Decreased ARID1A Expression Associates with Tumor Progression and Adverse Prognosis in Renal Cell Carcinoma

In this study, we aimed to evaluate association of ARID1A (AT-rich interacting domain-containing protein 1A) mutation and protein expression with clinicopathology and prognosis of renal cell carcinoma (RCC). Genomic Data Commons (GDC) showed ARID1A was one of the top-ten mutated genes found in kidney cancers and its mutations were found along its sequence. Interestingly, patients with ARID1A mutations had signicantly lower survival rate (38%; n=68) comparing to the non-mutated cases (58%; n=192). The results from OSkirc web tool revealed that patients with low expression of ARID1A had signicantly shorter overall survival and disease specic survival than those with high ARID1A expression. Immunohistochemistry revealed markedly decreased ARID1A expression in the RCC tissues (n=26), particularly in clear cell RCC (ccRCC) and chromophobe RCC (chRCC). Negative to weak ARID1A expression was signicantly associated with ccRCC (grade II) and chRCC subtypes, presence of comorbidity, and low eGFR levels. Finally, ARID1A protein was undetectable in 3/11 cases with ccRCC (grade II) and 2/6 chRCC cases, all of which had metastasis 1−50 months after surgical removal. In conclusion, decreased ARID1A expression is associated with the poor prognosis and metastasis of RCC and thus may serve as the prognostic marker of RCC, particularly ccRCC and chRCC subtypes. histopathology The subtype and the Fuhrman nuclear grading then evaluated. From a total of 26 RCC patients recruited, 17 clear cell RCC (ccRCC) Fuhrman nuclear grade Fuhrman nuclear grade II), chromophobe RCC (chRCC), papillary RCC (pRCC), one sarcomatoid RCC (sRCC).


Introduction
ARID1A encoding AT-rich interacting domain-containing protein 1A is one of the tumor suppressor genes, mutations of which have been found in various cancers, particularly ovarian clear cell carcinoma (57%), gastric cancer (29%), and bladder transitional cell carcinoma (13%) [1]. Several previous studies have investigated pathogenic mechanisms of defective ARID1A in the development and progression of cancers. One of these studies has shown that silencing of ARID1A in gastric cancer cells inhibited transcription of CDH-1 (encoding E-cadherin) and subsequently induced accumulation of β-catenin in the nucleus [2]. Such accumulation could enhance the Wnt/β-catenin pathway that regulates migration and invasion of gastric cancer cells [2]. A recent study has also demonstrated that inactivation of ARID1A in mice could facilitate colon tumorigenesis via defective APC/β-catenin and SWI/SNF pathways [3].
Decrease of ARID1A mRNA expression has been shown to be involved with promoter hypermethylation in breast invasive ductal carcinoma and squamous cell carcinoma [4,5]. Interestingly, a decrease (approximately 30-40 %) in ARID1A transcriptional activity has been observed in renal cell carcinoma (RCC) [6]. Moreover, the loss of ARID1A protein expression in tumor tissues has been observed in approximately 67% of patients with clear cell RCC (ccRCC) [7]. In addition, the loss of ARID1A protein expression in tumor tissues has been found to be associated with poor clinicopathological outcome of ccRCC patients, including large tumor size (>7 cm), high Fuhrman nuclear grade (III/IV), high pTNM stage (III) and relatively short progression-free interval [7,8]. These studies have implied that the loss of ARID1A protein expression in tumor tissues might serve as a prognostic marker in RCC.
Nevertheless, the relevance of ARID1A down-regulation in RCC has remained under-investigated. We thus evaluated the association of ARID1A gene mutation and ARID1A protein expression with clinicopathology and prognosis in various types of RCC, including ccRCC (grade I), ccRCC (grade II), chromophobe RCC (chRCC), papillary RCC (pRCC), and sarcomatoid RCC (sRCC).

Bioinformatics analysis
The mutated genes commonly found in kidney cancers were analyzed through the Genomic Data Commons (GDC) database (http://portal.gdc.cancer.gov/). All of The Cancer Genome Atlas (TCGA) projects related to kidney cancers, including TCGA-KIRC (kidney renal clear cell carcinoma), TCGA-KIRP (kidney renal papillary cell carcinoma), TCGA-KICH (kidney chromophobe) and TCGA-SARC (sarcoma), which recruited 943 patients (accessed on October 11, 2019), were analyzed. From this information the number of patients affected by each of the top-20 mutated genes was recorded. Additionally, the pro le of ARID1A mutations along the protein-coding regions and their frequency were investigated. Moreover, Kaplan-Meier curve and the log-rank test were conducted to estimate the overall survival (OS) of patients with mutated ARID1A compared to those without ARID1A mutations. In addition, the Online consensus Survival analysis for kidney renal clear cell carcinoma (KIRC), OSkirc web tool (http://bioinfo.henu.edu.cn/KIRC/KIRCList.jsp) was used to verify the prognostic outcomes of ARID1A gene in patients with KIRC, as recorded in a previous study [9]. The expression pro ling of ARID1A gene and clinical follow-up information of patients were accessed from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases. The Kaplan-Meier survival plots of overall survival (OS) and disease speci c survival (DSS) with hazard ratio (HR) and log-rank p-value were also examined.

RCC patients and tissues
RCC patients admitted to Sawanpracharak Hospital, Nakhon Sawan, Thailand for nephrectomy during 2013-2017 were recruited, whereas those with hereditary RCC syndrome were excluded. The participants were asked to give an informed consent. All study procedures were approved by the Human Ethic Review Board of Sawanpracharak Hospital and by Naresuan University Ethical Committee for Human Research (NU-IRB) and was conducted in accordance with the Declaration of Helsinki Principles. Clinical data, i.e., age, gender, tumor laterality, pathological subtype and staging (TNM and AJCC systems), tumor mass diameter, tumor invasion, metastasis, recurrence, and other complications, were extracted and analyzed.
Their paired renal tissues, including cancer and adjacent non-cancer areas, were then collected as formalin xed para n-embedded (FFPE) samples. To con rm RCC subtypes and the Fuhrman nuclear grading [10,11], in both cancer and adjacent non-cancer areas, hematoxylin and eosin (H&E) stained slides were evaluated by a renal pathologist.

Immunohistochemistry
Immunohistochemistry was performed on both cancer and adjacent non-cancer renal tissues. Brie y, 4µm-thick tissue sections were baked in a hot-air oven at 60°C for 1 h, depara nized with xylene, and rehydrated with graded series of ethanol/water. Thereafter, the tissue slides were immersed in 0.3% Triton-X in PBS-T (PBS containing 0.3% Tween-20; pH 7.4) for 40 min and then washed twice with PBS-T.
Antigen retrieval was performed by using heat-induced epitope retrieval (HIER) protocol with a buffer containing 10 mM Tris-Base, 1 mM EDTA and 0.05% Tween-20 (pH 8.0) at 97°C for 30 min. The tissue slides were then cooled down at 25°C for 20 min, washed twice with PBS-T, and incubated with 3% H 2 O 2 /methanol for 10 min. Non-speci c bindings were blocked with 5% BSA at 25°C for 30 min. After washing three times with PBS-T, the tissue sections were incubated with rabbit polyclonal anti-ARID1A antibody (Sigma-Aldrich; St. Louis, MO) (diluted 1:400) at 25°C overnight and then with biotinylated goat anti-rabbit secondary antibody (Dako Corp.; Kyoto, Japan) (diluted 1:200) at 37°C for 2 h. After washing with PBS-T three times, the sections were incubated with HRP-conjugated streptavidin (Dako Corp.) (diluted 1:200) at 25°C for 1 h. After other three washes, the immunoreactive protein was colorized with DAB (3, 3'-diaminobenzidine) peroxidase substrate (Vector Laboratories; Burlingame, CA) for 4 min. The section stained without primary antibody served as a negative control. The stained sections were imaged under an Olympus BX50 light microscope (Olympus; Tokyo, Japan).

Quantitative analysis of ARID1A protein expression
Three randomized locales per slide were evaluated by the renal pathologist and the investigators in a double-blind fashion. Allred scoring and grading system, which is commonly used for quantitative analysis of estrogen/progesterone receptor in breast cancer [12], was employed for quantitative analysis of ARID1A protein expression in cancer and adjacent non-cancer renal tissues obtained from all RCC patients. Brie y, the Allred score considered both proportional score (PS) and intensity score (IS) that measured the proportion of the ARID1A-stained cells and the ARID1A intensity, respectively. The PS ranged from 0 (no ARID1A-stained cells) to 5 (>2/3 ARID1A-stained cells in each eld), whereas the IS ranged from 0 (negative staining) to 3 (strong staining). Allred score was the summation of the PS and IS and was nally graded as negative (Allred score = 0-1), weak (Allred score = 2-3), moderate (Allred score = 4-5) and strong (Allred score = 6-8). If there was any discrepancy of the PS and IS among the investigators, the scores were reassessed to nd out the possible reasons for disagreement.
In addition, levels of ARID1A protein expression in various parts of the nephron, including glomerulus (Glom), proximal convoluted tubule (PCT), distal convoluted tubule (DCT) and collecting duct (CD), were evaluated in the adjacent non-cancer areas of ccRCC (grade I) versus ccRCC (grade II). The intensity of ARID1A (represented by DAB staining) was quantitated from at least 100 cells in each part of the nephron as aforementioned using Image J -Fiji analysis software (http:// ji.sc/Fiji).
Association of ARID1A protein expression with clinicopathology and progression-free survival According to the Allred four grading system described above, ARID1A protein expression was categorized into "negative to weak expression" and "moderate to strong expression". Such categorization was then analyzed for its association with clinicopathology (using Pearson's chi-square and Fisher's exact tests) and cumulative progression-free survival (PFS) within ve years after surgical removal (using Kaplan-Meier analysis and log-rank test). Finally, the univariate and multivariate analyses of PFS were analyzed by a clinical epidemiologist to evaluate the potential predictors of prognosis of patients with RCCs.

Statistical analysis
All quantitative data are presented as mean ± SEM unless stated otherwise. Comparisons of the data between the paired samples were done by paired Student's t-test, whereas comparisons of the unpaired samples were done by unpaired Student's t-test or by Mann-Whitney test (when the data did not distribute normally). The association between ARID1A protein expression and clinicopathology was analyzed by Pearson's chi-square and Fisher's exact tests, whereas the association between ARID1A protein expression and progression-free survival was evaluated by Kaplan-Meier analysis and log-rank test. All statistical analyses were done through the SPSS software (version 16.0) (SPSS; Chicago, IL). The univariate and multivariate analyses of progressive-free survival were analyzed using a binary logistic regression model, followed by the Gaussian model, through the Stata SE version 16 for Windows (StataCorp LP, TX). P values less than 0.05 were considered statistically signi cant.

Results
ARID1A was one of the top-ten mutated genes most frequently found in kidney cancers and its mutations were found along its sequence Bioinformatics analysis through the Genomic Data Commons (GDC) database using all of The Cancer Genome Atlas (TCGA) projects related to kidney cancers, including TCGA-KIRC (kidney renal clear cell carcinoma), TCGA-KIRP (kidney renal papillary cell carcinoma), TCGA-KICH (kidney chromophobe), and TCGA-SARC (sarcoma) showed that ARID1A was one of the top-ten mutated genes most frequently found in kidney cancers (Fig. 1A). A total of 32 mutations related to kidney cancers were found in ARID1A gene, accounting for 4.03% of all the mutated genes detected in all the affected cases. In addition, these 32 mutations in ARID1A gene, including 14 frameshift (43.75%), 14 missense mutations (43.75%), and 4 stop gained (12.5%) were found along its protein sequence (Fig. 1B). Interestingly, six of these mutations were found in the important domains, including ARID DNA-binding domain (PF01388) and SWI/SNF-like complex subunit BAF250/Osa (PF12031) domains (Fig. 1B). Moreover, patients with ARID1A mutations had signi cantly lower survival rate (38%; n=68) comparing to the non-mutated cases (58%; n=192) Histopathology and ARID1A protein expression in cancer vs. adjacent non-cancer areas The histopathology of cancer and adjacent non-cancer areas of the paired renal tissues collected from RCC patients, who were admitted at Sawanpracharak Hospital, Nakhon Sawan, Thailand for nephrectomy during 2013-2017, was examined with H&E staining. The subtype and the Fuhrman nuclear grading were then evaluated. From a total of 26 RCC patients recruited, 17 had clear cell RCC (ccRCC) (six with Fuhrman nuclear grade I and 11 with Fuhrman nuclear grade II), six had chromophobe RCC (chRCC), two had papillary RCC (pRCC), and only one had sarcomatoid RCC (sRCC).
Immunohistochemistry was performed to evaluate ARID1A protein expression in these paired renal tissues (Fig. 3A). The data showed that ARID1A protein was strongly expressed in the nuclei of lymphocytes, broblasts, intraglomerular cells, and tubular epithelial cells of proximal convoluted tubules and distal convoluted tubules in the adjacent non-cancer areas, whereas lighter ARID1A stainings were observed in these cells in the cancer area (Fig. 3A). Quantitative analysis using Allred scoring and grading system revealed that ARID1A Allred score was signi cantly decreased in cancer areas of all RCC cases (Fig. 3B). Comparing in each subtype of RCC, ARID1A Allred score was markedly decreased in cancer area of ccRCC (grade I), RCC (grade II) and chRCC, whereas there was no signi cant difference observed in pRCC and sRCC (Fig. 3B). Similarly, Allred grading revealed that almost all the adjacent non-cancer area (25/26 cases) had moderate to strong ARID1A expression, whereas almost all the cancer area of ccRCC (grade I), ccRCC (grade II) and chRCC (21/23 cases) had negative to weak ARID1A expression (Fig. 3C). However, the three cases with pRCC and sRCC had moderate to strong ARID1A expression (Fig. 3C).
The progression-free survival (PFS) of patients with moderate to strong ARID1A vs. negative to weak ARID1A expression The progression-free survival (PFS) of patients with moderate to strong ARID1A and negative to weak ARID1A expression was analyzed. All 9/9 RCC patients with moderate to strong ARID1A expression had progression-free survival rate of 100% (Fig. 3D). Log-rank test revealed a tendency of lower PFS in patients with negative to weak ARID1A expression (n=17) as compared to those with moderate to strong ARID1A expression (n=9) but the p value did not reach the statistically signi cant threshold (Fig. 3D).
Association of ARID1A protein expression and clinicopathological outcomes of patients Allred grading was then applied to compare clinicopathological outcomes of patients. There were 17 cases with "moderate to strong ARID1A expression" and nine cases with "negative to weak ARID1A expression". Their mean ages were comparable, whereas tumor mass diameter tended to be larger and progression-free duration tended to be shorter in patients with negative to weak ARID1A protein expression, although the statistically signi cant threshold was not reached (Table 1). Using Fisher's exact test, patients with negative to weak ARID1A expression had signi cantly greater number and percentage of ccRCC (grade II) and chRCC cases (p = 0.003), presence of comorbidity (p = 0.038), and low eGFR levels (p = 0.008) as compared to those with moderate to strong ARID1A expression (Table 2). However, their elderly (age ≥ 60 years), gender, tumor laterality, TNM stage and AJCC stage made no signi cant differences between the two groups ( Table 2). Univariate analysis revealed that negative expression of ARID1A in RCC tissues (29% of all patients; 95% CI: 0.07-0.52; p = 0.009), high AJCC stage (II-IV) (26% of all patients; 95% CI: 0.06-0.47; p = 0.011), and presentation with chronic kidney disease (31% of all patients; 95% CI: 0.08-0.54; p = 0.008) were associated with a short PFS (Table 3). Multivariate analysis revealed that negative expression of ARID1A in RCC tissues and other parameters were not associated with a short PFS of patients (Table 3). Furthermore, ARID1A protein was undetectable in 3/11 cases with ccRCC (grade II) and 2/6 chRCC cases, all of which had metastasis (to lung, brain, bone, and liver) 1-50 months after surgical removal.

Discussion
The present study investigated ARID1A gene mutation in kidney cancers on the Genome Data Commons (GDC). The results showed that ARID1A mutation was one of the top-ten mutated genes most common found in kidney cancers together with PBRM1 (Polybromo 1), VHL (von Hippel-Lindau), TP53 (tumor suppressor 53), SETD2 (SET domain-containing protein 2), etc. Among these, mutations of PBRM1 and VHL are the majority to cause ccRCC subtype [13,14]. Mutation of MET proto-oncogene and inactivation of SWI/SNF chromatin/histone modi ers are common in pRCC type I, whereas mutations of FH (fumarate hydratase), SETD2, and CDKN2A (cyclin dependent kinase inhibitor 2A) are more common in pRCC type II [13,14]. Chromophobe RCC is associated with mutations of PTEN (phosphatase and tensin homolog), TP53, and TERT (telomerase reverse transcriptase) mitochondrial genes [13,14]. Consistent with our present study, a recent study by Ricketts and colleagues [15] has shown that mutations of SWI/SNF complex genes, including PBRM1, ARID1A and SMARCA4, were found notably in ccRCC (47.1%), pRCC (53.0%) and chRCC (14.9%). It was thus plausible that ARID1A mutation might get involved in development and/or progression of RCC.
We further investigated mutation pro les of ARID1A gene in patients with kidney cancers. From a total of 32 mutations found, the majority came from frameshift (14/32) [19][20][21]. Due to these mutations found in ARID1A, the structural and functional complexes of ARID1A protein and SWI/SNF would be expected to be affected in cancer patients.
Although three common predictive and prognostic markers, including VHL, VEGF (vascular endothelial growth factor) and CAIX (carbonic anhydrase IX) have been validated in RCC, their uses in clinical routine are still regarded as controversial [22]. Recently, P4HB (Prolyl 4-hydroxylase, beta polypeptide) [23] and ve-proteins panel (APC; adenomatous polyposis coli, NOTCH1; neurogenic locus notch homolog protein 1, EYS; protein eyes shut homolog, lamin A and ARID1A) [24] have been proposed as novel, potential diagnostic and prognostic biomarkers for kidney renal clear cell carcinoma. In this study, we found that the patients with mutated ARID1A had a shorter overall survival than those without ARID1A mutations. The results from OSkirc web tool, which is a reliable and user-friendly analysis tool [9], also con rmed that the patients with low ARID1A expression had a signi cantly worse prognosis than those with high ARID1A expression. Consistently, several other studies have also reported that the patients with mutations or loss of ARID1A had a shorter disease-free survival and/or poorer overall outcome/survival [25][26][27]. Our results were in concordance with those reported previously indicating that ARID1A is an important tumor suppressor gene in RCC and may also serve as a prognostic biomarker in RCC patients.
Globally, most of the RCC cases are affected by three main RCC subtypes, including ccRCC (75%), pRCC (15%), and chRCC (5%) [28]. Sarcomatoid differentiation is distinct in a high-grade and poorly differentiated components and is found in only 5% of RCC [10]. In this present study, we recruited a variety of RCC subtypes and grades, including ccRCC (grade I), ccRCC (grade II), chRCC, pRCC and sRCC from patients with age of > 45 years following the recent classi cation system and histopathological descriptions [29]. To our knowledge, this is the rst evidence demonstrating ARID1A protein expression in sRCC.
Our histopathological data showed that ARID1A is localized mainly in the nuclei of lymphocytes, broblasts, intraglomerular cells and renal tubular epithelial cells in the non-neoplastic areas. By contrast, nuclear ARID1A expression was diminished in the cancer cells, especially in ccRCC and chRCC, whereas no signi cant changes were observed in pRCC and sRCC. Recent studies have demonstrated that nuclear ARID1A protein was generally found in lymphocytes, broblasts and endothelial cells in various normal tissues [30][31][32]. Therefore, expression of ARID1A in these cells is frequently used as a positive control to compare with that of cancer cells [30][31][32]. However, the precise mechanisms of the defective ARID1A expression in these cancer cells have not been thoroughly investigated. A variety of extracellular matrices, stromal cells (such as broblasts, mesenchymal cells, pericytes, endothelial cells and lymphatic vascular networks) and immune cells (such as lymphocytes, natural killer cells and tumor-associated macrophages) participate in enhancing cancer cell survival, growth, invasion and metastasis [33,34]. A recent evidence has suggested that cooperation of ARID1A and PIK3CA mutations enhanced proliferation of ovarian surface epithelial cells isolated from mice carrying mutant alleles by overproduction of interleukin-6 [35]. It is thus possible that ARID1A may help in the prevention of an in ammation-driven tumorigenesis in mouse ovarian clear-cell carcinoma [35]. Therefore, the interactions between the tumor microenvironment (de ned as the interplay between the nascent cancer cells and their surrounding environment) and ARID1A expression in these cancer cells and their effects on cancer initiation, progression and metastasis should be further investigated.
In our present study, the decrease/loss of ARID1A expression was commonly found in the nuclei of the cancer cells, especially in ccRCC and chRCC, when compared with the paired adjacent non-cancer tissues.
Such loss or decrease could be explained by mutations and molecular/epigenetic variations because the majority of the ARID1A mutations were the inactivation by nonsense and frameshift mutations throughout the gene that cloud lead to the loss of ARID1A protein expression [1,21,36]. Alternatively, inframe indel (insertion-deletion) mutations of ARID1A affecting the nuclear export signal could disturb the stability of ARID1A protein expression, like nonsense and frameshift mutations. Degradation of abnormal nuclear ARID1A is regulated by the nuclear ubiquitin-proteasome system [37]. Furthermore, epigenetic silencing might be also involved [4,5]. ARID1A gene promoter hypermethylation is recognized as the main etiologic factor for the decreasing ARID1A mRNA expression in many cancers. It has been shown that decreasing ARID1A mRNA expression is controlled by a repressive histone modi cation (H3K27Me3) in invasive breast cancers [4]. Promoter hypermethylation has been demonstrated to be related with low ARID1A mRNA expression in squamous cell carcinoma cell lines [5]. In kidney cancers, unmethylated CpG promoter of ARID1A gene has been reported [38]. However, the epigenetic analysis of ARID1A gene in renal cell carcinoma is still rarely conducted and further studies are needed to address the precise role of epigenetics in regulation of ARID1A expression.
Currently, there is no clearly speci c method for investigating ARID1A protein expression although some interpretation systems, such as the three staining grades [30] and Histo-score [39], had been used. In this study, we employed the Allred scoring and grading system to quantify ARID1A protein expression in the renal tissues. This strategy was also applied in a recent study evaluating ARID1A expression in breast cancer tissues [31]. The Allred scores of ARID1A protein expression in cancer tissues, particularly ccRCC (grade I), ccRCC (grade II) and chRCC, were signi cantly lower than in the paired non-cancer tissues, whereas those of pRCC and sRCC subtypes had no signi cant changes. Interestingly, a previous report has shown a similar results indicating that ARID1A protein expression was signi cantly decreased mostly in ccRCC [7]. In addition, the four-grade system allowed us to discriminate the RCC patients with negative to weak expression from those with moderate to strong expression. Previous studies have reported associations of the decrease/loss of ARID1A expression with clinical factors, such as the higher nuclear grade, larger tumor sizes, higher pTNM stage, and presence of metastatic of cancer in ccRCC patients [7,8]. In this study, we showed that the decrease/loss of ARID1A expression was signi cantly associated with ccRCC (grade II) and chRCC subtypes, presence of comorbidity, and low eGFR levels. As a result, the RCC patients with negative to weak ARID1A expression tended to have lower progression-free survival, consistent with another study in cervical cancer demonstrating that the patients with ARID1A loss had signi cantly lower survival [40].
In this study, statistical analysis by a binary logistic regression model and the Gaussian model was also performed to assess the prognostic variable to a progressive-free survival of patients. Univariated analysis revealed that negative ARID1A expression, high AJCC stage (II-IV), and presence of chronic kidney disease were signi cantly associated with the progressive-free survival, but negative ARID1A expression was not an independent prognostic factor for patients with RCCs in multivariate analysis. However, due to the small sample size, the prognostic signi cance of ARID1A expression in RCCs requires clari cation in further studies, using the Cox proportional hazard model with adequate sample size.
In summary, we have shown herein that ARID1A was one among the most common mutated genes found in kidney cancers. Immunohistochemistry demonstrated that ARID1A protein expression was markedly decreased in the RCC cancer tissues (particularly ccRCC and chRCC) as compared to the adjacent noncancer area. Additionally, level of ARID1A protein expression in the adjacent non-cancer renal tissues was signi cantly lower in ccRCC (grade II) than in ccRCC (grade I). Moreover, all of the RCC cases with negative ARID1A protein expression (3/11 cases with ccRCC (grade II) and 2/6 chRCC cases) had metastasis 1−50 months after the surgical removal. Finally, progression-free survival tended to be shorter in RCC patients with negative to weak ARID1A expression. Taken together, these data implicate that the defect/loss of ARID1A expression is associated with poor prognosis and metastasis of RCC and thus may serve as the prognostic marker of RCC, particularly ccRCC and chRCC subtypes.

Declarations
Con ict of interest statement  using log-rank test.