Open Access Research BMJ Open: first published as 10.1136/bmjopen-2012-000900 on 5 April 2012. Downloaded from Loss of epithelial membrane protein-2 expression confers an independent prognosticator in nasopharyngeal carcinoma: a cohort study Yi-Hsien Chen,1 Li-Ching Wu,2 Wen-Ren Wu,3 Hung-Jung Lin,1 Sung-Wei Lee,4 Ching-Yih Lin,5 Shih-Lun Chang,6 Nan-Haw Chow,7,8 Hsuan-Ying Huang,9 Chien-Feng Li,2,3,10,11 Han-Ping Hsu,12 Yow-Ling Shiue3 To cite: Chen Y-H, Wu L-C, ABSTRACT ARTICLE SUMMARY Wu W-R, et al. Loss of Objective: To evaluate the expression of epithelial epithelial membrane membrane protein-2 (EMP2) protein and its Article focus protein-2 expression confers clinicopathological associations in patients with - an independent Recent studies have suggested that EMP2 plays nasopharyngeal carcinoma. prognosticator in a tumour suppressor role in B cell lymphomas. nasopharyngeal carcinoma: Design: Retrospective population-based cohort study. - Immunoexpression of EMP2 was retrospectively a cohort study. BMJ Open Setting: This study was based on a biobank in Chi-Mei assessed in biopsies of 124 consecutive patients 2012;2:e000900. Medical Center (Tainan, Taiwan) from 1993 to 2002. with nasopharyngeal carcinoma. doi:10.1136/ Participants: Biopsies of 124 consecutive bmjopen-2012-000900 nasopharyngeal carcinoma patients without initial Key messages - Loss of EMP2 expression significantly correlates distant metastasis and treated with consistent < Prepublication history for with advanced primary tumour, nodal status and guidelines were assessed. Immunoexpressions of this paper is available online. AJCC stage. To view these files please EMP2 were analysed and the outcomes were correlated - In multivariate analyses, loss of EMP2 expres- visit the journal online (http:// with clinicopathological features and patient survivals. sion emerges as an independent prognosticator dx.doi.org/10.1136/ Primary and secondary outcome bmjopen-2012-000900). for worse disease-specific survival and local http://bmjopen.bmj.com/ measures: Immunoexpressions of EMP2 were recurrence-free survival. analyzed and the outcomes were correlated with Received 21 January 2012 clinicopathological features and patient survivals. Accepted 27 February 2012 Strengths and limitations of this study Results: Loss of EMP2 expression (49.2%) was - Significant correlation between loss of EMP2 This final article is available correlated with advanced primary tumour (p¼0.044), expression and several clinicopathologic vari- for use under the terms of nodal status (p¼0.045) and the 7th American Joint ables supported its potential role in nasopharyn- the Creative Commons Committee on Cancer stage (p¼0.027). In multivariate geal carcinomas. Attribution Non-Commercial analyses, loss of EMP2 expression emerged as an - The molecular mechanisms underlying EMP2 2.0 Licence; see independent prognosticator for worse disease-specific action require to be elucidated. on September 29, 2021 by guest. Protected copyright. http://bmjopen.bmj.com survival (DSS; p¼0.015) and local recurrence-free survival (LRFS; p¼0.030), along with the American non-keratinising carcinoma types, according Joint Committee on Cancer stages IIIeIV (p¼0.034, DSS; p¼0.023, LRFS). to current WHO tumour classification, although genetic and environmental factors Conclusions: Loss of EMP2 expression is common 124 and associated with adverse prognosticators and also play certain roles in pathogenesis. The might confer tumour aggressiveness through advances in diagnostic imaging, radiation hampering its interaction with specific membrane therapy and adjuvant chemotherapy of NPC protein(s) and hence the downstream signal have achieved better locoregional control, transduction pathway(s). while it appears less satisfactory in final treat- ment outcomes.56Even though being an important parameter, Tumour, Node, Metas- tasis staging still has space to improve in terms INTRODUCTION of providing the optimal prognostication to 157 For numbered affiliations see Nasopharyngeal carcinoma (NPC) is an the patients. Therefore, to identify end of article. endemic head and neck epithelial malignancy potential biomarkers with better correlation to in Southeastern Asia and Taiwan, strongly tumour growth and/or treatment outcomes in e 1e3 Correspondence to linked to Epstein Barr virus (EBV). The patients with NPC, subsequently, to aid in risk Professor Yow-Ling Shiue; latter association is especially authentic for stratification and perhaps development of [email protected] the differentiated and undifferentiated therapeutic targets, are indispensable. Chen Y-H, Wu L-C, Wu W-R, et al. BMJ Open 2012;2:e000900. doi:10.1136/bmjopen-2012-000900 1 EMP2 low expression in nasopharyngeal carcinoma BMJ Open: first published as 10.1136/bmjopen-2012-000900 on 5 April 2012. Downloaded from Human epithelial membrane protein-2 gene (EMP2), of EMP2 was scored by two pathologists (C-FL and mapped to chromosome 16, is highly conserved across H-YH) using a multiheaded microscope to reach e vertebrates.8 10 The expression pattern of EMP2 partially a consensus for each case without prior knowledge of overlaps to that of the peripheral myelin protein 22 clinical and follow-up information. The percentage of (PMP22, also known as the growth arrest-specific-3, tumour cells with EMP2 immunoexpression was GAS3) transcript. By containing the claudin domain and recorded for each specimen and loss of EMP2 expres- sharing approximately 40% amino acid identity with sion (negative) was defined in cases with staining #5% PMP22/GAS3,11 the EMP2 protein was detected as tumour cells (see the Statistical analysis section). a novel member of this four-transmembrane (tetraspan) superfamily.12 In humans, EMP2 has a discrete cell type Treatment and follow-up and tissue distribution, with high levels observed in the All 124 patients with follow-up for outcome have lung and moderate levels in the eye, heart, thyroid, received complete course of radiotherapy (RT, total dose uterus and intestine.11 13 14 Functionally, the best $7000 cGy) and also cisplatin-based chemotherapy in understood tetraspan proteins are connexins, which those of stage IIeIV diseases, based on the previously form the major structural element of gap junctions. published protocol.16 The method of RT was in general Connexins play important roles in the regulation of cell uniform within this period. All patients were regularly growth and differentiation. Cancer cells usually have monitored after RT until death or their last appointment downregulated levels of gap junctions, and several lines with the mean follow-up duration being 59.6 months of evidence suggest that loss of gap junctional intercel- (range: 4e117). lular communication is an important step in carcino- genesis. Re-expression of connexins in cancer cells causes Statistical analysis normalisation of cell growth control and reduced tumour Statistics were performed using SPSS V.14.0 software 2 growth.15 Accordingly, we aimed to systematically analyse (SPSS Inc). c Test was used to compare the EMP2 EMP2 immunoexpression in patients with NPC and expression status and various clinicopathological identified that loss of EMP2 expression is associated with parameters. The end points analysed were disease- adverse prognosticators, conferring to poor survivals. specific survival (DSS) and local recurrence-free survival (LRFS), calculated from the starting date of RT to the MATERIALS AND METHODS Patients and tumour specimens Table 1 Clinical pathological features of 124 The institutional review board approved the study by nasopharyngeal carcinomas using formalin-fixed tissue of NPC for this study (IRB100-09-003). Available paraffin-embedded tissue Variable n (%) http://bmjopen.bmj.com/ blocks were retrieved from 124 NPC patients who Gender underwent biopsy between January 1993 and December Male 95 (76.6) 2002. These patients were free of distant metastasis at Female 29 (23.4) initial presentation. The histological subtypes were Age (years) reappraised according to the current WHO classification <60 98 (79.0) $ and, the tumour staging was re-evaluated with the 7th 60 26 (21.0) American Joint Committee on Cancer (AJCC) system by Primary tumour (T) T1 30 (24.2) on September 29, 2021 by guest. Protected copyright. two pathologists, independently. T2 50 (40.3) Immunohistochemical staining and assessment of EMP2 T3 21 (16.9) T4 23 (18.5) expression m Nodal status (N) Tissue sections of 3 m thickness were cut onto N0 24 (19.4) precoated slides from paraffin-embedded tissue blocks N1 32 (25.8) and were next routinely deparaffinized with xylene and N2 48 (38.7) rehydrated with ethanol washes. Slides were heated by N3 20 (16.1) the microwave in a 10 mM citrate buffer (pH 6.0) for Stage 7 min to retrieve antigens. Endogenous peroxidase was I 7 (5.6) blocked with 3% H2O2. Slides were next washed by Tris- II 31 (25.0) buffered saline for 15 min and subsequently incubated III 46 (37.1) with a rabbit polyclonal primary antibody targeting IV 40 (32.2) Histological grade EMP2 (Atlas Antibodies, Stockholm, Sweden) at a dilu- Keratinising 5 (4.0) tion of 1:75 for 1 h. Primary antibodies were detected Non-keratinising/differentiated 54 (43.5) using the DAKO ChemMate EnVision Kit (K5001, Non-keratinising/undifferentiated 65 (52.4) Carpinteria, California, USA). The slides were incubated EMP2 expression level and developed with the secondary antibody for 30 min Positive (>5% tumour cells) 63 (50.8) and 3,3-diaminobenzidine for 5 min, followed by coun- Negative (#5% tumour cells) 61 (49.2) terstained using Gill’s haematoxylin. Immunoexpression 2 Chen Y-H, Wu L-C, Wu W-R, et al. BMJ Open 2012;2:e000900. doi:10.1136/bmjopen-2012-000900 EMP2 low expression in nasopharyngeal carcinoma BMJ Open: first published as 10.1136/bmjopen-2012-000900 on 5 April 2012. Downloaded from Figure 1 Immunohistochemically non-tumour respiratory epithelium (A) and those with squamous metaplasia (B) demonstrate diffuse and strong EMP2 immunoexpression, which can also be appreciated in representative non-keratinising carcinoma (C) but not in undifferentiated one (D). date of event developed. Patients lost to follow-up were showed characteristic EMP2 staining (>5% tumour cells; censored on the latest follow-up date.