[Downloaded free from http://www.cancerjournal.net on Sunday, May 10, 2015, IP: 78.188.217.115]
Original Article
Expression and clinical significance of aminopeptidase N/CD13 in non‑small cell lung cancer ABSTRACT Background: The objective of this study was to analyze the expression of aminopeptidase N/CD13 (APN/CD13) in non‑small cell lung cancer (NSCLC) and investigate its correlation with various clinical factors, including prognosis and efficacy of adjuvant chemotherapy. Materials and Methods: Using immunohistochemistry analysis, we analyzed the expression of CD13 in clinicopathologically characterized 127 NSCLC cases. The relationship between the expression levels of CD13 and clinical features was analyzed and presented. Statistical Analysis: The data were analyzed using statistical package for the social sciences software (Ver 13.0, IBM, USA). Those conforming to Gauss distribution criteria was represented as Mean ± SD and those not conforming to Gauss distribution criteria was represented as median (M). The overall survival was recorded from the date of surgery to the date of cancer‑specific death. APN/ CD13 expression levels and clinicopathological factors were analyzed by Chi‑square test or by Fisher’s exact test. The Kaplan‑Meier method was used to evaluate the probability of survival data and analyzed by Log rank test. Multivariate analysis was performed by Cox regression model. P < 0.05 was considered to be statistically significant. Results: APN/CD13 was mainly expressed in the cellular membrane of cancer cells in pulmonary adenocarcinoma and in the cellular membrane of interstitial cells in squamous carcinoma. Positive APN/CD13 was detected in 62.3% (43 of 69) squamous carcinoma patients and in 50% (29/58) adenocarcinoma patients. Expression of APN/CD13 was not correlated with age, gender, tumor, node, metastasis (TNM) stage, histological type and tumor size, but with TNM stage (P = 0.041) and lymph node metastasis status (P = 0.009). As indicated by Kaplan‑Meier survival curve, over‑expression of APN/CD13 was significantly correlated with the low survival rate. Cox regression analysis showed that APN/CD13 expression was an independent impact factor for the survival of lung adenocarcinoma patients receiving adjuvant chemotherapy (P = 0.006). Conclusions: Expression of APN/CD13 is a potential unfavorable factor to predict the efficacy and prognosis of post‑operative chemotherapy in NSCLC patients, especially in lung adenocarcinoma patients. KEY WORDS: Aminopeptidase N/CD13, immunohistochemistry, non‑small cell lung cancer, prognosis
INTRODUCTION Lung cancer is the leading cause of cancer‑related deaths in the world. Approximately 85% of all lung cancer cases are categorized as non‑small cell lung cancer (NSCLC).[1,2] Unfortunately, most NSCLC patients tend to present a more advanced stage of disease when first diagnosed due to its deep location and non‑specific symptoms. Therefore, it is of great value to search valuable biomarkers for early diagnosis, predict prognosis and develop novel therapeutic strategies. Aminopeptidase N (EC3.4.11.2, APN), a membrane‑bound glycoprotein whose N‑end amino acid is cut‑off, is a member of zinc‑bound metalloprotease super family. [3] Look et al. [4] confirmed that APN and CD13 were identically the
same protein by contrasting their complementary deoxyribonucleic acid clonal sequence. CD13 is expressed in many kinds of tissues and cells including intestine, proximal renal tubule, breasts, secretory epithelium of bronchial gland, colonal mucosa, and synaptic membrane of central nerve system[5‑7] and participates in extracellular matrix degradation, cell migration, angiogenesis and neoplasm invasion. For ovarian, pancreatic, or gastric cancer[8‑11] patients, CD13 expression is correlated with their prognosis. Meanwhile, both in vitro and animal studies of ovarian carcinoma[12,13] showed that over‑expression of CD13 could impair the sensitivity of cancer cells to cisplatin and that administering CD13 inhibitor or suppressing expression of CD13 protein could improve the sensitivity of cancer cells to paclitaxel alike to excision repair cross‑complementation group 1
Journal of Cancer Research and Therapeutics - January-March 2015 - Volume 11 - Issue 1
Quan Zhang, Jinghui Wang, Haiqing Zhang1, Dan Zhao1, Zongde Zhang2, Shucai Zhang Departments of Medical Oncology, 1 Pathology, and 2 Molecular Biology, Beijing Chest Hospital, Capital Medical University, Tongzhou District, Beijing, China For correspondence: Dr. Shucai Zhang, Department of Medical Oncology, Beijing Chest Hospital, Capital Medical University, 97 Machang Road, Tongzhou District, Beijing 101149, China. E‑mail: zhangshucai6304@ yahoo.com.cn
Access this article online Website: www.cancerjournal.net DOI: 10.4103/0973-1482.138007 PMID: *** Quick Response Code:
223
[Downloaded free from http://www.cancerjournal.net on Sunday, May 10, 2015, IP: 78.188.217.115]
Zhang, et al.: Expression and clinical significance of APN/CD13 in non‑small cell lung cancer
and beta‑tubulin III.[14] Furthermore several studies found that CD13 expressed in the tumor neovasculature and the tumor‑homing peptides containing Asn‑Gly‑Arg motif could interact with CD13.[15,16] Due to this property, these peptides have been exploited for ligand‑directed delivery of various drugs and particles to tumor vessels, in the attempt to increase their antitumor activity. Therefore, CD13‑target therapy may offer a new way for cancer chemotherapy.[17] In this study, we investigated expression of CD13 in NSCLC tissue and its correlations with gender, age, smoking, pathological type, Tumor, Node, Metastasis (TNM) stage, lesion features, and evaluated the relationship between CD13 expression and post‑operative prognosis with adjuvant chemotherapy in NSCLC patients. MATERIALS AND METHODS A total of 127 NSCLC patients with surgical treatment in our Hospital from 2002 to 2004 were enrolled following written informed consent. The pathological types of resected tumor were confirmed as either adenocarcinoma or squamous carcinoma. In all 127 patients, there were 91 male and 36 female with age ranging from 36 to 78 years (median, 57.9 years). A total of 58 patients were confirmed with adenocarcinoma and 69 squamous carcinoma. TNM staging following International Union Against Cancer 1997 were listed as follows: Stage I (35), Stage II (23), Stage IIIA (52) and Stage IIIB (17). All the surgical specimens were then fixed in 10% formalin, embedded in paraffin and made into 4 µm thick sections for immunohistochemical staining. All patients were treated with 4‑6 cycles of cisplatin/pacclitaxel or cisplatin/ vinorelbine as adjuvant chemotherapy. After the preliminary experiment was performed following the kit’s instruction, the positive section in preliminary experiment and sections omitting primary antibody treatment were respectively used as positive and negative control. Sections were deparaffininized in 100% xylene and re‑hydrated in descending ethanol series and water according to standard protocols before the antigen retrieval was performed under high pressure. The sections were then incubated in peroxidase inhibitor at 25°C for 5 min to block endogenous peroxidase activity, followed by incubation with 50 μl CD13 monoclonal antibody diluted to 1:5 with a final concentration of 20 μg/ml (Novocastra Company, Newcastle, UK) for overnight at 4°C. They were then incubated in sealant and in NovoLinkTM polymer respectively at 37°C for 15 min. Sections were processed further for microscopic examination. Independent pathologists respectively reviewed and scored the immunohistochemically stained tissue sections. They would discuss and decide the final result if their opinions were inconsistent. The results were assessed using the following semi‑quantitative scoring system: 0, color was light and consistent with that of the background; 1, color presented as 224
light yellow and a little darker than the background; 2, color presented as a heavy yellow and significantly darker than the background; 3, color presented as brown. 400 cancer cells were counted under the high power lens and the scoring system was established according to the percentage of the positive cells: 0, negative; 1, 75%. The product of the above two scores were used to assess the results. According to the published literature,[18] we defined that the score in the range of 4‑12 represented positive, below 4 negative. Statistical analysis The data were analyzed using Statistical Package for the Social Sciences 13.0 software. Those conforming to Gauss distribution criteria was represented as mean ± SD and those not conforming to Gauss distribution criteria was represented as median (M). The overall survival was recorded from the date of surgery to the date of cancer‑specific death. APN/ CD13 expression levels and clinicopathological factors were analyzed by Chi‑square test or by Fisher’s exact test. The Kaplan‑Meier method was used to evaluate the probability of survival data and analyzed by Log rank test. Multivariate analysis was performed by Cox regression model to study the effects of different clinicopathological factors (APN/CD13 expression, gender, age at surgery, pathological stage, tumor status, nodal status, metastatic status, histopathological types) on overall survival. P
Original Article
Expression and clinical significance of aminopeptidase N/CD13 in non‑small cell lung cancer ABSTRACT Background: The objective of this study was to analyze the expression of aminopeptidase N/CD13 (APN/CD13) in non‑small cell lung cancer (NSCLC) and investigate its correlation with various clinical factors, including prognosis and efficacy of adjuvant chemotherapy. Materials and Methods: Using immunohistochemistry analysis, we analyzed the expression of CD13 in clinicopathologically characterized 127 NSCLC cases. The relationship between the expression levels of CD13 and clinical features was analyzed and presented. Statistical Analysis: The data were analyzed using statistical package for the social sciences software (Ver 13.0, IBM, USA). Those conforming to Gauss distribution criteria was represented as Mean ± SD and those not conforming to Gauss distribution criteria was represented as median (M). The overall survival was recorded from the date of surgery to the date of cancer‑specific death. APN/ CD13 expression levels and clinicopathological factors were analyzed by Chi‑square test or by Fisher’s exact test. The Kaplan‑Meier method was used to evaluate the probability of survival data and analyzed by Log rank test. Multivariate analysis was performed by Cox regression model. P < 0.05 was considered to be statistically significant. Results: APN/CD13 was mainly expressed in the cellular membrane of cancer cells in pulmonary adenocarcinoma and in the cellular membrane of interstitial cells in squamous carcinoma. Positive APN/CD13 was detected in 62.3% (43 of 69) squamous carcinoma patients and in 50% (29/58) adenocarcinoma patients. Expression of APN/CD13 was not correlated with age, gender, tumor, node, metastasis (TNM) stage, histological type and tumor size, but with TNM stage (P = 0.041) and lymph node metastasis status (P = 0.009). As indicated by Kaplan‑Meier survival curve, over‑expression of APN/CD13 was significantly correlated with the low survival rate. Cox regression analysis showed that APN/CD13 expression was an independent impact factor for the survival of lung adenocarcinoma patients receiving adjuvant chemotherapy (P = 0.006). Conclusions: Expression of APN/CD13 is a potential unfavorable factor to predict the efficacy and prognosis of post‑operative chemotherapy in NSCLC patients, especially in lung adenocarcinoma patients. KEY WORDS: Aminopeptidase N/CD13, immunohistochemistry, non‑small cell lung cancer, prognosis
INTRODUCTION Lung cancer is the leading cause of cancer‑related deaths in the world. Approximately 85% of all lung cancer cases are categorized as non‑small cell lung cancer (NSCLC).[1,2] Unfortunately, most NSCLC patients tend to present a more advanced stage of disease when first diagnosed due to its deep location and non‑specific symptoms. Therefore, it is of great value to search valuable biomarkers for early diagnosis, predict prognosis and develop novel therapeutic strategies. Aminopeptidase N (EC3.4.11.2, APN), a membrane‑bound glycoprotein whose N‑end amino acid is cut‑off, is a member of zinc‑bound metalloprotease super family. [3] Look et al. [4] confirmed that APN and CD13 were identically the
same protein by contrasting their complementary deoxyribonucleic acid clonal sequence. CD13 is expressed in many kinds of tissues and cells including intestine, proximal renal tubule, breasts, secretory epithelium of bronchial gland, colonal mucosa, and synaptic membrane of central nerve system[5‑7] and participates in extracellular matrix degradation, cell migration, angiogenesis and neoplasm invasion. For ovarian, pancreatic, or gastric cancer[8‑11] patients, CD13 expression is correlated with their prognosis. Meanwhile, both in vitro and animal studies of ovarian carcinoma[12,13] showed that over‑expression of CD13 could impair the sensitivity of cancer cells to cisplatin and that administering CD13 inhibitor or suppressing expression of CD13 protein could improve the sensitivity of cancer cells to paclitaxel alike to excision repair cross‑complementation group 1
Journal of Cancer Research and Therapeutics - January-March 2015 - Volume 11 - Issue 1
Quan Zhang, Jinghui Wang, Haiqing Zhang1, Dan Zhao1, Zongde Zhang2, Shucai Zhang Departments of Medical Oncology, 1 Pathology, and 2 Molecular Biology, Beijing Chest Hospital, Capital Medical University, Tongzhou District, Beijing, China For correspondence: Dr. Shucai Zhang, Department of Medical Oncology, Beijing Chest Hospital, Capital Medical University, 97 Machang Road, Tongzhou District, Beijing 101149, China. E‑mail: zhangshucai6304@ yahoo.com.cn
Access this article online Website: www.cancerjournal.net DOI: 10.4103/0973-1482.138007 PMID: *** Quick Response Code:
223
[Downloaded free from http://www.cancerjournal.net on Sunday, May 10, 2015, IP: 78.188.217.115]
Zhang, et al.: Expression and clinical significance of APN/CD13 in non‑small cell lung cancer
and beta‑tubulin III.[14] Furthermore several studies found that CD13 expressed in the tumor neovasculature and the tumor‑homing peptides containing Asn‑Gly‑Arg motif could interact with CD13.[15,16] Due to this property, these peptides have been exploited for ligand‑directed delivery of various drugs and particles to tumor vessels, in the attempt to increase their antitumor activity. Therefore, CD13‑target therapy may offer a new way for cancer chemotherapy.[17] In this study, we investigated expression of CD13 in NSCLC tissue and its correlations with gender, age, smoking, pathological type, Tumor, Node, Metastasis (TNM) stage, lesion features, and evaluated the relationship between CD13 expression and post‑operative prognosis with adjuvant chemotherapy in NSCLC patients. MATERIALS AND METHODS A total of 127 NSCLC patients with surgical treatment in our Hospital from 2002 to 2004 were enrolled following written informed consent. The pathological types of resected tumor were confirmed as either adenocarcinoma or squamous carcinoma. In all 127 patients, there were 91 male and 36 female with age ranging from 36 to 78 years (median, 57.9 years). A total of 58 patients were confirmed with adenocarcinoma and 69 squamous carcinoma. TNM staging following International Union Against Cancer 1997 were listed as follows: Stage I (35), Stage II (23), Stage IIIA (52) and Stage IIIB (17). All the surgical specimens were then fixed in 10% formalin, embedded in paraffin and made into 4 µm thick sections for immunohistochemical staining. All patients were treated with 4‑6 cycles of cisplatin/pacclitaxel or cisplatin/ vinorelbine as adjuvant chemotherapy. After the preliminary experiment was performed following the kit’s instruction, the positive section in preliminary experiment and sections omitting primary antibody treatment were respectively used as positive and negative control. Sections were deparaffininized in 100% xylene and re‑hydrated in descending ethanol series and water according to standard protocols before the antigen retrieval was performed under high pressure. The sections were then incubated in peroxidase inhibitor at 25°C for 5 min to block endogenous peroxidase activity, followed by incubation with 50 μl CD13 monoclonal antibody diluted to 1:5 with a final concentration of 20 μg/ml (Novocastra Company, Newcastle, UK) for overnight at 4°C. They were then incubated in sealant and in NovoLinkTM polymer respectively at 37°C for 15 min. Sections were processed further for microscopic examination. Independent pathologists respectively reviewed and scored the immunohistochemically stained tissue sections. They would discuss and decide the final result if their opinions were inconsistent. The results were assessed using the following semi‑quantitative scoring system: 0, color was light and consistent with that of the background; 1, color presented as 224
light yellow and a little darker than the background; 2, color presented as a heavy yellow and significantly darker than the background; 3, color presented as brown. 400 cancer cells were counted under the high power lens and the scoring system was established according to the percentage of the positive cells: 0, negative; 1, 75%. The product of the above two scores were used to assess the results. According to the published literature,[18] we defined that the score in the range of 4‑12 represented positive, below 4 negative. Statistical analysis The data were analyzed using Statistical Package for the Social Sciences 13.0 software. Those conforming to Gauss distribution criteria was represented as mean ± SD and those not conforming to Gauss distribution criteria was represented as median (M). The overall survival was recorded from the date of surgery to the date of cancer‑specific death. APN/ CD13 expression levels and clinicopathological factors were analyzed by Chi‑square test or by Fisher’s exact test. The Kaplan‑Meier method was used to evaluate the probability of survival data and analyzed by Log rank test. Multivariate analysis was performed by Cox regression model to study the effects of different clinicopathological factors (APN/CD13 expression, gender, age at surgery, pathological stage, tumor status, nodal status, metastatic status, histopathological types) on overall survival. P
