Urologic Oncology: Seminars and Original Investigations 32 (2014) 1272–1276
Original article
A novel marker ADAM17 for clear cell renal cell carcinomas: Implication for patients’ prognosis Guorong Li, M.D., Ph.D.a,e,*, Fabien Forest, M.D.b, Gang Feng, Ph.D., M.D.c, Anne Gentil-Perret, M.D.b, Michel Péoc'h, M.D., Ph.D.b, Michèle Cottier, M.D., Ph.D.d,e, Nicolas Mottet, M.D., Ph.D.a,e a
Department of Urology, North Hospital, CHU de Saint-Etienne, University of Jean-Monnet, Saint-Etienne, France Department of Pathology, North Hospital, CHU de Saint-Etienne, University of Jean-Monnet, Saint-Etienne, France c Clinical Genetics Laboratory, Yijishan Hospital of Wannan Medical College, Wuhu, China d Department of Cytology, North Hospital, CHU de Saint-Etienne, University of Jean-Monnet, Saint-Etienne, France e LINA, EA 4624, Faculty of Medicine, University of Jean-Monnet, Saint-Etienne, France
b
Received 10 April 2014; received in revised form 22 May 2014; accepted 23 May 2014
Abstract Introduction: A disintegrin and metalloproteinase-17 (ADAM17) plays an important role in biological activity in different cancers. Its expression and prognostic value have not been studied in clear cell renal cell carcinoma (cRCC). The objective of this study was to explore the prognostic value of ADAM17 in patients with cRCC. Materials and methods: A total of 131 patients with cRCC were studied. There were 90 men and 41 women, with an average age of 67 years (range: 34–93 y). There were 110 patients with localized disease and 21 patients with metastatic disease. The expression of ADAM17 was evaluated by immunohistochemistry with a monoclonal antibody. The follow-up varied from 4.2 to 184 months, with a median of 72 months for patients with localized disease. Kaplan-Meier with a log-rank test was performed to compare the progression-free survival after surgery. The univariate and multivariate analyses were performed to examine the significance of ADAM17 expression for the patient’s progression-free survival. Results: The ADAM17 expression was found in 109/131 tumors. The ADAM17 expression was found in 20/21 metastatic tumors and in 89/110 localized tumors. Regarding patients with localized tumors, 31 patients experienced a recurrence or death during follow-up. The Kaplan-Meier analysis revealed that the high expression of ADAM17 was associated with a reduced progression-free survival (P ¼ 0.005). The univariate logistic regression analysis indicated that the high expression of ADAM17 was associated with the disease progression (hazard ratio ¼ 2.826; 95% CI: 1.324–6.034; P ¼ 0.007). The high expression of ADAM17 remained a significant factor for decreased progression-free survival in multivariate analysis. Conclusion: ADAM17 was frequently expressed in cRCC. The high expression of ADAM17 was correlated with a worse outcome for patients with cRCC. ADAM17 is a new biomarker for the management of patients with cRCC. r 2014 Elsevier Inc. All rights reserved.
Keywords: ADAM17; Prognosis; Renal cell carcinoma; Immunohistochemistry
1. Introduction A disintegrin and metalloproteinase-17 (ADAM17) is a member of the ADAM family, which plays an important This research was financed by the CHU de Saint-Etienne (AOL 0908126), France. * Corresponding author. Tel.: þ33-47-782-8814; fax: þ33-47-782-8464. E-mail address: [email protected] (G. Li). http://dx.doi.org/10.1016/j.urolonc.2014.05.011 1078-1439/r 2014 Elsevier Inc. All rights reserved.
role in biological activities of malignant lesion through epidermal growth factor receptor (EGFR)/phosphoinositide 3-kinase/protein kinase B (AKT) pathway [1,2]. This matrix metalloproteinaselike enzyme is involved in the release of several ligands. All the ligands released by ADAM17 have been implicated in tumor formation or progression or both. These ligands include transforming growth factor (TGF)-α, amphiregulin, heparin-binding epidermal growth factor, epiregulin, and tumor necrosis factor (TNF)-α. Another
G. Li et al. / Urologic Oncology: Seminars and Original Investigations 32 (2014) 1272–1276
function of ADAM17 is its implication in tumor formation or progression [2]. ADAM17-catalyzed shedding of TGF-α was found to be necessary for murine tumor formation [3] whereas inhibition of ADAM17 prohibited the malignancy formation [4]. ADAM17 was recently demonstrated to be overexpressed in several cancer tissues. The incidence of renal cell carcinoma (RCC) is increasing steadily. Despite the fact that small tumors are found frequently, one-third of patients with renal cancer present a metastatic disease at the time of diagnosis, and 30% to 40% of patients with localized renal cancer will develop a metastasis after surgery [5]; 40% of patients with RCC will die from metastasis [6]. These data indicate that prognosis prediction is a major issue for follow-up of patients. At present, the prediction of prognosis relies on solely the clinical stage and grade. Efficient prognostic markers are urgently needed to guide the surveillance and to find the at-risk patients for targeted therapy. Like other cancers, the process of formation and progression in renal carcinoma is through complicated molecular interactions. Many pathways are involved in this process. Several models have suggested the evidence that ADAM17 may participate in the tumorigenesis of renal cancer or other renal disease [7,8]. However, there has been no information concerning its prognostic value in clear cell RCC (cRCC). Therefore, we conducted a clinical study to explore its prognostic value in cRCC. To our best knowledge, this is the first study exploring the clinical significance of ADAM17 in cRCC. 2. Materials and methods 2.1. Patients and tumor samples A total of 131 patients with cRCC were included. There were 90 men and 41 women, with an average age of 67 years (range: 34–93 y). The tumors were staged according to Union for International Cancer Control/American Joint Commission on Cancer TNM staging system and graded according to Fuhrman criteria [9,10]. All patients benefited regularly from the surveillance after surgery. The surveillance included the image examination, blood creatinine level measurement, and physical examination. The histological slides were reviewed for the confirmation of diagnosis of clear cell histology and of grade determination by a urologic pathologist (A.G.P.). The corresponding blocks were used for the immunohistochemical study. Consent was obtained from each patient for the future study of the tumor tissues when the patients were hospitalized. This research was approved by the local ethics committee. 2.2. Immunohistochemical staining for ADAM17 Immunohistochemical staining was performed as per our previous study [11]. Paraffin-embedded, 5-μm thick tissue
1273
sections from all primary tumors were stained for the ADAM17 protein using a primary mouse monoclonal antibody (Abcam, ab57484; Cambridge, UK). Slides were deparaffinized through a series of xylene baths. Rehydration was performed through graded alcohols. Endogenous peroxidase activity was blocked in ChemMate PeroxidaseBlocking solution (DakoCytomation) 3 times every 5 minutes. The sections were then passed through the automated immuno-staining machine for a standard immunohistochemical protocol. Diaminobenzidine was used as a chromogen, and commercial hematoxylin was used for counterstaining. For each immunohistochemical staining, positive and negative controls were used. ADAM17 expression was evaluated at the membrane and cytoplasmic level. The percentages of positive cells (high Z 85%, moderate 50%–85%, weak o 50%, and absence ¼ 0%) were counted to define an expression level. Immunohistochemical analysis was performed by 2 urologic pathologists (F.F. and M.P.) in a blinded manner with respect to the clinical data concerning the subjects. For heterogeneous staining, ADAM17-expressing cells were counted in several representative high-power microscopic fields. Divergent scores were then discussed and decided on a multiheaded microscope. Finally, a high or low expression level was given to each sample. 2.3. Statistical analysis Survival was calculated by the Kaplan-Meier method, and the resulting curves were compared using the log-rank test. The univariate logistic regression analysis and the multivariate analysis by the Cox regression model were performed. P o 0.05 was considered to be statistically significant. 3. Results 3.1. Patient characteristics and survival The stage and grade of patients’ tumors are summarized in Table 1. The follow-up varied from 4.2 to 184 months, with a median of 72 months for patients with localized tumors. For patients with localized disease, 31 patients experienced a recurrence or death during the follow-up. 3.2. ADAM17 expression in cRCC: Pattern of expression The ADAM17 was found in 109/131 tumors. The expression of ADAM17 was located in the membrane (Fig. 1). Expression in the cytoplasm could be found in 20 tumors in combination with expression in the membrane. The distribution of high expression of ADAM17 in the different stage and grade is summed in Table 1. The high expression of ADAM17 was found in 13/39 T1a and in 15/26 T1b tumors. The expression of ADAM17 was found
1274
G. Li et al. / Urologic Oncology: Seminars and Original Investigations 32 (2014) 1272–1276
Table 1 Patients' characteristics and ADAM17 expression Characteristics
No. (%)
ADAM17 expression High
Low
85 (64.9) 46 (35.1)
43 22
42 24
Age, y Z68 o67
54 (41.2) 77 (58.8)
28 36
26 41
T classification T1 T2 T3
65 (49.6) 8 (6.1) 58 (44.3)
28 4 34
37 4 24
Fuhrman grade 1 2 3 4
25 59 39 8
15 30 22 5
10 29 17 3
Total patients Sex Male Female
131
(19) (45) (29.7) (6.3)
epidermal growth factor, and epiregulin [1,2]. In renal cancer, an experiment showed that ectodomain shedding of CA9 was regulated by TNF-α-converting enzyme/ ADAM17 [15]. Another experiment showed that ADAM17 was involved in tumor formation of renal cancer [7]. This experiment found that the metalloprotease ADAM17 played a pivotal role in several acquired tumor cell capabilities by mediating the availability of soluble TGF-α, an EGFR ligand, and thus the establishment of a key autocrine signaling pathway [7]. Silencing of ADAM17 in human renal carcinoma cell lines corrected critical features associated with cancer cells, including growth autonomy, tumor inflammation, and tissue invasion. Highly malignant renal carcinoma cancer cells failed to form in vivo tumors in the absence of ADAM17. From the aforementioned 2 experiments, we can imagine that ADAM17 actively participates in biological activity of renal cancer. Therefore, the study of ADAM17 expression in cRCC is meaningful. Nevertheless, immunohistochemical study of ADAM17 in patients with
in 20/21 metastatic tumors and in 89/110 localized tumors. Among the positive staining of ADAM17 for the localized tumors, the high, moderate, and weak expressions was found respectively in 52/110, 26/110, and 11/110 cases. 3.3. Correlation of ADAM17 expression with progression-free survival for localized patients The Kaplan-Meier analysis of progression-free survival was shown in Fig. 2. During the follow-up, 31 events of death or metastasis were found for the 110 patients with localized tumors. The Kaplan-Meier analysis revealed that the high expression of ADAM17 was associated with a reduced progression-free survival (P ¼ 0.005). By using the univariate logistic regression analysis, the high expression of ADAM17 was associated with a shorter progression-free survival (hazard ratio ¼ 2.826; 95% CI: 1.324–6.034; P ¼ 0.007). ADAM17 expression and T category remained significant factors for decreased progression-free survival in the multivariate Cox regression analysis (Table 2). 4. Discussion ADAM17 has been found to be up-regulated in many cancers. ADAM17 is becoming a new cancer diagnostic and prognostic biomarker [12–14]. It exerts active roles in malignancy formation and progression. ADAM17 was originally identified by its ability to release membranebound TNF-α from its precursor. ADAM17 is thus sometimes referred to as TNF-α-converting enzyme [4]. Other growth factors shown to be released by ADAM17 include the EGFR ligands, TGF-α, amphiregulin, heparin-binding
Fig. 1. Illustration of immunohistochemical staining of tumor cells in cRCC: (A) membrane staining (anti-ADAM17, 60) and (B) negative staining. (Color version of figure is available online.)
G. Li et al. / Urologic Oncology: Seminars and Original Investigations 32 (2014) 1272–1276
Fig. 2. Progression-free survival according to ADAM17 expression. The high expression of CXCR4 was associated with a reduced progression-free survival (P ¼ 0.005, log-rank test).
cRCC has been rare. In fact, we failed in finding any report concerning its immunohistochemical staining in cRCC. In the present study, we used a monoclonal antibody to study its expression in cRCC. We found that ADAM17 was located mainly in the membrane of tumor cells. ADAM17 was frequently expressed in metastatic cRCC as well as in localized cRCC. Taken together, we assume that ADAM17 signaling is an important event during malignancy transformation and progression of cRCC. The prediction of prognosis of patients with cRCC is based only on the pathological stage and grade. The biomarkers are urgently needed to guide for patient surveillance after surgery. Unfortunately, there have been no biomarkers that can be used in clinic for cRCC. Because
Table 2 Univariate and multivariate analyses for the patient's progression-free survival Univariate analysis HR
95% CI
Multivariate analysis P value HR
95% CI
1275
ADAM17 plays an important role in biological activity of cancers, it is reasonable to explorer its prognostic value in cRCC. We found that the high expression of ADAM17 was a predictive factor of progression-free survival for patients with localized cRCC. Therefore, ADAM17 may be a new biomarker for the surveillance after surgery for patients with localized cRCC. Some articles have been recently published on the prognostic value of ADAM17 in other cancers. It seems that a high expression of ADAM17 is associated with poor outcome in patients with various cancers [12–14]. We believe that ADAM17 is a new promising prognostic marker in patients with cRCC. ADAM17 is a new therapeutic target in cancer [16]. Another clinical implication of ADAM17 signaling in cRCC may be its therapeutical potential. Since 2006, molecular targeted therapy has changed treatment strategy for metastatic renal cancer [17]. The targeted therapy showed better results over the traditional immunotherapy [18]. The targeted therapy provides a 50% response rate. At present, there are 6 targeted agents approved for treatment in metastatic RCC: sorafenib, sunitinib, pazopanib, bevacizumab (in combination with interferon alpha), temsirolimus, and everolimus [17]. ADAM17 pathway provides a new cancer targeted therapy [16]. Serveral clinical trials are undergoing in other cancers. Therefore, ADAM17 may be a new target for treatment of patients with metastatic cRCC.
5. Conclusion We found that ADAM17 expression was located principally in the membrane of tumor cells in patients with cRCC. It was frequently expressed in metastatic cRCC as well as in localized cRCC. The high expression of ADAM17 was a predictive factor of worse outcome. The expression of ADAM17 level may be used to predict the prognosis of patients with cRCC. More studies are needed to confirm our results.
P value
Acknowledgment
Sex Male Female
1 0.825 0.349–1.947 0.660
Age, y Z68 o67
1 0.606 0.263–1.390 0.237
T category T1 þ T2 1 T3 3.309 1.603–6.832 0.001
We thank the technicians and Master students of pathology department for their skillful work of immunohistochemical staining.
1 2.317 1.021–4.831 0.034
Fuhrman 1þ2 3þ4
1 2.608 1.282–5.304 0.008
1 1.569 0.703–3.504 0.271
ADAM17 Low High
1 2.826 1.324–6.034 0.007
1 2.221 1.013–5.297 0.036
HR ¼ hazard ratio.
References [1] Arribas J, Bech-Serra JJ, Santiago-Josefat B. ADAMs, cell migration and cancer. Cancer Metastasis Rev 2006;25:57–68. [2] Duffy MJ, McKiernan E, O0 Donovan N, et al. Role of ADAMs in cancer formation and progression. Clin Cancer Res 2009;15:1140–4. [3] Borrell-Pages M, Rojo F, Albanell J, et al. TACE is required for the activation of the EGFR by TGF-alpha in tumors. EMBO J 2003;22:1114–24. [4] Kenny PA, Bissell MJ. Targeting TACE-dependent EGFR ligand shedding in breast cancer. J Clin Invest 2007;117:337–45.
1276
G. Li et al. / Urologic Oncology: Seminars and Original Investigations 32 (2014) 1272–1276
[5] Lam JS, Leppert JT, Figlin RA, et al. Role of molecular markers in the diagnosis and therapy of renal cell carcinoma. Urology 2005;66:1–9. [6] Kim SP, Weight CJ, Leibovich BC, et al. Outcomes and clinicopathologic variables associated with late recurrence after nephrectomy for localized renal cell carcinoma. Urology 2011;78: 1101–6. [7] Franovic A, Robert I, Smith K, et al. Multiple acquired renal carcinoma tumor capabilities abolished upon silencing of ADAM17. Cancer Res 2006;66:8083–90. [8] Melenhorst WB, Visser L, Timmer A, et al. ADAM17 upregulation in human renal disease: a role in modulating TGF-alpha availability? Am J Physiol Renal Physiol 2009;297:F781–90. [9] Moch H, Artibani W, Delahunt B, et al. Reassessing the current UICC/ AJCC TNM staging for renal cell carcinoma. Eur Urol 2009;56:636–43. [10] Fuhrman SA, Lasky LC, Limas C. Prognostic significance of morphologic parameters in renal cell carcinoma. Am J Surg Pathol 1982;6:655–63. [11] Li G, Barthelemy A, Feng G, et al. S100A1: a powerful marker to differentiate chromophobe renal cell carcinoma from renal oncocytoma. Histopathology 2007;50:642–7.
[12] McGowan PM, McKiernan E, Bolster F, et al. ADAM-17 predicts adverse outcome in patients with breast cancer. Ann Oncol 2008;19:1075–81. [13] McGowan PM, Ryan BM, Hill AD, et al. ADAM-17 expression in breast cancer correlates with variables of tumor progression. Clin Cancer Res 2007;13:2335–43. [14] Shou ZX, Jin X, Zhao ZS. Upregulated expression of ADAM17 is a prognostic marker for patients with gastric cancer. Ann Surg 2012;256:1014–42. [15] Zatovicova M, Sedlakova O, Svastova E, et al. Ectodomain shedding of the hypoxiainduced carbonic anhydrase IX is a metalloproteasedependent process regulated by TACE/ADAM17. Br J Cancer 2005;93:1267–76. [16] Duffy MJ, Mullooly M, O0 Donovan N, et al. The ADAMs family of proteases: new biomarkers and therapeutic targets for cancer? Clin Proteomic 2011;8:9. [17] Coppin C, Kollmannsberger C, Le L, et al. Targeted therapy for advanced renal cell cancer (RCC): a Cochrane systematic review of published randomized trials. BJU Int 2011;108:1556–63. [18] Motzer RJ, Hutson TE, Tomczak P, et al. Overall survival and updated results for sunitinib compared with interferon alfa in patients with metastatic renal cell carcinoma. J Clin Oncol 2009;27:3584–90.
Original article
A novel marker ADAM17 for clear cell renal cell carcinomas: Implication for patients’ prognosis Guorong Li, M.D., Ph.D.a,e,*, Fabien Forest, M.D.b, Gang Feng, Ph.D., M.D.c, Anne Gentil-Perret, M.D.b, Michel Péoc'h, M.D., Ph.D.b, Michèle Cottier, M.D., Ph.D.d,e, Nicolas Mottet, M.D., Ph.D.a,e a
Department of Urology, North Hospital, CHU de Saint-Etienne, University of Jean-Monnet, Saint-Etienne, France Department of Pathology, North Hospital, CHU de Saint-Etienne, University of Jean-Monnet, Saint-Etienne, France c Clinical Genetics Laboratory, Yijishan Hospital of Wannan Medical College, Wuhu, China d Department of Cytology, North Hospital, CHU de Saint-Etienne, University of Jean-Monnet, Saint-Etienne, France e LINA, EA 4624, Faculty of Medicine, University of Jean-Monnet, Saint-Etienne, France
b
Received 10 April 2014; received in revised form 22 May 2014; accepted 23 May 2014
Abstract Introduction: A disintegrin and metalloproteinase-17 (ADAM17) plays an important role in biological activity in different cancers. Its expression and prognostic value have not been studied in clear cell renal cell carcinoma (cRCC). The objective of this study was to explore the prognostic value of ADAM17 in patients with cRCC. Materials and methods: A total of 131 patients with cRCC were studied. There were 90 men and 41 women, with an average age of 67 years (range: 34–93 y). There were 110 patients with localized disease and 21 patients with metastatic disease. The expression of ADAM17 was evaluated by immunohistochemistry with a monoclonal antibody. The follow-up varied from 4.2 to 184 months, with a median of 72 months for patients with localized disease. Kaplan-Meier with a log-rank test was performed to compare the progression-free survival after surgery. The univariate and multivariate analyses were performed to examine the significance of ADAM17 expression for the patient’s progression-free survival. Results: The ADAM17 expression was found in 109/131 tumors. The ADAM17 expression was found in 20/21 metastatic tumors and in 89/110 localized tumors. Regarding patients with localized tumors, 31 patients experienced a recurrence or death during follow-up. The Kaplan-Meier analysis revealed that the high expression of ADAM17 was associated with a reduced progression-free survival (P ¼ 0.005). The univariate logistic regression analysis indicated that the high expression of ADAM17 was associated with the disease progression (hazard ratio ¼ 2.826; 95% CI: 1.324–6.034; P ¼ 0.007). The high expression of ADAM17 remained a significant factor for decreased progression-free survival in multivariate analysis. Conclusion: ADAM17 was frequently expressed in cRCC. The high expression of ADAM17 was correlated with a worse outcome for patients with cRCC. ADAM17 is a new biomarker for the management of patients with cRCC. r 2014 Elsevier Inc. All rights reserved.
Keywords: ADAM17; Prognosis; Renal cell carcinoma; Immunohistochemistry
1. Introduction A disintegrin and metalloproteinase-17 (ADAM17) is a member of the ADAM family, which plays an important This research was financed by the CHU de Saint-Etienne (AOL 0908126), France. * Corresponding author. Tel.: þ33-47-782-8814; fax: þ33-47-782-8464. E-mail address: [email protected] (G. Li). http://dx.doi.org/10.1016/j.urolonc.2014.05.011 1078-1439/r 2014 Elsevier Inc. All rights reserved.
role in biological activities of malignant lesion through epidermal growth factor receptor (EGFR)/phosphoinositide 3-kinase/protein kinase B (AKT) pathway [1,2]. This matrix metalloproteinaselike enzyme is involved in the release of several ligands. All the ligands released by ADAM17 have been implicated in tumor formation or progression or both. These ligands include transforming growth factor (TGF)-α, amphiregulin, heparin-binding epidermal growth factor, epiregulin, and tumor necrosis factor (TNF)-α. Another
G. Li et al. / Urologic Oncology: Seminars and Original Investigations 32 (2014) 1272–1276
function of ADAM17 is its implication in tumor formation or progression [2]. ADAM17-catalyzed shedding of TGF-α was found to be necessary for murine tumor formation [3] whereas inhibition of ADAM17 prohibited the malignancy formation [4]. ADAM17 was recently demonstrated to be overexpressed in several cancer tissues. The incidence of renal cell carcinoma (RCC) is increasing steadily. Despite the fact that small tumors are found frequently, one-third of patients with renal cancer present a metastatic disease at the time of diagnosis, and 30% to 40% of patients with localized renal cancer will develop a metastasis after surgery [5]; 40% of patients with RCC will die from metastasis [6]. These data indicate that prognosis prediction is a major issue for follow-up of patients. At present, the prediction of prognosis relies on solely the clinical stage and grade. Efficient prognostic markers are urgently needed to guide the surveillance and to find the at-risk patients for targeted therapy. Like other cancers, the process of formation and progression in renal carcinoma is through complicated molecular interactions. Many pathways are involved in this process. Several models have suggested the evidence that ADAM17 may participate in the tumorigenesis of renal cancer or other renal disease [7,8]. However, there has been no information concerning its prognostic value in clear cell RCC (cRCC). Therefore, we conducted a clinical study to explore its prognostic value in cRCC. To our best knowledge, this is the first study exploring the clinical significance of ADAM17 in cRCC. 2. Materials and methods 2.1. Patients and tumor samples A total of 131 patients with cRCC were included. There were 90 men and 41 women, with an average age of 67 years (range: 34–93 y). The tumors were staged according to Union for International Cancer Control/American Joint Commission on Cancer TNM staging system and graded according to Fuhrman criteria [9,10]. All patients benefited regularly from the surveillance after surgery. The surveillance included the image examination, blood creatinine level measurement, and physical examination. The histological slides were reviewed for the confirmation of diagnosis of clear cell histology and of grade determination by a urologic pathologist (A.G.P.). The corresponding blocks were used for the immunohistochemical study. Consent was obtained from each patient for the future study of the tumor tissues when the patients were hospitalized. This research was approved by the local ethics committee. 2.2. Immunohistochemical staining for ADAM17 Immunohistochemical staining was performed as per our previous study [11]. Paraffin-embedded, 5-μm thick tissue
1273
sections from all primary tumors were stained for the ADAM17 protein using a primary mouse monoclonal antibody (Abcam, ab57484; Cambridge, UK). Slides were deparaffinized through a series of xylene baths. Rehydration was performed through graded alcohols. Endogenous peroxidase activity was blocked in ChemMate PeroxidaseBlocking solution (DakoCytomation) 3 times every 5 minutes. The sections were then passed through the automated immuno-staining machine for a standard immunohistochemical protocol. Diaminobenzidine was used as a chromogen, and commercial hematoxylin was used for counterstaining. For each immunohistochemical staining, positive and negative controls were used. ADAM17 expression was evaluated at the membrane and cytoplasmic level. The percentages of positive cells (high Z 85%, moderate 50%–85%, weak o 50%, and absence ¼ 0%) were counted to define an expression level. Immunohistochemical analysis was performed by 2 urologic pathologists (F.F. and M.P.) in a blinded manner with respect to the clinical data concerning the subjects. For heterogeneous staining, ADAM17-expressing cells were counted in several representative high-power microscopic fields. Divergent scores were then discussed and decided on a multiheaded microscope. Finally, a high or low expression level was given to each sample. 2.3. Statistical analysis Survival was calculated by the Kaplan-Meier method, and the resulting curves were compared using the log-rank test. The univariate logistic regression analysis and the multivariate analysis by the Cox regression model were performed. P o 0.05 was considered to be statistically significant. 3. Results 3.1. Patient characteristics and survival The stage and grade of patients’ tumors are summarized in Table 1. The follow-up varied from 4.2 to 184 months, with a median of 72 months for patients with localized tumors. For patients with localized disease, 31 patients experienced a recurrence or death during the follow-up. 3.2. ADAM17 expression in cRCC: Pattern of expression The ADAM17 was found in 109/131 tumors. The expression of ADAM17 was located in the membrane (Fig. 1). Expression in the cytoplasm could be found in 20 tumors in combination with expression in the membrane. The distribution of high expression of ADAM17 in the different stage and grade is summed in Table 1. The high expression of ADAM17 was found in 13/39 T1a and in 15/26 T1b tumors. The expression of ADAM17 was found
1274
G. Li et al. / Urologic Oncology: Seminars and Original Investigations 32 (2014) 1272–1276
Table 1 Patients' characteristics and ADAM17 expression Characteristics
No. (%)
ADAM17 expression High
Low
85 (64.9) 46 (35.1)
43 22
42 24
Age, y Z68 o67
54 (41.2) 77 (58.8)
28 36
26 41
T classification T1 T2 T3
65 (49.6) 8 (6.1) 58 (44.3)
28 4 34
37 4 24
Fuhrman grade 1 2 3 4
25 59 39 8
15 30 22 5
10 29 17 3
Total patients Sex Male Female
131
(19) (45) (29.7) (6.3)
epidermal growth factor, and epiregulin [1,2]. In renal cancer, an experiment showed that ectodomain shedding of CA9 was regulated by TNF-α-converting enzyme/ ADAM17 [15]. Another experiment showed that ADAM17 was involved in tumor formation of renal cancer [7]. This experiment found that the metalloprotease ADAM17 played a pivotal role in several acquired tumor cell capabilities by mediating the availability of soluble TGF-α, an EGFR ligand, and thus the establishment of a key autocrine signaling pathway [7]. Silencing of ADAM17 in human renal carcinoma cell lines corrected critical features associated with cancer cells, including growth autonomy, tumor inflammation, and tissue invasion. Highly malignant renal carcinoma cancer cells failed to form in vivo tumors in the absence of ADAM17. From the aforementioned 2 experiments, we can imagine that ADAM17 actively participates in biological activity of renal cancer. Therefore, the study of ADAM17 expression in cRCC is meaningful. Nevertheless, immunohistochemical study of ADAM17 in patients with
in 20/21 metastatic tumors and in 89/110 localized tumors. Among the positive staining of ADAM17 for the localized tumors, the high, moderate, and weak expressions was found respectively in 52/110, 26/110, and 11/110 cases. 3.3. Correlation of ADAM17 expression with progression-free survival for localized patients The Kaplan-Meier analysis of progression-free survival was shown in Fig. 2. During the follow-up, 31 events of death or metastasis were found for the 110 patients with localized tumors. The Kaplan-Meier analysis revealed that the high expression of ADAM17 was associated with a reduced progression-free survival (P ¼ 0.005). By using the univariate logistic regression analysis, the high expression of ADAM17 was associated with a shorter progression-free survival (hazard ratio ¼ 2.826; 95% CI: 1.324–6.034; P ¼ 0.007). ADAM17 expression and T category remained significant factors for decreased progression-free survival in the multivariate Cox regression analysis (Table 2). 4. Discussion ADAM17 has been found to be up-regulated in many cancers. ADAM17 is becoming a new cancer diagnostic and prognostic biomarker [12–14]. It exerts active roles in malignancy formation and progression. ADAM17 was originally identified by its ability to release membranebound TNF-α from its precursor. ADAM17 is thus sometimes referred to as TNF-α-converting enzyme [4]. Other growth factors shown to be released by ADAM17 include the EGFR ligands, TGF-α, amphiregulin, heparin-binding
Fig. 1. Illustration of immunohistochemical staining of tumor cells in cRCC: (A) membrane staining (anti-ADAM17, 60) and (B) negative staining. (Color version of figure is available online.)
G. Li et al. / Urologic Oncology: Seminars and Original Investigations 32 (2014) 1272–1276
Fig. 2. Progression-free survival according to ADAM17 expression. The high expression of CXCR4 was associated with a reduced progression-free survival (P ¼ 0.005, log-rank test).
cRCC has been rare. In fact, we failed in finding any report concerning its immunohistochemical staining in cRCC. In the present study, we used a monoclonal antibody to study its expression in cRCC. We found that ADAM17 was located mainly in the membrane of tumor cells. ADAM17 was frequently expressed in metastatic cRCC as well as in localized cRCC. Taken together, we assume that ADAM17 signaling is an important event during malignancy transformation and progression of cRCC. The prediction of prognosis of patients with cRCC is based only on the pathological stage and grade. The biomarkers are urgently needed to guide for patient surveillance after surgery. Unfortunately, there have been no biomarkers that can be used in clinic for cRCC. Because
Table 2 Univariate and multivariate analyses for the patient's progression-free survival Univariate analysis HR
95% CI
Multivariate analysis P value HR
95% CI
1275
ADAM17 plays an important role in biological activity of cancers, it is reasonable to explorer its prognostic value in cRCC. We found that the high expression of ADAM17 was a predictive factor of progression-free survival for patients with localized cRCC. Therefore, ADAM17 may be a new biomarker for the surveillance after surgery for patients with localized cRCC. Some articles have been recently published on the prognostic value of ADAM17 in other cancers. It seems that a high expression of ADAM17 is associated with poor outcome in patients with various cancers [12–14]. We believe that ADAM17 is a new promising prognostic marker in patients with cRCC. ADAM17 is a new therapeutic target in cancer [16]. Another clinical implication of ADAM17 signaling in cRCC may be its therapeutical potential. Since 2006, molecular targeted therapy has changed treatment strategy for metastatic renal cancer [17]. The targeted therapy showed better results over the traditional immunotherapy [18]. The targeted therapy provides a 50% response rate. At present, there are 6 targeted agents approved for treatment in metastatic RCC: sorafenib, sunitinib, pazopanib, bevacizumab (in combination with interferon alpha), temsirolimus, and everolimus [17]. ADAM17 pathway provides a new cancer targeted therapy [16]. Serveral clinical trials are undergoing in other cancers. Therefore, ADAM17 may be a new target for treatment of patients with metastatic cRCC.
5. Conclusion We found that ADAM17 expression was located principally in the membrane of tumor cells in patients with cRCC. It was frequently expressed in metastatic cRCC as well as in localized cRCC. The high expression of ADAM17 was a predictive factor of worse outcome. The expression of ADAM17 level may be used to predict the prognosis of patients with cRCC. More studies are needed to confirm our results.
P value
Acknowledgment
Sex Male Female
1 0.825 0.349–1.947 0.660
Age, y Z68 o67
1 0.606 0.263–1.390 0.237
T category T1 þ T2 1 T3 3.309 1.603–6.832 0.001
We thank the technicians and Master students of pathology department for their skillful work of immunohistochemical staining.
1 2.317 1.021–4.831 0.034
Fuhrman 1þ2 3þ4
1 2.608 1.282–5.304 0.008
1 1.569 0.703–3.504 0.271
ADAM17 Low High
1 2.826 1.324–6.034 0.007
1 2.221 1.013–5.297 0.036
HR ¼ hazard ratio.
References [1] Arribas J, Bech-Serra JJ, Santiago-Josefat B. ADAMs, cell migration and cancer. Cancer Metastasis Rev 2006;25:57–68. [2] Duffy MJ, McKiernan E, O0 Donovan N, et al. Role of ADAMs in cancer formation and progression. Clin Cancer Res 2009;15:1140–4. [3] Borrell-Pages M, Rojo F, Albanell J, et al. TACE is required for the activation of the EGFR by TGF-alpha in tumors. EMBO J 2003;22:1114–24. [4] Kenny PA, Bissell MJ. Targeting TACE-dependent EGFR ligand shedding in breast cancer. J Clin Invest 2007;117:337–45.
1276
G. Li et al. / Urologic Oncology: Seminars and Original Investigations 32 (2014) 1272–1276
[5] Lam JS, Leppert JT, Figlin RA, et al. Role of molecular markers in the diagnosis and therapy of renal cell carcinoma. Urology 2005;66:1–9. [6] Kim SP, Weight CJ, Leibovich BC, et al. Outcomes and clinicopathologic variables associated with late recurrence after nephrectomy for localized renal cell carcinoma. Urology 2011;78: 1101–6. [7] Franovic A, Robert I, Smith K, et al. Multiple acquired renal carcinoma tumor capabilities abolished upon silencing of ADAM17. Cancer Res 2006;66:8083–90. [8] Melenhorst WB, Visser L, Timmer A, et al. ADAM17 upregulation in human renal disease: a role in modulating TGF-alpha availability? Am J Physiol Renal Physiol 2009;297:F781–90. [9] Moch H, Artibani W, Delahunt B, et al. Reassessing the current UICC/ AJCC TNM staging for renal cell carcinoma. Eur Urol 2009;56:636–43. [10] Fuhrman SA, Lasky LC, Limas C. Prognostic significance of morphologic parameters in renal cell carcinoma. Am J Surg Pathol 1982;6:655–63. [11] Li G, Barthelemy A, Feng G, et al. S100A1: a powerful marker to differentiate chromophobe renal cell carcinoma from renal oncocytoma. Histopathology 2007;50:642–7.
[12] McGowan PM, McKiernan E, Bolster F, et al. ADAM-17 predicts adverse outcome in patients with breast cancer. Ann Oncol 2008;19:1075–81. [13] McGowan PM, Ryan BM, Hill AD, et al. ADAM-17 expression in breast cancer correlates with variables of tumor progression. Clin Cancer Res 2007;13:2335–43. [14] Shou ZX, Jin X, Zhao ZS. Upregulated expression of ADAM17 is a prognostic marker for patients with gastric cancer. Ann Surg 2012;256:1014–42. [15] Zatovicova M, Sedlakova O, Svastova E, et al. Ectodomain shedding of the hypoxiainduced carbonic anhydrase IX is a metalloproteasedependent process regulated by TACE/ADAM17. Br J Cancer 2005;93:1267–76. [16] Duffy MJ, Mullooly M, O0 Donovan N, et al. The ADAMs family of proteases: new biomarkers and therapeutic targets for cancer? Clin Proteomic 2011;8:9. [17] Coppin C, Kollmannsberger C, Le L, et al. Targeted therapy for advanced renal cell cancer (RCC): a Cochrane systematic review of published randomized trials. BJU Int 2011;108:1556–63. [18] Motzer RJ, Hutson TE, Tomczak P, et al. Overall survival and updated results for sunitinib compared with interferon alfa in patients with metastatic renal cell carcinoma. J Clin Oncol 2009;27:3584–90.
