Pathology – Research and Practice 210 (2014) 285–290
Contents lists available at ScienceDirect
Pathology – Research and Practice journal homepage: www.elsevier.com/locate/prp
Original Article
Expression of CD9 and CD82 in clear cell renal cell carcinoma and its clinical significance Hyeong Ju Kwon a , Sun Young Min a , Min Jee Park a , Cheol Lee a , Jeong Hwan Park a , Ji Youn Chae a , Kyung Chul Moon a,b,∗ a b
Department of Pathology, Seoul National University College of Medicine, Seoul, Republic of Korea Kidney Research Institute, Medical Research Center, Seoul National University College of Medicine, Seoul, Republic of Korea
a r t i c l e
i n f o
Article history: Received 10 September 2013 Received in revised form 7 January 2014 Accepted 14 January 2014 Keywords: CD9 CD82 Clear cell renal cell carcinoma Prognosis
a b s t r a c t CD9 and CD82, members of the tetraspanin family, act as metastasis suppressors in many human malignant tumors, but the role of these molecules is not well known in clear cell renal cell carcinoma (CCRCC). This study was designed to evaluate the immunohistochemical expression of CD9 and CD82 in 644 cases of CCRCC and to determine the clinicopathologic and prognostic significance of their expression. The percentage of positive tumor cells was evaluated, and the expression was classified into 2 categories: low expression (less than 10% positive cells) or high expression (more than 10% positive cells) for CD9 expression and negative (no positive cells) or positive for CD82 expression. CD9 high expression was found in 303 (47.0%) patients, and CD82 positivity was found in 98 (15.2%) patients. High expression of CD9 was statistically associated with older patients (p = 0.003). The cases showing positive immunoreactivity for CD82 exhibited a high stage (p < 0.001) and high nuclear grade (p < 0.001). The overall, cancer-specific and progression-free survival rates were significantly higher in patients with a CD82-negative profile compared to patients with a CD82-positive profile (p < 0.001). Although the biological function of CD82 in CCRCC remains unclear, the CCRCC patients with CD82 positive expression show poor prognosis. © 2014 Elsevier GmbH. All rights reserved.
Introduction Renal cell carcinoma (RCC) is the most prevalent adult renal epithelial cancer, accounting for approximately 90% of all renal malignancies [1]. RCC is a heterogeneous disease with distinct clinical, pathologic, and molecular characteristics and can be classified into several subtypes, i.e., clear cell, chromophobe, papillary, collecting duct, and other rare subtype. Clear cell RCC (CCRCC) is the most common cancer of the kidney, representing 70% of renal cell carcinomas and CCRCC tends to have a worse prognosis compared with other RCCs [2]. In spite of advances in diagnosis, especially improved imaging techniques and the incidental diagnosis of many tumors with imaging tests for unrelated complaints, approximately 30% of patients have metastatic disease at the time of diagnosis. In addition, approximately 20% of patients undergoing surgery will have a relapse and develop metastases [1]. The stage, Fuhrman grade, histological subtype and many histological features, such
∗ Corresponding author at: Department of Pathology and Kidney Research Institute, Medical Research Center, Seoul National University College of Medicine, Chongno-gu, Yongon-dong, Seoul 110-799, Republic of Korea. Tel.: +82 2 2072 1767; fax: +82 2 743 5530. E-mail addresses: [email protected], [email protected] (K.C. Moon). 0344-0338/$ – see front matter © 2014 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.prp.2014.01.004
as vascular invasion, necrosis and sarcomatoid differentiation, are well-known prognostic factors for RCC [3]. Tetraspanins are a family of cell surface membrane proteins that contain four transmembrane domains. These proteins combine with a variety of adhesion molecules, receptors and other transmembrane proteins to form clusters known as the tetraspanin web [4]. There are 33 tetraspanin members in humans, and tetraspanins are expressed in all cell types, each of which usually expresses multiple superfamily members [5], which are involved in the coordination of intercellular and intercellular processes, including cell adhesion, proliferation, signal transduction, differentiation and migration [4]. Tetraspanins are related to cell adhesion and for this reason, we can speculate that they may be associated with cancer metastasis. Indeed, it has recently been reported that the expression level of tetraspanins is associated with the metastasis of some cancers. CD9 and CD82, members of the tetraspanin family, act as a metastasis suppressors by inhibiting tumor cell motility [6]. The decrease of CD9 expression has been reported to be associated with the progression of breast, non-small cell carcinoma, stomach, colon and prostate cancer [6]. Moreover, the decrease of CD9 expression correlated with poor prognosis in some cancers, including colon, lung, breast, and ovarian carcinoma [6]. The decrease of CD82 expression also has been reported to be associated with
286
H.J. Kwon et al. / Pathology – Research and Practice 210 (2014) 285–290
the progression, metastasis or poor prognosis of breast cancer, gastrointestinal adenocarcinoma, penile squamous cell carcinoma, uterine carcinoma, prostate cancer and thyroid cancer [6–8]. Recently, it has been reported that CD9 is always expressed in chromophobe RCC and papillary RCC and expressed in almost half of all CCRCCs [9]. CD82 is expressed in most chromophobe RCCs and yet is not expressed in most oncocytomas and papillary and clear cell RCCs [10]. But the relationship between CD9 or CD82 expression and CCRCC metastasis or prognosis has not been reported. In a previous study [3], we investigated the expression of one of the tetraspanin superfamily members, CD151, in CCRCC and found that a high expression of CD151 is associated with metastasis and poor prognosis in CCRCC. Accordingly, we have speculated whether CD9 or CD82 is also associated with metastasis and poor prognosis in CCRCC. Materials and methods Patients and tissue specimens For the retrospective analysis, we collected the records of 644 clear cell renal cell carcinoma patients who had undergone radical or partial nephrectomy between 1995 and 2005 at the Seoul National University Hospital (Seoul, South Korea). The study was approved by the Seoul National University Hospital institutional review board. Samples were obtained from 473 men and 171 women, with a median age of 55.8 years (range 24–84 years). The median follow-up period was 88.1 months (range 2–223 months). The mean tumor size was 5.6 cm (range 0.8–22 cm). The clinicopathologic information was extracted from the medical records, pathologic reports and HE slide review. The histology type, tumor stage and nuclear grade were determined by pathologic review. The tumor stage was based on pTNM staging by the 2010 American Joint Committee on Cancer (AJCC), and nuclear grading was performed according to Fuhrman et al. [11]. The recurrence or metastasis of RCC was determined by clinical, radiological and pathological findings. The incidence of disease-related deaths was obtained from the medical records or death certificates. Tissue microarray and immunohistochemistry The H&E slides of all cases were reviewed, and the area that showed sufficient and fresh tumor with no hemorrhage or necrosis was selected in each case. One representative core tissue (2 mm in diameter) was obtained from individual paraffin blocks and placed in a new paraffin block using a trephine apparatus (Superbiochips Laboratories, Seoul, Korea). Additional 110 non-neoplastic renal parenchymal sections adjacent to the RCC from the patients were also included in the tissue microarray blocks. Immunohistochemical staining was performed using the Bond-Max Autostainer (Leica Microsystems, IL, USA). After heat-induced antigen retrieval, mouse monoclonal anti-human CD9 antibody (NovoCastra, Newcastle Upon Tyne, UK, dilution 1:250) and mouse monoclonal anti-human CD82 antibody (NovoCastra, dilution 1:50) were incubated with the samples for 15 min. The binding of the primary antibody was detected using the Bond Polymer Refine Detection kit (Leica Microsystems) according to the manufacturer’s instructions. The CD9 and CD82 immunoreactivity was scored by estimating the percentage of tumor cells with clear cytoplasmic or membranous immunostaining. The CD9 scores were dichotomized as low (0–10% positive cells) versus high (more than 10% positive cells), whereas the CD82 scores were dichotomized as negative (no positive cells) versus positive. The results were evaluated independently by two pathologists who were blinded to the outcome and score of the other observer.
Statistical analysis Statistical analyses were performed using SPSS software (version 19.0; SPSS Inc., Chicago, IL, USA). The correlations between the CD9 and CD82 expression and clinicopathologic characteristics were analyzed by Pearson’s X2 test. The overall survival (OS) was defined as the time interval between the primary radical or partial nephrectomy and the last follow-up or death. The cancer-specific survival (CSS) was defined as the time interval from the date of primary radical or partial nephrectomy to time of last follow-up or cancer-related death. The progression-free survival (PFS) period was defined as the time interval between the primary radical or partial nephrectomy and the last follow-up or evidence of recurrence or metastasis. The survival curves were plotted using the Kaplan–Meier method, and significance was determined using the univariate log-rank test. The parameters that were significant by the univariate analysis (p < 0.05) were entered into a multivariate Cox regression model to evaluate the increment statistical power and independence of prognostic impact. All of the statistical tests were two sided and p values less than 0.05 were considered statistically significant. Results CD9 and CD82 expression in CCRCC CD9 was present in the cytoplasm and/or membrane, and of the 644 CCRCC samples, 341 (53.0%) showed low CD9 immunopositivity, and 303 (47.0%) showed high immunopositivity (Fig. 1a and b). CD82 was also observed in the cytoplasm and/or membrane (Fig. 1c and d) and 546 (84.8%) cases showed negative staining for CD82 and 98 (15.2%) samples showed positive staining. In the non-neoplastic renal tissue specimens, all 110 sections showed no positivity for CD82, and CD9 was focally expressed in non-neoplastic renal tubular epithelial cells. Correlation between CD9, CD82 expression and clinicopathologic parameters To study the relationship between CD9 and, CD82 expression and clinicopathologic parameters, we analyzed 644 CCRCC cases (Table 1). Expression of CD9 was not statistically associated with any clinicopathologic parameters except patients’ age (p = 0.006). However positive immunostaining of CD82 was significantly associated with a high TNM stage (p < 0.001), high nuclear grade (p < 0.001), high pT (p < 0.001) and high pN category (p = 0.005). Correlation between CD9 and CD82 expression and prognosis After univariate analysis (Table 2), no prognostic implication was found between CD9 expression and the overall (p = 0.844), cancer-specific (p = 0.484) or progression-free survival (p = 0.829) in patients with CCRCC (Fig. 2a, c, e). The overall, cancer-specific and progression-free survival rates were significantly better in CD82 negative group compared with CD82 positive group (p < 0.001, Fig. 2b, d, f). Other conventional prognostic parameters, including the stage and nuclear grade were significantly correlated with the overall, cancer-specific survival and progression-free survival (p < 0.001). Multivariate analysis using the Cox proportional hazards model revealed that the stage (p < 0.001), nuclear grade (p < 0.001), and CD82 expression (p = 0.024) were independent prognostic factors of the overall survival in patients with CCRCC (Table 3). Conversely, CD82 expression was not an independent prognostic factor for the cancer-specific survival (p = 0.169) and progression-free survival (p = 0.322).
H.J. Kwon et al. / Pathology – Research and Practice 210 (2014) 285–290
287
Fig. 1. Immunohistochemical analysis of CD9 and CD82 expression in CCRCC: (A) CD9 low and (B) CD9 high expression. (C) CD82 negative and (D) CD82 positive expression. CD9 and CD82 expressed in cytoplasm and/or membrane of tumor cells. Original magnification ×200.
Table 1 Clinicopathologic characteristics of patients with CCRCC and correlations between CD9, CD82 expression and clinicopathologic parameters. CD82neg pT category pT1, pT2 pT3, pT4 pN category pN0 pN1 pM category pM0 pM1 Stage I, II III, IV Nuclear grade 1, 2 3, 4 Sex M F Age ≤55 years >55 years
CD82pos
p-Value
CD9low
CD9high
281 60
238 65
328 13
298 5
314 27
278 25
272 69
226 77
195 146
150 153
396 150
77 21
179 162
126 177
Contents lists available at ScienceDirect
Pathology – Research and Practice journal homepage: www.elsevier.com/locate/prp
Original Article
Expression of CD9 and CD82 in clear cell renal cell carcinoma and its clinical significance Hyeong Ju Kwon a , Sun Young Min a , Min Jee Park a , Cheol Lee a , Jeong Hwan Park a , Ji Youn Chae a , Kyung Chul Moon a,b,∗ a b
Department of Pathology, Seoul National University College of Medicine, Seoul, Republic of Korea Kidney Research Institute, Medical Research Center, Seoul National University College of Medicine, Seoul, Republic of Korea
a r t i c l e
i n f o
Article history: Received 10 September 2013 Received in revised form 7 January 2014 Accepted 14 January 2014 Keywords: CD9 CD82 Clear cell renal cell carcinoma Prognosis
a b s t r a c t CD9 and CD82, members of the tetraspanin family, act as metastasis suppressors in many human malignant tumors, but the role of these molecules is not well known in clear cell renal cell carcinoma (CCRCC). This study was designed to evaluate the immunohistochemical expression of CD9 and CD82 in 644 cases of CCRCC and to determine the clinicopathologic and prognostic significance of their expression. The percentage of positive tumor cells was evaluated, and the expression was classified into 2 categories: low expression (less than 10% positive cells) or high expression (more than 10% positive cells) for CD9 expression and negative (no positive cells) or positive for CD82 expression. CD9 high expression was found in 303 (47.0%) patients, and CD82 positivity was found in 98 (15.2%) patients. High expression of CD9 was statistically associated with older patients (p = 0.003). The cases showing positive immunoreactivity for CD82 exhibited a high stage (p < 0.001) and high nuclear grade (p < 0.001). The overall, cancer-specific and progression-free survival rates were significantly higher in patients with a CD82-negative profile compared to patients with a CD82-positive profile (p < 0.001). Although the biological function of CD82 in CCRCC remains unclear, the CCRCC patients with CD82 positive expression show poor prognosis. © 2014 Elsevier GmbH. All rights reserved.
Introduction Renal cell carcinoma (RCC) is the most prevalent adult renal epithelial cancer, accounting for approximately 90% of all renal malignancies [1]. RCC is a heterogeneous disease with distinct clinical, pathologic, and molecular characteristics and can be classified into several subtypes, i.e., clear cell, chromophobe, papillary, collecting duct, and other rare subtype. Clear cell RCC (CCRCC) is the most common cancer of the kidney, representing 70% of renal cell carcinomas and CCRCC tends to have a worse prognosis compared with other RCCs [2]. In spite of advances in diagnosis, especially improved imaging techniques and the incidental diagnosis of many tumors with imaging tests for unrelated complaints, approximately 30% of patients have metastatic disease at the time of diagnosis. In addition, approximately 20% of patients undergoing surgery will have a relapse and develop metastases [1]. The stage, Fuhrman grade, histological subtype and many histological features, such
∗ Corresponding author at: Department of Pathology and Kidney Research Institute, Medical Research Center, Seoul National University College of Medicine, Chongno-gu, Yongon-dong, Seoul 110-799, Republic of Korea. Tel.: +82 2 2072 1767; fax: +82 2 743 5530. E-mail addresses: [email protected], [email protected] (K.C. Moon). 0344-0338/$ – see front matter © 2014 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.prp.2014.01.004
as vascular invasion, necrosis and sarcomatoid differentiation, are well-known prognostic factors for RCC [3]. Tetraspanins are a family of cell surface membrane proteins that contain four transmembrane domains. These proteins combine with a variety of adhesion molecules, receptors and other transmembrane proteins to form clusters known as the tetraspanin web [4]. There are 33 tetraspanin members in humans, and tetraspanins are expressed in all cell types, each of which usually expresses multiple superfamily members [5], which are involved in the coordination of intercellular and intercellular processes, including cell adhesion, proliferation, signal transduction, differentiation and migration [4]. Tetraspanins are related to cell adhesion and for this reason, we can speculate that they may be associated with cancer metastasis. Indeed, it has recently been reported that the expression level of tetraspanins is associated with the metastasis of some cancers. CD9 and CD82, members of the tetraspanin family, act as a metastasis suppressors by inhibiting tumor cell motility [6]. The decrease of CD9 expression has been reported to be associated with the progression of breast, non-small cell carcinoma, stomach, colon and prostate cancer [6]. Moreover, the decrease of CD9 expression correlated with poor prognosis in some cancers, including colon, lung, breast, and ovarian carcinoma [6]. The decrease of CD82 expression also has been reported to be associated with
286
H.J. Kwon et al. / Pathology – Research and Practice 210 (2014) 285–290
the progression, metastasis or poor prognosis of breast cancer, gastrointestinal adenocarcinoma, penile squamous cell carcinoma, uterine carcinoma, prostate cancer and thyroid cancer [6–8]. Recently, it has been reported that CD9 is always expressed in chromophobe RCC and papillary RCC and expressed in almost half of all CCRCCs [9]. CD82 is expressed in most chromophobe RCCs and yet is not expressed in most oncocytomas and papillary and clear cell RCCs [10]. But the relationship between CD9 or CD82 expression and CCRCC metastasis or prognosis has not been reported. In a previous study [3], we investigated the expression of one of the tetraspanin superfamily members, CD151, in CCRCC and found that a high expression of CD151 is associated with metastasis and poor prognosis in CCRCC. Accordingly, we have speculated whether CD9 or CD82 is also associated with metastasis and poor prognosis in CCRCC. Materials and methods Patients and tissue specimens For the retrospective analysis, we collected the records of 644 clear cell renal cell carcinoma patients who had undergone radical or partial nephrectomy between 1995 and 2005 at the Seoul National University Hospital (Seoul, South Korea). The study was approved by the Seoul National University Hospital institutional review board. Samples were obtained from 473 men and 171 women, with a median age of 55.8 years (range 24–84 years). The median follow-up period was 88.1 months (range 2–223 months). The mean tumor size was 5.6 cm (range 0.8–22 cm). The clinicopathologic information was extracted from the medical records, pathologic reports and HE slide review. The histology type, tumor stage and nuclear grade were determined by pathologic review. The tumor stage was based on pTNM staging by the 2010 American Joint Committee on Cancer (AJCC), and nuclear grading was performed according to Fuhrman et al. [11]. The recurrence or metastasis of RCC was determined by clinical, radiological and pathological findings. The incidence of disease-related deaths was obtained from the medical records or death certificates. Tissue microarray and immunohistochemistry The H&E slides of all cases were reviewed, and the area that showed sufficient and fresh tumor with no hemorrhage or necrosis was selected in each case. One representative core tissue (2 mm in diameter) was obtained from individual paraffin blocks and placed in a new paraffin block using a trephine apparatus (Superbiochips Laboratories, Seoul, Korea). Additional 110 non-neoplastic renal parenchymal sections adjacent to the RCC from the patients were also included in the tissue microarray blocks. Immunohistochemical staining was performed using the Bond-Max Autostainer (Leica Microsystems, IL, USA). After heat-induced antigen retrieval, mouse monoclonal anti-human CD9 antibody (NovoCastra, Newcastle Upon Tyne, UK, dilution 1:250) and mouse monoclonal anti-human CD82 antibody (NovoCastra, dilution 1:50) were incubated with the samples for 15 min. The binding of the primary antibody was detected using the Bond Polymer Refine Detection kit (Leica Microsystems) according to the manufacturer’s instructions. The CD9 and CD82 immunoreactivity was scored by estimating the percentage of tumor cells with clear cytoplasmic or membranous immunostaining. The CD9 scores were dichotomized as low (0–10% positive cells) versus high (more than 10% positive cells), whereas the CD82 scores were dichotomized as negative (no positive cells) versus positive. The results were evaluated independently by two pathologists who were blinded to the outcome and score of the other observer.
Statistical analysis Statistical analyses were performed using SPSS software (version 19.0; SPSS Inc., Chicago, IL, USA). The correlations between the CD9 and CD82 expression and clinicopathologic characteristics were analyzed by Pearson’s X2 test. The overall survival (OS) was defined as the time interval between the primary radical or partial nephrectomy and the last follow-up or death. The cancer-specific survival (CSS) was defined as the time interval from the date of primary radical or partial nephrectomy to time of last follow-up or cancer-related death. The progression-free survival (PFS) period was defined as the time interval between the primary radical or partial nephrectomy and the last follow-up or evidence of recurrence or metastasis. The survival curves were plotted using the Kaplan–Meier method, and significance was determined using the univariate log-rank test. The parameters that were significant by the univariate analysis (p < 0.05) were entered into a multivariate Cox regression model to evaluate the increment statistical power and independence of prognostic impact. All of the statistical tests were two sided and p values less than 0.05 were considered statistically significant. Results CD9 and CD82 expression in CCRCC CD9 was present in the cytoplasm and/or membrane, and of the 644 CCRCC samples, 341 (53.0%) showed low CD9 immunopositivity, and 303 (47.0%) showed high immunopositivity (Fig. 1a and b). CD82 was also observed in the cytoplasm and/or membrane (Fig. 1c and d) and 546 (84.8%) cases showed negative staining for CD82 and 98 (15.2%) samples showed positive staining. In the non-neoplastic renal tissue specimens, all 110 sections showed no positivity for CD82, and CD9 was focally expressed in non-neoplastic renal tubular epithelial cells. Correlation between CD9, CD82 expression and clinicopathologic parameters To study the relationship between CD9 and, CD82 expression and clinicopathologic parameters, we analyzed 644 CCRCC cases (Table 1). Expression of CD9 was not statistically associated with any clinicopathologic parameters except patients’ age (p = 0.006). However positive immunostaining of CD82 was significantly associated with a high TNM stage (p < 0.001), high nuclear grade (p < 0.001), high pT (p < 0.001) and high pN category (p = 0.005). Correlation between CD9 and CD82 expression and prognosis After univariate analysis (Table 2), no prognostic implication was found between CD9 expression and the overall (p = 0.844), cancer-specific (p = 0.484) or progression-free survival (p = 0.829) in patients with CCRCC (Fig. 2a, c, e). The overall, cancer-specific and progression-free survival rates were significantly better in CD82 negative group compared with CD82 positive group (p < 0.001, Fig. 2b, d, f). Other conventional prognostic parameters, including the stage and nuclear grade were significantly correlated with the overall, cancer-specific survival and progression-free survival (p < 0.001). Multivariate analysis using the Cox proportional hazards model revealed that the stage (p < 0.001), nuclear grade (p < 0.001), and CD82 expression (p = 0.024) were independent prognostic factors of the overall survival in patients with CCRCC (Table 3). Conversely, CD82 expression was not an independent prognostic factor for the cancer-specific survival (p = 0.169) and progression-free survival (p = 0.322).
H.J. Kwon et al. / Pathology – Research and Practice 210 (2014) 285–290
287
Fig. 1. Immunohistochemical analysis of CD9 and CD82 expression in CCRCC: (A) CD9 low and (B) CD9 high expression. (C) CD82 negative and (D) CD82 positive expression. CD9 and CD82 expressed in cytoplasm and/or membrane of tumor cells. Original magnification ×200.
Table 1 Clinicopathologic characteristics of patients with CCRCC and correlations between CD9, CD82 expression and clinicopathologic parameters. CD82neg pT category pT1, pT2 pT3, pT4 pN category pN0 pN1 pM category pM0 pM1 Stage I, II III, IV Nuclear grade 1, 2 3, 4 Sex M F Age ≤55 years >55 years
CD82pos
p-Value
CD9low
CD9high
281 60
238 65
328 13
298 5
314 27
278 25
272 69
226 77
195 146
150 153
396 150
77 21
179 162
126 177
