|Year : 2015 | Volume
| Issue : 1 | Page : 223-228
Expression and clinical significance of aminopeptidase N/CD13 in non-small cell lung cancer
Quan Zhang1, Jinghui Wang1, Haiqing Zhang2, Dan Zhao2, Zongde Zhang3, Shucai Zhang1
1 Department of Medical Oncology, Beijing Chest Hospital, Capital Medical University, Tongzhou District, Beijing, China
2 Department of Pathology, Beijing Chest Hospital, Capital Medical University, Tongzhou District, Beijing, China
3 Department of Molecular Biology, Beijing Chest Hospital, Capital Medical University, Tongzhou District, Beijing, China
|Date of Web Publication||16-Apr-2015|
Department of Medical Oncology, Beijing Chest Hospital, Capital Medical University, Tongzhou District, Beijing
Source of Support: None, Conflict of Interest: None
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.
Keywords: Aminopeptidase N/CD13, immunohistochemistry, non-small cell lung cancer, prognosis
|How to cite this article:|
Zhang Q, Wang J, Zhang H, Zhao D, Zhang Z, Zhang S. Expression and clinical significance of aminopeptidase N/CD13 in non-small cell lung cancer. J Can Res Ther 2015;11:223-8
|How to cite this URL:|
Zhang Q, Wang J, Zhang H, Zhao D, Zhang Z, Zhang S. Expression and clinical significance of aminopeptidase N/CD13 in non-small cell lung cancer. J Can Res Ther [serial online] 2015 [cited 2019 Sep 17];11:223-8. Available from: http://www.cancerjournal.net/text.asp?2015/11/1/223/138007
| > 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). , 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 (EC126.96.36.199, APN), a membrane-bound glycoprotein whose N-end amino acid is cut-off, is a member of zinc-bound metalloprotease super family.  Look et al.  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 ,,] and participates in extracellular matrix degradation, cell migration, angiogenesis and neoplasm invasion. For ovarian, pancreatic, or gastric cancer ,,, patients, CD13 expression is correlated with their prognosis. Meanwhile, both in vitro and animal studies of ovarian carcinoma , 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 and beta-tubulin III.  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. , 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. 
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 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, <10%; 2, 11-15%; 3, 15-75%; 4, >75%. The product of the above two scores were used to assess the results. According to the published literature,  we defined that the score in the range of 4-12 represented positive, below 4 negative.
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 <0.05 was considered to be statistically significant.
| > Results|| |
CD13 protein was mainly expressed in cellular membrane, little in cytoplasm, as shown by immunohistochemistry. Among 127 cases of NSCLC tissue specimens, 72 showed positive CD13 expression, the positive rate being 56.7%. For squamous carcinoma, CD13 was mainly expressed in the interstitial cells, the positive rate being 62.3%. However, CD13 was mainly expressed in the parenchymal cells, the positive rate being 50% among adenocarcinoma cases [Figure 1] and [Figure 2]. APN was also observed on granulocytes, monocytes and macrophages in adjacent normal tissue of NSCLC patients, on Langhans type giant cells in lung tissue of lung tuberculosis patients and in connective tissue (especially just beneath the basement membrane of the bronchial epithelium) in normal lung tissue of patients with bronchiectasis [Figure 3],[Figure 4]a nd [Figure 5].
|Figure 2: Expression of CD13 in squamous cell lung carcinoma (a) ×100, (b) ×400|
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|Figure 3: Expression of CD13 in adjacent normal tissue in non-small cell lung cancer patients (a) ×100, (b) ×400|
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|Figure 4: Expression of CD13 on Langhans type giant cells in lung tissue of lung tuberculosis patients (a) ×100, (b) ×400|
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|Figure 5: Expression of CD13 in normal lung tissue of patients with bronchiectasis (a) ×100, (b) ×400|
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The correlations between clinicopathological/demographic characteristics (age, gender, smoking and clinical staging) and the expression of CD13 in NSCLC study subjects were summarized in [Table 1]. No significant association was observed between CD13 expression and patient's age, gender, smoking, tumor size (T classification) and status of distant metastasis (M classification) in 127 NSCLC cases. However, CD13 expression was positively correlated with the status of lymph node metastasis (N classification) (P = 0.009) and clinical stage (P = 0.041) in NSCLC patients [Table 2].
|Table 1: Clinicopathologic features of patient samples and expression of CD13 in NSCLC|
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|Table 2: Correlation between the clinicopathologic/ demographic characteristics and expression of CD13 in NSCLC|
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The correlations between clinicopathological, demographic characteristics (age, gender, smoking, tumor size, lymph node metastasis, clinical staging, histopathological types, and CD13 expression) and post-operative 5 year survival rate in NSCLC study subjects were summarized in [Table 3]. Patients with CD13 negative expression seemed to have higher post-operative 5 year survival rate. Significant difference was observed in either squamous or adecarcinoma patients.
|Table 3: The association between patient characteristics and overall survival|
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The median survival time (MST) of total enrolled 127 patients following adjuvant chemotherapy was 48 months. Among 69 patients with squamous carcinoma, 43 patients with positive CD13 expression showed a MST of 42 months. This was significantly shorter than those with negative CD13 expressions (61 months, P = 0.016) [Figure 6]. Cox regression analysis showed that TNM stage were the independent factors to impact on the post-operative survival of squamous carcinoma patients with adjuvant chemotherapy [Table 4]. However, among 58 patients with adenocarcinoma, 29 patients with positive CD13 expression showed a significantly shorter MST (33 months) than that of 29 patients with negative CD13 expression (61 months, P = 0.001) [Figure 7]. Cox regression analysis showed that TNM stage and CD13 expression were the independent factors to impact on survival of lung adenocarcinoma patients with adjuvant chemotherapy [Table 5].
|Figure 6: Effect of CD13 expression on the survival of squamous carcinoma patients with adjuvant chemotherapy|
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|Figure 7: Effect of CD13 expression on the survival of lung adenocarcinoma patients with adjuvant chemotherapy|
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|Table 4: Multivariate Cox analysis of overall survival of 69 patients with squamous carcinoma|
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|Table 5: Multivariate Cox analysis of overall survival of 58 patients with adenocarcinoma|
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| > Discussion|| |
APN or CD13 is directly digested by protease and participated in diverse pathophysiological procedures such as invasion and metastasis of cancer cells, angiogenesis, immunoregulation, signal transduction and viral infection.  In the present study, total 127 squamous cell lung carcinoma and adenocarcinoma surgical specimens were subject to immunohistochemical examination. In squamous carcinoma tissues, CD13 was mainly expressed in interstitial cells, seldom in parenchyma cells. However, CD13 was mainly expressed in parenchyma cells among adenocarcinoma tissues. CD13 was expressed mainly on cellular membrane, seldom in cytoplasm in both squamous carcinoma and adenocarcinoma tissues due to the molecular structure of the protein.
Tokuhara et al.  studied the expression conditions of CD13 in 27 lung cancer cell lines by using flow cytometry and real-time polymerase chain reaction (RT-PCR). Their RT-PCR results indicated that adenocarcinoma cell lines presented positive CD13 expression, confirming the findings of flow cytometry. Tokuhara et al.  investigated expression of CD10 and CD13 in NSCLC and demonstrated that CD10 messenger ribonucleic acid (mRNA) and CD13 mRNA expressed in both squamous carcinoma and adenocarcimona tissues were correlated with the prognosis of the disease. Ichimura et al.  analyzed the tissue arrays of American population by using immunohistochemistry and revealed that CD13 was expressed in the interstitium of squamous carcinoma, but in the parenchyma of adenocarcinoma and adenosquamous carcinoma. They also investigated CD13 expression in Japanese patients with squamous cell lung carcinoma and confirmed that positive CD13 was located in the fibroblasts and vascular endothelial cells at interstitial tissues. It seems that the immunohistochemical staining of CD13 could be used to distinguish squamous cell lung carcinoma from adenocarcinoma.
It is recognized that cancer cells and adhered histocytes can release diverse proteases to participate in the degradation of basilar membrane and extracellular matrix, thus promoting the invasion and metastasis of malignancy.  As early as 1993, Saiki et al.  revealed the effect of CD13 in tumor: CD13 enhanced invasion of malignancy and degradation of extracellular matrix. Chang et al.  also demonstrated that CD13 could enhance the invasive ability of lung cancer cells through interacting with tumor-associated surface antigen L6. Saiki et al.  indicated that the malignant cells with more highly expressed CD13 could degrade collagen protein intravenous more effectively, thus impairing the basilar membrane and extracellular matrix and promoting the invasion of the malignancy. In this study, it was demonstrated that CD13 expression was correlated with lymph node status. The higher the N stage in which the patients were located, the higher the CD13 positive rate was. This indicated that CD13 played an important role in the invasion and metastasis of NSCLC. However, further analysis based on the pathological type showed a different result. For squamous carcinoma, CD13 expression was significantly correlated with lymph node status. However, it was not the case for adenocarcinoma. This may be due to the expression position of CD13 in the two pathological types of lung cancers. In squamous carcinoma, CD13 was mainly expressed in the fibroblasts and vascular endothelial cells in the interstitial tissues, which, when compared with the condition in adenocarcinoma, may account for enhanced capacity for the invasion and metastasis of the malignancy.
In recent studies conducted in solid tumors such as NSCLC, pancreatic, ovarian, or colonal cancer, ,,, the correlations between CD13 expression and prognosis were suggested, indicating that over-expression of CD13 in cancer tissue represented poor prognosis. In vitro and animal studies on ovarian cancer , showed that over-expression of CD13 could impair the sensitivity of ovarian cancer cells to Cisplatin, a chemotherapeutic drug. CD13 inhibitor could enhance the sensitivity of ovarian cancer cells to Paclitaxel, another chemotherapeutic drug. As demonstrated by two double-blinded prospective trials, , adjuvant medication of a strong CD13 inhibitor (Bestatin) could prolong the survival of patients with either resectable or unresectable lung squamous carcinoma. The study findings indicated that the survival of patients with low expression of CD13 was longer than those with high expression of CD13 no matter in squamous carcinoma or adenocarcinoma group. This may be explained by: (a) CD13 over-expression decreased the sensitivity of cancer cells to Cisplatin and anti-microtubule reagents; (b) Expressed CD13 helped the invasion and metastasis of cancer cells and promoted the recurrence of the tumor, hence affecting the prognosis. Therefore, expression of CD13 in tissues was an effective variable to evaluate the prognosis of NSCLC patients under post-operative adjuvant chemotherapy. CD13 inhibitor may be used to enhance the sensitivity of cancer cells to chemotherapeutics, suppress angiogenesis of cancers by affecting the CD13 expression and offer clinical benefit to NSCLC patients.
| > Conclusion|| |
Our study demonstrated that: (a) CD13 is mainly expressed in the interstitial cells of squamous carcinoma and in the parenchyma cells of adenocarcinoma in the tissue of NSCLC; (b) expression of CD13 is significantly correlated with the lymph node metastasis status and stage of NSCLC; (c) CD13 may be an important prognostic factor for NSCLC: over-expression of CD13 indicates poor prognosis of NSCLC; (d) Expression of CD13 is an independent impact factor to lung adenocarcinoma patients. The work demonstrated that inhibition of CD13 activity is a new approach for improving the therapeutic efficacy of chemotherapy for NSCLC patients. Due to the limited sample size of investigational subjects, further clinical research would be required to confirm these findings and establish further the CD13 role as a reliable clinical biomarker for predicting the patient outcomes of NSCLC.
| > Acknowledgment|| |
This study was initiated and performed by the Department of Medical Oncology, Beijing Chest Hospital, Capital Medical University. The secretarial assistance at the Department of Pathology and Department of Molecular Biology, is greatly appreciated.
| > References|| |
Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011;61:69-90.
Non-Small Cell Lung Cancer Collaborative Group. Chemotherapy in non-small cell lung cancer: A meta-analysis using updated data on individual patients from 52 randomised clinical trials. Non-small Cell Lung Cancer Collaborative Group. BMJ 1995;311:899-909.
Antczak C, De Meester I, Bauvois B. Ectopeptidases in pathophysiology. Bioessays 2001;23:251-60.
Look AT, Ashmun RA, Shapiro LH, Peiper SC. Human myeloid plasma membrane glycoprotein CD13 (gp150) is identical to aminopeptidase N. J Clin Invest 1989;83:1299-307.
Ashmun RA, Look AT. Metalloprotease activity of CD13/aminopeptidase N on the surface of human myeloid cells. Blood 1990;75:462-9.
Riemann D, Kehlen A, Langner J. CD13 - Not just a marker in leukemia typing. Immunol Today 1999;20:83-8.
Alliot F, Rutin J, Leenen PJ, Pessac B. Pericytes and periendothelial cells of brain parenchyma vessels co-express aminopeptidase N, aminopeptidase A, and nestin. J Neurosci Res 1999;58:367-78.
Tokuhara T, Hattori N, Ishida H, Hirai T, Higashiyama M, Kodama K, et al
. Clinical significance of aminopeptidase N in non-small cell lung cancer. Clin Cancer Res 2006;12:3971-8.
Ikeda N, Nakajima Y, Tokuhara T, Hattori N, Sho M, Kanehiro H, et al
. Clinical significance of aminopeptidase N/CD13 expression in human pancreatic carcinoma. Clin Cancer Res 2003;9:1503-8.
Hashida H, Takabayashi A, Kanai M, Adachi M, Kondo K, Kohno N, et al
. Aminopeptidase N is involved in cell motility and angiogenesis: Its clinical significance in human colon cancer. Gastroenterology 2002;122:376-86.
Surowiak P, Drag M, Materna V, Suchocki S, Grzywa R, Spaczyñski M, et al
. Expression of aminopeptidase N/CD13 in human ovarian cancers. Int J Gynecol Cancer 2006;16:1783-8.
van Hensbergen Y, Broxterman HJ, Rana S, van Diest PJ, Duyndam MC, Hoekman K, et al
. Reduced growth, increased vascular area, and reduced response to cisplatin in CD13-overexpressing human ovarian cancer xenografts. Clin Cancer Res 2004;10:1180-91.
Yamashita M, Kajiyama H, Terauchi M, Shibata K, Ino K, Nawa A, et al
. Involvement of aminopeptidase N in enhanced chemosensitivity to paclitaxel in ovarian carcinoma in vitro
and in vivo
. Int J Cancer 2007;120:2243-50.
Zhang S, Li Q, Zhang Q, Wang J, Zhang H, Zhang Z, et al
. Expression of ERCC1 and class III ß-tubulin in resected non-small cell lung cancer and its correlation with platinum-based adjuvant chemotherapy. Int J Biol Markers 2010;25:141-9.
Curnis F, Cattaneo A, Longhi R, Sacchi A, Gasparri AM, Pastorino F, et al
. Critical role of flanking residues in NGR-to-isoDGR transition and CD13/integrin receptor switching. J Biol Chem 2010;285:9114-23.
Ito S, Miyahara R, Takahashi R, Nagai S, Takenaka K, Wada H, et al
. Stromal aminopeptidase N expression: Correlation with angiogenesis in non-small-cell lung cancer. Gen Thorac Cardiovasc Surg 2009;57:591-8.
Wickström M, Larsson R, Nygren P, Gullbo J. Aminopeptidase N (CD13) as a target for cancer chemotherapy. Cancer Sci 2011;102:501-8.
Grujiæ M, Renko M. Aminopeptidase inhibitors bestatin and actinonin inhibit cell proliferation of myeloma cells predominantly by intracellular interactions. Cancer Lett 2002;182:113-9.
Amãlinei C, Cãruntu ID, Bãlan RA. Biology of metalloproteinases. Rom J Morphol Embryol 2007;48:323-34.
Tokuhara T, Adachi M, Hashida H, Ishida H, Taki T, Higashiyama M, et al
. Neutral endopeptidase/CD10 and aminopeptidase N/CD13 gene expression as a prognostic factor in non-small cell lung cancer. Jpn J Thorac Cardiovasc Surg 2001;49:489-96.
Ichimura E, Yamada M, Nishikawa K, Abe F, Nakajima T. Immunohistochemical expression of aminopeptidase N (CD13) in human lung squamous cell carcinomas, with special reference to bestatin adjuvant therapy. Pathol Int 2006;56:296-300.
Chambers AF, Matrisian LM. Changing views of the role of matrix metalloproteinases in metastasis. J Natl Cancer Inst 1997;89:1260-70.
Saiki I, Fujii H, Yoneda J, Abe F, Nakajima M, Tsuruo T, et al
. Role of aminopeptidase N (CD13) in tumor-cell invasion and extracellular matrix degradation. Int J Cancer 1993;54:137-43.
Chang YW, Chen SC, Cheng EC, Ko YP, Lin YC, Kao YR, et al
. CD13 (aminopeptidase N) can associate with tumor-associated antigen L6 and enhance the motility of human lung cancer cells. Int J Cancer 2005;116:243-52.
Ichinose Y, Genka K, Koike T, Kato H, Watanabe Y, Mori T, et al
. Randomized double-blind placebo-controlled trial of bestatin in patients with resected stage I squamous-cell lung carcinoma. J Natl Cancer Inst 2003;95:605-10.
Furuse K, Fukuoka M, Genka K, Kato H, Tsubura E, Ohta M, et al
. Double blind controlled study of ubenimex (Bestatin) against squamous cell lung cancer - A multicenter cooperative study. Gan To Kagaku Ryoho 1993;20:1187-94.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]