Integrin α7 is overexpressed and correlates with higher pathological grade, increased T stage, advanced TNM stage as well as worse survival in clear cell renal cell carcinoma patients: A retrospective study

Abstract Objective This study aimed to explore the association of integrin α7 with clinicopathological characteristics and overall survival (OS) in clear cell renal cell carcinoma (ccRCC) patients. Methods 179 ccRCC patients who underwent nephrectomy were included in this retrospective study. Tumor tissue and paired adjacent tissue specimens of patients were obtained. Immunohistochemistry assay was performed to detect integrin α7 expression. OS was calculated with the median follow‐up duration of 91.0 months (range: 3.0‐116.0 months). Results Integrin α7 was highly expressed in tumor tissue compared to paired adjacent tissue (P < .001), and tumor integrin α7 high expression was correlated with higher pathological grade (P = .004), increased T stage (P = .017), and advanced TNM stage (P = .033). Kaplan–Meier curve showed that patients with integrin α7 high expression (mean OS = 69.8, 95%CI: 60.5‐79.1 months) presented with worse OS compared to patients with integrin α7 low expression (mean OS = 101.8, 95%CI: 96.0‐107.7 months; P < .001). Multivariate Cox's regression analysis further disclosed that tumor integrin α7 high expression independently predicted poor OS (P < .001). Conclusion Integrin α7 is upregulated and correlates with higher pathological grade, increased T stage, and advanced TNM stage, meanwhile it also acts as a valuable prognostic factor for worse survival in ccRCC patients.

there are still 20%-40% ccRCC patients developing local recurrence or distant metastasis after surgery. 2,4 Hence, investigation of additional biomarkers to supervise disease development and progression is imperative in ccRCC patients.
Integrins, one family of cell adhesion molecules, are heterodimers consisting of an alpha and a beta subunit, which play important roles in many physiological and pathological processes via regulating cell-cell or cell-matrix adhesion. 5 Integrin α7, belonging to the integrin family of adhesion molecule, is localized on chromosome 12p13 and consists of at least 27 exons spanning a region of about 22.5 kb. 6 According to accumulating evidences, integrin α7 serves as an important regulator in tumor progression of several carcinomas, including prostate cancer, esophageal squamous cell carcinoma (OSCC), and glioblastoma. [5][6][7][8] In addition, integrin α7 also has been described as a functional cancer stem cells (CSCs) surface marker, and its knockdown could effectively decrease the stemness of cancer cells, such as OSCC, whereas the information about the role of integrin α7 in RCC, particular in ccRCC, is still rarely known. 6 Considering the key role of integrin α7 in tumor progression and its effect on CSC properties in different carcinomas, we hypothesized that integrin α7 might play a critical role in ccRCC.
Thus, this study aimed to explore the association of integrin α7 with clinicopathological characteristics and overall survival (OS) in ccRCC patients.

| Tissue specimens and data collection
Tumor tissue and paired adjacent tissue specimens of patients were obtained from Tumor Specimen storeroom of hospital.
Baseline characteristics of patients were collected from medical records, which included age, gender, tumor location, pathological grade, tumor size, T stage, N stage, and TNM stage. In addition, survival data were also collected from patients' follow-up records, and the median follow-up duration was 91.0 months (range: 3.0-116.0 months).

| Immunohistochemistry assay
Firstly, formalin-fixed and paraffin-embedded specimens of tumor tissue and paired adjacent tissue were serially sectioned at 4 μm thickness, and the sections were deparaffinized with xylene, rehydrated with ethanol, microwaved in 0.01 mol/L sodium citrate buffer (pH 6.0) for antigen retrieval, and incubated with H 2 O 2 at 37°C for 10 minutes. Then 1.5% normal goat serum was used for blocking under 37°C for 20 minutes. Subsequently, the sections were incu- and proportion score were used to calculate the total score. Two investigators blinded to the type of tissues performed these procedures. For staining in tumor tissue and paired adjacent tissue, it was visually scored and quantified according to a semiquantitative scoring system. 9 Briefly, the intensity of immunostaining was scored as: 0 = negative (no staining), 1 = weak (light yellow), 2 = moderate (yellow), and 3 = strong (brown); and the proportion of immune-positive cells was scored as: 1 = <10%, 2 = 10% ~ 50%, 3 = 51% ~ 75%, and 4 = more than 75%. The two scores of the corresponding sample were multiplied to obtain a total score ranging from 0 to 12 points.
High expression of integrin α7 was defined as total score ≥3, and low expression of integrin α7 was defined as total score <3. All procedures in this experiment were performed by one specialist from the Laboratory Department of our hospital who were blinded to the type of tissues and patients' information. In addition, the IHC was scored by two clinic pathologists with rich experience from the Pathological Department of our hospital who were blinded to the type of tissues and patients' information as well. When the opinion of these two clinic pathologists was different, the third clinic pathologist with rich experience from the Pathological Department of our hospital who were blinded to the type of tissues and patients' information would score the results of IHC.

| Statistical analysis
Statistical analysis was performed by SPSS 19.0 software (IBM), and graph making was accomplished using GraphPad Prism 7.00 (GraphPad Inc). Normal distributed continuous variables were presented as mean value ± standard deviation; skewed distributed continuous variables were presented as median (25th-75th quantiles); and categorized variables were presented as count (percentage). Comparison of percentage was determined by Chi-square test or Wilcoxon rank sum test. Survival analysis was performed using Kaplan-Meier (K-M) curve and Log-rank test.
Factors affecting OS were determined by univariate and multivariate Cox's proportional hazards regression analyses with Forward Stepwise (Conditional) method. P value < .05 was considered as significant.

| Study flow
A total of 415 patients with RCC who underwent nephrectomy were screened, while 204 cases were excluded, among which 79 cases were with tissue samples unavailable, 65 cases were without complete medical records or follow-up data, 31 cases were with other types of RCC other than ccRCC, 18 cases were with TNM stage IV or distant metastasis, seven cases had a history of malignant tumors other than ccRCC, and four cases were with secondary ccRCC (Figure 1). Subsequently, 211 ccRCC cases were eligible, whereas 32 cases were excluded, including 28 cases who could not be contacted to get informed consents and four cases who were reluctant to provide the written informed consents. Finally, the remaining 179 ccRCC cases were included in the analysis.

| Association of tumor integrin α7 expression with tumor characteristics
Tumor integrin α7 high expression was correlated with higher pathological grade (P = .004), increased T stage (P = .017), and advanced TNM stage (P = .033; Table 2). However, no correlation was found between tumor integrin α7 expression and other characteristics (All P > .05).

F I G U R E 2
Integrin α7 expression between tumor tissue and adjacent tissue. Immunohistochemistry assay revealed that integrin α7 was highly expressed in tumor tissue (A). The percentage of patients with integrin α7 high expression in tumor tissue was increased than that in adjacent tissue (B  (Figure 3). Tumor integrin α7 high expression was correlated with worse OS in ccRCC patients (P < .001).

| D ISCUSS I ON
In the present study, we observed that (1) Integrin α7 was highly expressed in tumor tissue, and its high expression was associated with advanced cancer features. (2) Tumor integrin α7 high expression independently predicted poor OS in ccRCC patients.
Integrin α7, a member of the extracellular matrix binding proteins, contributes to the interaction of relevant cell-cell and cell-extracellular matrix in a wide range of cellular processes, which also involves in the processes of tumorigenesis and tumor progression in different malignancies. 5,7 A majority of previous studies have focused on the function of integrin α7 on cell activities in different carcinomas and disclosed its tumor promoter role in these cancers. For example, integrin α7 interacts with S100P to promote cells migration and cells invasion in lung carcinoma. 10 Another mechanistic study discloses that integrin α7 induces cells migration and invasion abilities via the activation of epithelial-mesenchymal transition (EMT) in OSCC. 6 In addition, parts of previous studies have revealed that integrin α7 possesses effect on regulating stemness of cancer cells. For instance, integrin α7 effectively promotes the stemness of OSCC cells through regulating the focal adhesion kinase (FAK)-mediated pathway in OSCC. 6 Therefore, these previous evidences suggest that integrin α7 appears to be a promoter in the pathological processes of several carcinomas due to its effect on cell activities and stemness of cancer cells.
In clinical trials, there is limited information about the association of integrin α7 with disease conditions in carcinomas, among which one previous study reveals that integrin α7 is related to poor differentiation and lymph node metastasis in OSCC patients, while little is known about the participation of integrin α7 in RCC patients, particularly ccRCC patients. 6 In consideration of the underlying mechanism and influence of integrin α7 in different carcinomas as well as its correlation with the stemness of cancer cells, we suspected that integrin α7 might exert an influence on tumor progression in ccRCC patients. Thus, we evaluated the association of integrin α7 with clinicopatholgocial features in ccRCC patients, and we found that integrin α7 was highly expressed in tumor tissue, and its high expression was associated with Although some interesting results were discovered in this study, some limitations still existed. Firstly, this is a retrospective study, and we screened all patients who had completed records regarding OS, but not DFS, and the effect of integrin α7 on DFS in ccRCC patients was not investigated due to the loss of DFS records. Secondly, the sample size in this study was relatively small, which might cause poor statistical power. Thirdly, all patients enrolled in this study were from our hospital, which might result in regional bias.
In a word, integrin α7 is upregulated in tumor tissue, and it correlates with higher pathological grade, increased T stage, and advanced TNM stage; meanwhile, it also acts as a valuable prognostic factor for worse survival in ccRCC patients.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.