Potential therapeutic target and independent prognostic marker of TROP2 in laryngeal squamous cell carcinoma

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1 ORIGINAL ARTICLE Potential therapeutic target and independent prognostic marker of TROP2 in laryngeal squamous cell carcinoma Hao Wu, PhD, 1 Huiming Xu, MS, 1 Shu Zhang, MS, 2 Xudong Wang, PhD, 3 Huijun Zhu, 2 Huilin Zhang, MD, 4 Jin Zhu, PhD, 5 * Jianfei Huang, MD, PhD 2 * 1 Department of Otorhinolaryngology/Head and Neck Surgery, Nantong University Hospital, Nantong, Jiangsu, China, 2 Department of Clinical Pathology, Nantong University Hospital, Nantong, Jiangsu, China, 3 Department of Surgical Comprehensive Laboratory, Nantong University Hospital, Nantong, Jiangsu, China, 4 Department of Clinical Pathology, Nanjing Maternity and Child Health Care Hospital, Nanjing, Jiangsu, China, 5 Department of Pathology, Key Laboratory of Antibody Technique of Ministry of Health, Huadong Medical Institute of Biotechniques, Nanjing, Jiangsu, China. Accepted 10 July 2012 Published online 18 September 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI /hed ABSTRACT: Background. The human trophoblastic cell surface antigen 2 (TROP2) gene is associated with the development of malignancies, but its expression in laryngeal squamous cell carcinoma (SCC) and its relationship with clinical characteristics of the disease remain undetermined. Methods. Expression of TROP2 protein was detected by immunohistochemistry with a self-made anti-trop2 antibody in laryngeal SCC tissue microarrays. Results. Elevated expression of TROP2 was detected in laryngeal SCC tissues compared with adjacent noncancerous tissues. TROP2 expression in laryngeal SCC was related to tumor differentiation (p ¼.0001) and lymph node metastasis (p ¼.0352). Cox regression analyses confirmed that TROP2 expression (p ¼.015), lymph node metastasis (p ¼.001), degree of differentiation (p ¼.002), tumor site (p ¼.021), and T classification (p ¼.003) were independent prognostic factors. Conclusions. TROP2 can be used as an independent prognostic indicator for laryngeal SCC. VC 2012 Wiley Periodicals, Inc. Head Neck 35: , 2013 KEY WORDS: TROP2, laryngeal neoplasms, squamous cell carcinoma, prognosis, tumor marker INTRODUCTION Laryngeal squamous cell carcinoma (SCC) is a common type of head and neck malignancy, and it is the second most common malignancy in the respiratory tract. 1 3 Tumor stage, pathologic differentiation, primary site, and lymph node metastasis of laryngeal SCC are correlated with prognosis. These indexes, however, are insufficient for precise assessment of the tumor. Therefore, a thorough investigation of potential molecular biomarkers in predicting laryngeal SCC prognosis is of great significance. 4 6 Human trophoblastic cell surface antigen 2 (TROP2) gene protein is a single-transmembrane surface glycoprotein, which participates in regulation of intracellular concentration of calcium ions. 7,8 TROP2 was first discovered in human trophoblast cells 9,10 and found to be associated with regulation of cyclin D1 and protein kinase C (PKC) levels, which activate the mitogen-activated protein *Corresponding author: J. Zhu, Department of Pathology, Key Laboratory of Antibody Technique of Ministry of Health, Huadong Medical Institute of Biotechniques, Nanjing, China. zjsimmons@yahoo.com.cn; J. Huang, Department of Clinical Pathology, Nantong University Affiliated Hospital, Nantong, China. ntyydoctor@126.com Contract grant sponsor: Science and Technique Development Fund, Nanjing, Jiangsu, China; contract grant number: ; contract grant sponsor: Medical Science and Technique Development Fund, Nanjing, Jiangsu, China; contract grant number: ZKX09015; contract grant sponsor: Doctoral Fund, Nantong University, Nantong, Jiangsu, China; contract grant number: kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway to increase expression of cyclin D1 and cyclin E. In that case, it adjusts the growth of tumor cells and favors the invasion and metastasis of the malignancy. 11,12 Previous studies have reported low-level or no expression of TROP2 in normal human tissues, but high expression in malignant ones, which is relevant to the development, invasion, lymph node metastasis, and prognosis of various respiratory and alimentary tract tumors However, TROP2 expression and its prognostic role in laryngeal SCC have not yet been explained. We detected the expression of TROP2 mrna in fresh laryngeal SCC tissues via 1-step quantitative reverse transcription polymerase chain reaction (RT-PCR; qpcr). In addition, we prepared tissue microarrays of laryngeal SCC, and determined the expression of TROP2 in laryngeal SCC and corresponding adjacent tissues using immunohistochemistry with self-made anti-trop2 antibody, 21 and analyzed its relationship with clinicopathologic parameters of patients with laryngeal SCC. MATERIALS AND METHODS Clinical information and preparation of tissue microarrays All hospitalized patients between January 2002 and May 2009 were enrolled from the Department of Otolaryngology/Head and Neck Surgery, Nantong University Hospital, China. All patients were diagnosed with HEAD & NECK DOI /HED OCTOBER

2 WU ETAL. laryngeal SCC in accord with the World Health Organization (WHO) criteria 22 and TNM (tumor, node, metastasis) classification (Union Internationale Contre le Cancer [UICC] 2002) and underwent total or partial laryngectomy and neck dissection (unilateral or bilateral, radical or functional, based on clinical and surgical findings). Nodal metastasis was confirmed by postoperative histologic examination. In this study, 109 paraffin-fixed tissue samples of laryngeal SCC and 28 control samples were prepared as tissue microarrays. Another 15 fresh laryngeal SCCs and surrounding normal tissues were collected as controls. None of the patients had received preoperative radiotherapy or chemotherapy. Complete follow-up data until May 2010 were documented in all these cases. Tissue microarrays were produced by Shanghai Xinchao Bio-tech Co. Ltd. The representative cancer area was labeled in specific paraffin blocks in accord with hematoxylin and eosin staining results. A tissue array needle was inserted to obtain a 2-mm-diameter tissue sample, with 1 core for each sample. The tissue was then sequentially aligned into the preprepared blank paraffin blocks. The tissue microarrays were cut into 4-lm sections and placed on tissue microarray specific adhesive-coated glass slides. Ethical approval to perform this study was obtained from the Human Research Ethics Committee of the local hospital. One-step quantitative polymerase chain reaction The primers of TROP2 (NM_002353) and endogenous control GAPDH (NM_002046) were designed with the software Primer Premier 5.0. All primers were synthesized by Shanghai Sangon Biological Engineering Technology and Services. Total RNA was extracted from 15 laryngeal SCC samples and their adjacent peritumoral tissues using the TRIzol reagent (Invitrogen, Carlsbad, CA). Expressions of TROP2 and GAPDH were determined by real-time PCR with IQ5 (Bio-Rad, Hercules, CA) using SensiMix One-Step Kit based SYBR Green method (Quantace, London, UK). The primers for TROP2 were as follows: forward primer 5 0 -TAT TAC CTG GAC GAG ATT CCCC-3 0 and reverse primer 5 0 -CCC CGA CTT TCT CCG GTTG-3 0, for GAPDH, forward primer TGC ACC ACCAAC TGC TTA GC-3 0 and reverse primer 3 0 -GGC ATG GAC TGT GGT CAT GAG-5 0. Amplification conditions consisted of 30 minutes at 42 C for reverse transcription and 2 minutes at 94 C for Taq activation, followed by 35 cycles of 95 C for 20 seconds, 56 C for 20 seconds, and elongation at 72 C for 30 seconds. Each measurement was performed in triplicate. Immunohistochemical staining and scoring The tissue microarray slides for immunohistochemistry were deparaffinized, and peroxidase was quenched with methanol and 3% H 2 O 2 for 15 minutes. For antigen retrieval, the sections were boiled under pressure in citrate buffer, ph 6.0, for 3 minutes. Self-made anti-trop2 monoclonal antibody 21 was added and incubated, containing 1% bovine serum albumin, for 1 hour. Following washing with PBS, sections were incubated with horseradish peroxidase conjugated goat anti-mouse antibody (Dako Cytomation, Carpinteria, CA) for 15 minutes and then washed. The color was developed by a 15-minute incubation with diaminobenzidine solution (Kem-En-Tec Diagnostics, Taastrup, Denmark), and sections were weakly counterstained with hematoxylin. Negative control reactions used PBS instead of the primary antibody. The results of immunohistochemistry were determined with a double-blind method and the staining results were evaluated under an optical microscope by 2 pathologists. Discordant cases were reevaluated under a double-headed microscope to achieve a consensus. In the case of disagreement, the slides were reviewed by a third pathologist until a consensus score was established. Expression levels of TROP2 protein were assessed by observing the incidence and staining intensity of immunohistochemically positive cells, as described previously. 23,24 The incidence of positive cells was scored as follows: negative as 0; 1% to 10% positive cells as 1 point; 10% to 50% positive cells as 2 points; and >50% positive cells as 3 points. Staining intensity was scored as: no color as 0; yellow for weak positive as 1 point; light brown for medium positive as 2 points; and brown for strong positive as 3 points. The 2 components were produced to obtain an overall expression score, as follows: 0 as ( ); 1 3 as (þ); 4 6 as(þþ); and 9 as (þþþ). Statistical analysis The SPSS15.0 software package was used for statistical analysis. Statistical analyses were performed with a paired t test for comparison of TROP2 mrna expression. A rank-sum test was used for analysis of the distinction between TROP2 expression in laryngeal SCC tissues and in adjacent noncancerous tissues, as well as the correlation between TROP2 and clinicopathologic parameters. Survival curves (overall and disease-free survival) were calculated using the Kaplan Meier method, and the logrank test was used for analysis. Multivariate analysis was performed using Cox proportional hazards model; the risk ratio and its 95% confidence interval were recorded for each marker. Probability values < 0.05 were considered statistically significant in all of the analyses. RESULTS Patient demographics and tumor clinicopathologic characteristics Among 109 patients (107 men and 2 women; mean age, 60.8 years; age range, years), 28 patients (25.7%) presented with supraglottic laryngeal SCC and 81 (74.3%) with glottic laryngeal SCC. The distribution of T classification was as follows: 72 patients (66.1%) had small (T1 2) tumors and 37 (33.9%) had local advanced (T3 4) tumors. Nineteen cases (17.4%) were complicated with lymph node metastasis. Regarding the histologic differentiation, there were 51 patients (46.8%) with well-differentiated, 49 (45.0%) with moderately differentiated, and 9 (8.2%) with poorly differentiated tumors. In terms of distribution of TNM classification, 13 patients (11.9%) were found with stage I, 52 (47.7%) with stage II, 33 (30.3%) with stage III, and 11 (10.1%) with stage IV tumors. Patients were divided into 2 groups by the total exposure (pack-years) to cigarette smoking 25 : 41 patients (37.6%) with <60 pack-years were grouped 1374 HEAD & NECK DOI /HED OCTOBER 2013

3 PROGNOSTIC MARKER OF TROP2 IN LARYNGEAL SCC significant inverse correlation between positive expression of TROP2 protein and degree of differentiation of carcinoma (p ¼.0001) or lymphatic metastasis (p ¼.0352). In contrast, no significant correlation was found between sex, age, smoking, tumor site, T classification, and TNM classification. FIGURE 1. TROP2 mrna expression in laryngeal squamous cell carcinoma (SCC) and adjacent nontumorous tissues with a different level for the 1-step qpcr result. Expression of TROP2 mrna in laryngeal SCC tissues ( ) was higher than that in adjacent nontumorous tissues ( ) (t ¼ 1.75, p ¼.008), when compared with the GAPDH internal control. together and the other group consisted of 68 patients (62.4%) with >60 pack-years. All 109 cases of laryngeal SCC shared an average follow-up time of months; median follow-up time of 35.1 months; 5-year overall survival of 67.2% (41/61); and 5-year disease-free survival of 59.0% (36/61). Results of quantitative polymerase chain reaction qpcr was used to detect expression of TROP2 mrna in laryngeal SCC and corresponding adjacent tissues. Expression of TROP2 mrna in laryngeal SCC tissues ( ) was higher than that in adjacent nontumorous tissues ( ) (t ¼ 1.75, p ¼.008), when compared with GAPDH internal control mrna (Figure 1). Immunohistochemistry results TROP2 expressed in the cytoplasmic membrane was presented as brown particles, with no positive signals detected in stroma and most adjacent noncancerous tissues (Figure 2). Comparison between TROP2 protein expression rate of 89.0% (97/109) in tumor tissues and 50.0% (14/28) in adjacent noncancerous tissues was of statistical significance (Z ¼ 5.765, p ¼.0000). The immunohistochemistry results confirmed higher expression of TROP2 in laryngeal SCC tissues than that in the noncancerous tissues, which was consistent with the qpcr results mentioned earlier. Relationship between TROP2 protein expression and clinical parameters The relationship between levels of TROP2 protein and the clinicopathologic parameters of laryngeal SCC is shown in Table 1. The rank-sum test analysis showed a Correlation of TROP2 protein expression and clinicopathologic parameters with lifespan Univariate analysis showed correlation of smoking (p ¼.0274), tumor site (p ¼.0035), tumor differentiation (p ¼.0079), T classification (p ¼.0009), TNM classification (p ¼.0008), lymph node metastasis (p <.0001) and positive TROP2 status (p ¼.0030) with lifespan of patients with laryngeal SCC. A multivariate Cox proportional hazards regression model was used to analyze smoking, tumor site, lymph node metastasis, histologic differentiation, T classification, TNM classification, and positive TROP2 expression. Positive TROP2 expression (p ¼.002), tumor site (p ¼.021), lymph node metastasis (p ¼.004), poor differentiation (p ¼.001), T classification (p ¼.003), and TNM classification (p ¼.015) were independent prognostic factors for poor prognosis in laryngeal SCC (Table 2). Kaplan Meier survival curves showed that patients with positive TROP2 expression had a shorter survival time (overall and disease-free survival) than those with negative CHOP2 expression (Figure 3). DISCUSSION Lipinski et al 9,26 discovered the relationship between TROP2 protein and embryonic development and neoplastic growth, via the study of normal and neoplastic human trophoblasts. Previous studies 13,15 20 have demonstrated high TROP2 expression in various carcinoma tissues (oral, colon, pancreatic, gastric, and ovarian carcinoma) and low or no expression in normal tissues. Additionally, high expression of TROP2 is related to the development, progression, and nodal metastasis of the malignancy, which suggests TROP2 as a prognostic marker of carcinoma. 13,17 20 In the present study, we detected expression of TROP2 mrna in fresh laryngeal carcinoma and adjacent noncancerous tissues using qpcr and found a significantly higher level in carcinoma tissues, which was consistent with the study of Ornachi et al, 15 in which the expression of TROP2 mrna in colorectal cancer tissues was detected. For further proof, we prepared tissue microarrays with laryngeal carcinoma specimens. Immunohistochemistry demonstrated higher TROP2 protein expression in laryngeal SCC than that in adjacent noncancerous tissues, which indicated that TROP2 promoted development of laryngeal carcinoma. This was similar to the study of Fong et al 13 on buccal cavity squamous cell carcinoma. Previous studies have clarified a close relationship between the degree of histologic differentiation of laryngeal carcinoma and patient survival rates Well-differentiated tumors have a limited ability for invasion, but once they have infiltrated the adjacent vessels or nerves, prognosis is probably poor. 30 It has been demonstrated previously that lymph node metastasis and patient survival rates are associated. 31 Our study demonstrated the HEAD & NECK DOI /HED OCTOBER

4 WU ETAL. FIGURE 2. Hematoxylin eosin staining and expression pattern of TROP2 protein in laryngeal SCC and adjacent nontumorous tissues with tissue microarray sections. (A1) Well-differentiated laryngeal SCC tissue pattern with hematoxylin eosin staining. (A2, A3) Positive immunostaining of TROP2 in laryngeal SCC tissue. (B1) Moderately differentiated laryngeal SCC tissue pattern with hematoxylin eosin staining. (B2, B3) Expression pattern of TROP2 in laryngeal SCC with strongly positive immunohistochemical reaction. (C1) Tumor-adjacent tissue pattern with hematoxylin eosin staining. (C2) Tumor-adjacent tissue pattern with weakly positive immunohistochemistry staining of TROP2. (C3) Squamous epithelium of adjacent nontumorous tissue with negative immunohistochemical staining of TROP2 (original magnification 40 in A1, A2, B1, B2, C1, and C2; 400 in A3, B3, and C3). association of TROP2 protein expression with laryngeal SCC histologic differentiation and lymph node metastasis, which was consistent with the immunohistochemical study of pancreatic cancer by Fong and colleagues. 16 In that study, TROP2 protein was overexpressed in tumor tissues in 55% of the patients, which was associated with lymph node metastasis and degree of tumor malignancy, and further resulted in poor prognosis. Bignotti et al 20 analyzed patients with ovarian cancer by qpcr and immunohistochemistry, and confirmed that TROP2 is an independent prognostic factor. These studies have indicated the possible promotion of tumor invasion and metastasis by TROP2. Fornaro et al 32 have suggested that the TROP2 gene may be a cell surface signal transduction factor that regulates proliferation of tumor cells. Ripani et al 7 have found that TROP2 is a calcium-channel associated membrane protein, and specific antibody cross-linking of TROP2 leads to a 2 to 3 times higher level of cytoplasmic calcium ions. TROP2 regulates intracellular calcium concentration through the cytoplasmic tail region and PKC, which has a major role in the signal transduction process. Phosphorylation at serine residue 303 or 1376 HEAD & NECK DOI /HED OCTOBER 2013

5 PROGNOSTIC MARKER OF TROP2 IN LARYNGEAL SCC TABLE 1. Relationship between TROP2 expression and clinical parameters. Clinical parameter n Total positives (%) þ þþ þþþ Z value* p value* Sex Male (88.8) Female 2 2 (100) Age, y < (84.4) (92.2) Smoking (pack-years) < (82.9) (92.6) Tumor site Supraglottis (89.3) Glottis (88.9) Histologic differentiation Well (78.4) Moderately and poorly (98.3) T classification T1 T (88.9) T3 T (89.2) Lymph node metastasis No (87.8) Yes (94.7) TNM classification I II (89.2) III IV (88.6) * The Z and p values were calculated by rank-sum test., þ, þþ and þþþ were defined in immunohistochemical staining and scoring of materials and methods. receptor activation leads to release of calcium ions from the endoplasmic reticulum. This initiates positive feedback that activates more PKC, as well as the Raf pathway and nuclear factor-jb, which leads to increased gene transcription and cell proliferation. 8 The above-mentioned studies have indicated an important role for TROP2 expression in progression of laryngeal SCC. TABLE 2. Parameter Univariate and multivariate analyses of prognostic variables. Univariate* Multivariate p value p value Risk ratio 95% CI Sex Male/Female.5724 Age, y <60/ Smoking (pack-years) <60/ Tumor site Supraglottis/Glottis Histologic differentiation Well/Mod and poorly T classification T1 2/T Lymph node metastasis No/Yes TNM classification I II/III IV TROP2 Negative ( )/Positive (þ þþþ) * Statistical analyses were performed by log-rank test. Statistical analyses were performed by Cox regression model. In our study, the Kaplan Meier analysis demonstrated that the lifespan of patients with positive TROP2 expression was shorter than that of patients with negative expression. Univariate and multivariate analyses showed that positive TROP2 expression, smoking, degree of tumor differentiation, tumor site, T classification, TNM classification, and lymph node metastasis were correlated with lifespan of patients with laryngeal SCC. Among these factors, positive TROP2 expression, degree of tumor differentiation, tumor site, T classification, TNM classification, and lymph node metastasis were independent prognostic factors affecting laryngeal cancer. This confirms the regulatory effect of TROP2 in tumor cell growth, invasion, and metastasis found in previous studies, 11,12,33 and the poor prognosis in TROP2-positive patients. 13,17 20 There were 2 limitations to the present study that need to be acknowledged. First, the sample size was small and, in particular, only a small number of patients had lymph node metastasis. Second, most samples stained positive for TROP2, making comparison with TROP2-negative samples difficult. CONCLUSIONS Elevated expression of TROP2 in laryngeal SCC tissues has a close correlation with the pathologic differentiation and nodal metastasis of the tumor. TROP2 may play a role as a prognostic indicator of survival in patients with laryngeal SCC. The self-made anti-trop2 antibody can be used in targeted therapy of laryngeal SCC, which may merit further investigation. HEAD & NECK DOI /HED OCTOBER

6 WU ETAL. FIGURE 3. Survival of patients with laryngeal SCC by expression of CHOP2, with the Kaplan Meier method. (A) Overall survival rate in patients with positive expression of TROP2 (deep green line) was significantly lower than that in patients with negative TROP2 expression (blue line). (B) Disease-free survival rate in patients with positive expression of TROP2 (deep green line) was significantly lower than that in patients with negative TROP2 expression (blue line). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] REFERENCES 1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, CA Cancer J Clin 2009;59: Hoffman HT, Porter K, Karnell LH, et al. Laryngeal cancer in the United States: changes in demographics, patterns of care, and survival. Laryngoscope 2006;116: Argiris A, Eng C. Epidemiology, staging, and screening of head and neck cancer. 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