and cystectomy [3, 4]. However, non-muscle-invasive urothelial carcinoma has a high risk of recurrence and a substantial risk of progression [5], and muscle-invasive carcinoma is associated with a high mortality, despite aggressive treatment [3, 6]. Hence, there is a great need for additional biomarkers to predict the risk of recurrence and progression into muscle invasive carcinoma for patients with early stage tumours.

Loss of expression of the membrane-cytoskeletal linking protein ezrin was initially demonstrated to be associated with tumour progression and poor prognosis in patients with T1G3 tumours treated with non-maintenance BCG (n = 92) [7]. In another recent study comprising 104 tumours of different stages and grades, loss of membranous ezrin expression was found to correlate with higher grade and stage, and invasiveness, but the associations with disease progression and survival were not reported [8]. The protein ezrin is closely related to two other membrane associated proteins, radixin and moesin, all three together named ERM proteins. All these proteins are important for regulation of cell adhesion and in the linkage between membrane proteins and the cortical cytoskeleton, thus affecting cell survival, migration and invasion, all factors contributing to tumour progression and development [9‐11]. ERM proteins are inactive in the cytoplasm, and activated by binding to the cell membrane [10]. Ezrin is expressed in a variety of cancers [12] and the prognostic value of ezrin expression seems to differ in different cancer forms. In several cancer forms, high expression of ezrin has been associated with more aggressive tumours [13‐19], whereas in serous ovarian carcinoma, lost expression of ezrin correlated with a worse prognosis [20], similar to the findings in bladder cancer [7].

The aim of this study was to further evaluate the utility of ezrin as a prognostic biomarker in two independent patient cohorts comprising a total number of 442 cases. Given previous findings of an in vitro interaction of ezrin with podocalyxin-like protein (PODXL), an established mediator of metastasis [21], and our recent results demonstrating that membranous PODXL expression is an independent predictor of tumour progression and poor prognosis in urothelial bladder cancer [22], we also examined the correlations between tumour-specific expression of ezrin and PODXL.

The results from this study demonstrate that loss of membranous ezrin expression in urothelial bladder cancer is strongly associated with a more aggressive tumour phenotype; i.e. higher grade and more advanced tumour stage, and an impaired survival. To our best knowledge, the expression of ezrin in urothelial bladder cancer has only been described in two previous studies; one selected cohort of T1G3 tumours (n = 98) treated with non-maintenance BCG [7], and another unselected cohort (n = 104). In the former study, while correlations of ezrin expression with stage and grade could not be performed, the authors found no associations between ezrin expression and age, sex, substaging, tumour size, focality or the presence of CIS, but that reduced ezrin expression was an independent predictor of progression into muscle-invasive disease and shorter disease- specific survival [7]. In the latter study, loss of ezrin was found to correlate with higher grade and T-stage, and with muscularis propria invasion, but hazard ratios for risk of progression or association with survival were not presented [8].

Thus, the results from our study further validate previous findings of strong significant associations between loss of membranous ezrin expression and more advanced T-stage and high tumour grade in urothelial bladder cancer. Moreover, this study is the first to report the prognostic value of ezrin expression in tumours representing all stages and grades, whereby reduced ezrin expression was found to be associated with a significantly reduced 5-year OS in both examined cohorts, and with a significantly reduced DSS in cohort II. Of note, ezrin expression only retained an independent prognostic value for 5-year OS in the smaller cohort, and neither for 5-year OS nor for DSS in the larger, clinically more well-characterized, cohort. Therefore, further validation of the prognostic value of ezrin expression in additional patient cohorts is warranted. Nevertheless, since tumour stage may be difficult to determine in TURB-specimens, the strong link between loss of ezrin expression and advanced tumour stage found here indicates that assessment of ezrin may be an important surrogate marker for bladder cancer patients at risk of having progressive disease.

In contrast to the findings by Palou et al. [7], we were not able to demonstrate an association between reduced ezrin expression and risk of progression into muscle-invasive disease in pTa-pT1 or pT1-tumours (information only available in cohort II). It should however be pointed out that the number of patients having received BCG-treatment in our study was too small, and since all patients in the study by Palou had received non-maintenance BCG-treatment [7], a potential treatment predictive effect of ezrin cannot be ruled out, and should be taken into consideration in future studies.

The observed significant association between reduced ezrin expression and female sex in both cohorts is noteworthy, not least since the distribution of tumour grades did not differ between sexes in any of the cohorts, and a significant association between female sex and more advanced T-stage could only be found in cohort II. While the risk of bladder cancer is considerably higher in men, there is data indicating an impaired survival from bladder cancer in women [25]. These findings indicate that ezrin may be a relevant investigative biomarker in studies related to the molecular pathological epidemiology of urothelial bladder cancer, in particular studies addressing the influence of sex hormones and reproductive factors on cancer risk and survival.

Despite use of different antibodies for detection of ezrin expression in the two previous studies [7, 8] and the present, the results were concordant, which further supports the utility of ezrin as a prognostic and, potentially treatment predictive, biomarker in urothelial bladder cancer. Immunohistochemistry has several advantages compared with other assays, e.g. gene expression analyses, since it is comparatively cheap, can readily be adopted into clinical protocols, and, most importantly, allows for assessment of biomarkers in relation to their subcellular location. In our study, no prognostic value or correlation with clinicopathological factors could be demonstrated for cytoplasmic ezrin expression, which is also in line with previous findings [7, 8]. The lack of prognostic value for cytoplasmic ezrin expression is also in agreement with the observation of ERM proteins only being active when bound to the cell membrane and not when located in the cytoplasm [10].

The herein observed inverse association between membranous expression of ezrin and PODXL does not provide evidence of, but may well indicate, a functional link between these proteins in urothelial bladder cancer. This hypothesis is further supported by the previously demonstrated ability of PODXL to form complex with ezrin in breast and prostate cancer cells in vitro, thereby inducing phosphorylation of ezrin and changes in its subcellular location, in turn leading to an increased migration and invasion [21]. In light of the apparently contrasting prognostic value and intercorrelation of ezrin and PODXL expression in urothelial bladder cancer, it will be of interest to investigate the existence of a negative functional cooperativity between these proteins in this cancer form.

Of note, although cutoffs selected by CRT-analysis in our study varied somewhat between the cohorts and according to the endpoint, ranging from 12.5% to 27.5% membranous positivity, they still landed closely to the prognostic cutoff determined as the median percentage of ezrin expression at 20% in the study by Palou et al. [7], although all tumours in their study were pT1G3. A different approach was used in the study by Athanasopoulou et al., where four categories of a combined score of percentage and intensity of membranous ezrin immunoreactivity was applied in the statistical analyses [8]. Moreover, in that study, nearly all (103/104) tumours were reported to have positive membranous ezrin expression [8]. Future studies, preferably in the prospective setting, are warranted to determine optimal cutoffs for the potential use of ezrin as a biomarker in clinical practice.

In summary, the results from this study demonstrate that reduced membranous ezrin expression in urothelial bladder cancer is associated with more advanced tumours and a reduced survival. These findings suggest that ezrin may be a useful prognostic biomarker and possibly aid in tailoring the treatment of patients with non-muscle-invasive carcinoma of the bladder. Further studies are warranted in order to confirm the utility of ezrin as a prognostic biomarker in clinical practice.