ORIGINAL RESEARCH Desirée Hao, MD1,2 Tien Phan, MD2,4 Evaluation of E-cadherin, β-catenin Amanda Jagdis, MD3 Jodi E. Siever, MSc5 and vimentin protein expression using Alexander C. Klimowicz, PhD6 Janessa J. Laskin, MD3,7 quantitative immunohistochemistry in Thomas A. Thomson, MD8 M. Sarah Rose, PhD9 nasopharyngeal carcinoma patients Stephanie K. Petrillo, MSc6 Anthony M. Magliocco, MD10 Harold Y. Lau, MD2,4 Abstract Purpose: 1 Department of Medical Oncology, Tom Baker Aberrant expression of proteins involved in epithelial-to-mesenchymal transition Cancer Centre, Calgary, Alberta, Canada have been described in various cancers. In this retrospective study, we sought to evaluate 2 Faculty of Medicine, University of Calgary, E-cadherin, β-catenin and vimentin protein expression in non-metastatic nasopharyngeal Calgary, Alberta, Canada (NPC) patients treated with curative intent, examine their relationship with each other, 3 Department of Internal Medicine, Faculty of and with clinical outcome measures. Medicine, University of British Columbia, Van- Methods: couver, British Columbia, Canada Pre-treatment formalin-fixed paraffin-embedded biopsies of 140 patients treated 4 Department of Radiation Oncology, Tom between January 2000 and December 2007 were assembled into a tissue microarray Baker Cancer Centre, Calgary, Alberta, Canada (TMA). Automated quantitative immunohistochemistry (AQUA®) was performed on 5 Senior Analyst/Biostatistics, Research & In- sequential TMA sections stained with fluorescent-labeled antibodies against E-cadherin, novation, Population, Public, and Aboriginal β-catenin and vimentin. Cox proportional hazards regression was used to estimate the ef- Health, Alberta Health Services fect of cytoplasmic vimentin, cytoplasmic E-cadherin, β-catenin nuclear/cytoplasmic ratio 6 Department of Pathology, Tom Baker Cancer expression on overall survival and disease-free survival. Centre, Calgary, Alberta, Canada Results: 7 Department of Medical Oncology, British The average age of the patients was 51.7 years (SD=12.1; range 18-85), 66% were Columbia Cancer Agency – Vancouver male, 71% had a KPS ≥ 90% at the start of treatment and 65% had stage III/IV disease. 8 Pathologist, British Columbia Cancer Agency After adjusting for performance status, WHO and stage, high E-cadherin levels over the – Vancouver and Faculty of Medicine, Univer- 75th percentile were found to produce a significantly increased risk for both a worse over- sity of British Columbia all survival (HR = 2.53, 95% CI 1.21, 5.27) and disease free survival (DFS; HR = 2.14, 9 Rho - Sigma Scientific Consultants Calgary, 95%CI 1.28, 3.59). Vimentin levels over the first quartile produced an increased risk for a Calgary, AB, Canada worse DFS (HR = 2.21, 95% CI 1.11, 4.38). No association was seen between β-catenin 10 Department of Anatomic Pathology, H. Lee and survival. Moffitt Cancer Center, Tampa, Florida, U.S.A. Conclusion: In this cohort of NPC patients, higher levels of E-cadherin and higher levels Manuscript submitted 24th April, 2014 of vimentin were associated with worse outcomes. Further work is needed to understand Manuscript accepted 9th September, 2014 the role of these epithelial mesenchymal transition proteins in NPC. Clin Invest Med 2014; 37 (5): E320-E330. Correspondence to: Desirée Hao Department of Medical Oncology Tom Baker Cancer Centre 1331 29th St NW Calgary, Alberta, Canada, T2N 4N2 E-Mail: [email protected] © 2014 CIM Clin Invest Med • Vol 37, no 5, October 2014 E320 Hao et al. E-cadherin, β-catenin and vimentin protein expression in nasopharyngeal carcinoma Epithelial mesenchymal transition (EMT) which underlies curative intent radiation ± platinum chemotherapy at British embryonic development, increasingly appears to be critical in Columbia Cancer Agency and Tom Baker Cancer Centre were the process of tumour invasion and metastases [1]. Although identified with retrievable biopsy samples, either from the pri- EMT is regulated by a complex interplay of signaling pathways, mary site or from a nodal metastasis. Tissue microarrays a hallmark of EMT is the down regulation of E-cadherin, mis- (TMAs) were constructed from duplicate 0.6 mm cores of pre- localization of β-catenin and nuclear expression of vimentin treatment formalin-fixed paraffin-embedded specimens. Fluo- [2]. E-cadherin is a transmembrane glycoprotein that is in- rescent immunohistochemistry was performed on sequential volved in mediating cell–cell adhesion between adjacent TMA sections that were co-stained with antibodies against E- epithelial cells in various tissues [3, 4]. Loss of E-cadherin has cadherin (1:200, Rabbit clone 24E10, Cell Signaling, Dan- been related to tumour aggressiveness and increased rates of vers, MA) or β-catenin (1:1000 mouse clone B-cat-1, Dako metastasis in breast, gastric, hepatocellular, bladder and pros- Cytomation, Glostrup, Denmark) and vimentin (MAB2105 tate cancer [5-10]. β-catenin is a multi-functional protein in- rat; R&D Systems, Minneapolis, MN) and pan-cytokeratin volved in the cadherin-mediated cell-cell adhesion system [11]. (AE1/AE3 or rabbit anti-cow wide spectrum screening anti- It associates with the cytoplasmic portion of E-cadherin and body, respectively; Dako Cytomation). Antibody signals was the actin cytoskeleton via α-catenin to form the adherens junc- amplified and visualized using a TSA-Plus CY5 (E-caderin and tion [12, 13]. β-catenin is also an important effector of Wnt β-catenin) or Cy3 (vimentin) Tyramide Signal Amplification signaling in the nucleus, where it regulates the expression of kit (PerkinElmer, Waltham, MA) and pan-cytokeratin was genes involved in cell growth. Unbound cytoplasmic β-catenin visualized using a goat anti-primary Alexa 555 conjugated sec- is targeted for proteosomal degradation in the absence of Wnt ondary antibody (Invitrogen, Carlsbad, CA). After staining, signaling. E-cadherin is thought to antagonize β-catenin sig- TMA slides were mounted in ProLong Gold mounting media naling by binding to it, thus keeping the level of cytoplasmic β- with diamidino-phenylindole to visualize the nucleus (Invitro- catenin low and unavailable for downstream activation of the gen). TMAs were then scanned using a HistoRx PM-2000 Wnt pathway [14, 15]. Loss of E-cadherin expression has been image analysis platform. AQUAnalysis™ software (version associated with increased localization of β-catenin to the nu- 2.3.4.1) was used to create a "mask" to isolate the epithelial/ cleus [15, 16]. Vimentin is a type III intermediate filament tumour cells within the tissue cores. E-cadherin, β-catenin or protein normally found in mesenchymal cells and is expressed vimentin pixel intensity was measured within the tumour area in epithelial cells that are migrating during embryogenesis, or- (tAQUA), cytoplasmic tumour area (cAQUA) or the nuclear ganogenesis, wound healing and tumour invasion [17]. The tumour area (nAQUA). vimentin promoter is a target of the β-catenin/T-cell factor pathway, suggesting that the functional regulation of epithelial Statistical Analysis cells is involved in tumour invasion and/or metastasis [18]. The nuclear/cytoplasm β-catenin ratio (n/cBC) was obtained Since nasopharyngeal cancers (NPC) commonly invade by dividing the nAQUA scores by the cAQUA scores [26]. For surrounding tissue or metastasize to cervical lymph nodes early vimentin and E-cadherin, cAQUA scores were used for analysis in the natural history of the disease, we sought to characterize (tumour cell cytoplasmic vimentin and tumour cell cytoplas- the protein expression of E-cadherin, β-catenin and vimentin, mic E-cadherin, respectively), and both were Z-score standard- three proteins involved in EMT, in non-metastatic NPC pa- ized between the Calgary and the Vancouver cohorts. All three tients treated with curative intent. We explored the relation- biomarkers were stratified into quartiles with the highest cate- ship among E-cadherin, β-catenin and vimentin, and their rela- gory considered to have the highest expression of the protein. tionship with clinical outcome measures. Categorical variables were expressed as a frequency and percentage; patient age was expressed as mean ± standard de- Materials and Methods viation. The correlation between the biomarkers was assessed Study Population, TMA Construction and Analysis using the Spearman rank correlation coefficient. The relation- ship between each biomarker and other variables (T-stage, N- This study was approved by the University of Calgary Conjoint stage, WHO class, and grade) was assessed using ANOVA after Health Research Ethics Board and the University of British appropriate transformations were applied to achieve an ap- Columbia, British Columbia Cancer Agency (BCCA) Re- proximately Normal distribution or the Kruskal Wallis non- search Ethics Board. Between January 2000 and December parametric test when an appropriate transformation could not 2007, 137 patients with non-metastatic NPC treated with be found. © 2014 CIM Clin Invest Med • Vol 37, no 5, October 2014 E321 Hao et al. E-cadherin, β-catenin and vimentin protein expression in nasopharyngeal carcinoma TABLE 1. Demographic and treatment characterristics for the overall sample Included Excluded Characteristic Patients Patients p (N = 137) (N=142) Age in years (Mean (SD)) 52 (12.1) 52 (13.3) 0.453 No. (%) Male gender 91 (66%) 99 (70%) 0.555 Stage I/II 48 (35%) 60 (43%) 0.819 III/IV 89 (65%) 81 (57%) KPS≥ 90% 97 (71%) 120 (84%) 0.006 WHO type 1 16 (12%) 11 (8%) 2 38 (29%) 30 (22%) 0.164 3 79 (59%) 97 (70%) RT protocol 3D conformal(66-70 Gy, 33-35 fractions) 89 (67%) 101 (75%) 0.183 IMRT(70 Gy, 33-35 fractions) 43 (33%) 34 (25%) Treatment modality RT only 80 (58%) 97 (68%) CCRT 38 (28%) 31 (22%) 0.222 Adjuvant chemotherapy 19 (14%) 14 (10%) T stage T1 49 (36%) 55 (39%) T2 39 (29%) 43 (30%) 0.709 T3 21 (15%) 16 (11%) T4 28 (20%) 28 (20%) N stage N0 44 (32%) 45 (32%) N1 36 (26%) 33 (23%) 0.864 N2/3 57 (42%) 64 (45%) 2 year locoregional control rate 113 (82%) 126 (89%) 0.136 Total Death 36 (26%) 34 (24%) 0.811 NPC-related 35/36 (97%) 32/24 (94%) Note: Denominator varies slightly due to missingg data.