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Egyptian Journal of Pathology:
doi: 10.1097/01.XEJ.0000429926.01489.93
Original Articles

Expression of p21 and PTEN in renal cell carcinoma in correlation with the different clinicopathological parameters

El Deeb, Nevine M.F.a; Hamza, Mervat A.a; Abou Youssef, Tamer M.b; Ashour, Guheina A.R.c

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Author Information

Departments of aPathology

bUrosurgery, Faculty of Medicine, University of Alexandria, Alexandria, Egypt

cMinistry of Health, Benghazi, Libya

Correspondence to Nevine M.F. El Deeb, MD, Department of Pathology, Faculty of Medicine, University of Alexandria, 21521 Alexandria, Egypt Tel: +20 111 254 6116; fax: +002 03 4847426; e-mail: nevineldeeb@yahoo.com

Received January 10, 2013

Accepted February 15, 2013

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Abstract

Objective: The aim of this study was to investigate the expression of p21 and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in renal cell carcinoma (RCC) and to evaluate their correlation with clinicopathological variables.

Materials and methods: Using immunohistochemistry, we studied the expression of p21 and PTEN in a tissue microarray of 50 cases of RCC, including different histological subtypes. Marker expression was correlated to clinicopathological variables.

Results: p21 nuclear immunostaining was observed in 72% of cases. Higher p21 expression was significantly associated with higher stage RCCs (P=0.022) and with worse patient outcome (P=0.050). Higher expression of p21 was observed in metastatic RCC and in RCC with venous invasion; however, this relation was not statistically significant (P=0.330 and 0.414, respectively). PTEN was expressed in 80% of cases. Papillary RCC showed a significantly higher expression of PTEN compared with clear cell RCC (P=0.003). There was an inverse correlation between the levels of expression of p21 and PTEN, but this was not statistically significant (P=0.095).

Conclusion: Assessment of p21 and PTEN expression in RCC may aid prediction of the biologic behavior of the tumor and identification of the appropriate candidates for targeted therapy. Higher p21 expression could be considered a poor prognostic factor for RCC. Papillary RCC show higher PTEN expression compared with clear cell RCC, and thus may less likely benefit from the mammalian target of rapamycin inhibitor therapy.

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Introduction

Renal cell carcinoma (RCC) is a highly aggressive tumor and is the most lethal of all urologic malignancies. Approximately 25% of patients present with metastatic disease and up to 40% of patients experience recurrence following surgery for clinically localized disease (Lam et al., 2009). The significant disparity in the survival of patients with localized versus metastatic disease, lack of effective systemic therapy, and the highly variable natural history of RCC highlight the need to identify a reliable set of molecular markers of progression as well as targets for novel therapies (Weiss et al., 2007). Tumor stage and nuclear grade are the main traditional pathological prognostic factors in RCC. However, finding better prognostic markers remains a major goal in order to optimize patient selection for specific therapeutic approaches (Bukowski et al., 2004).

p21 is a cell cycle-regulating and apoptosis-regulating protein with multiple and seemingly diverse functions in normal and tumor cells. It has been shown to have both proapoptotic and antiapoptotic functions (Park et al., 2008). Because of its placement in the tumor suppressor pathway downstream of p53, this protein has received considerable attention as a possible conveyer of the p53 repair and proapoptotic signals (Weiss et al., 2007). The antiapoptotic function of p21 allows cells with damaged DNA to survive while repairs to their DNA can be made. However, this same mechanism, which is beneficial in the case of normal cells, can also result in failure of chemotherapy by allowing cells with damaged DNA to repair the damage induced by chemotherapy. In this manner, p21 may contribute toward tumor progression (Roninson, 2002) and attenuation of this protein may have potential in the treatment of malignancy (Park et al., 2008).

p21 has been suggested to be a novel therapeutic target as well as a prognostic marker in RCC (Weiss et al., 2007). It has been proposed that p21 plays a major role in the mechanisms by which tumors deficient in phosphatase and tensin homolog deleted on chromosome 10 (PTEN) acquire resistance to chemotherapy (Lin et al., 2007).

PTEN is one of the most frequently inactivated tumor suppressors in human cancer and its deregulation has also been implicated in other diseases (Leslie and Downes, 2004). The PTEN gene encodes a dual lipid and tyrosine phosphatase that controls signaling through the phosphatidylinositol-3 kinase (PI3K)/Akt pathway, preventing cells from growing and dividing too rapidly and acting as a tumor suppressor protein that is commonly mutated or deleted in human cancers (Lin et al., 2007). As RCC is a malignancy associated with frequent treatment failures when metastatic, and because RCC and other cancers lacking PTEN often resist conventional chemotherapy, the mechanism by which PTEN contributes toward chemotherapy failure is of high clinical importance and may lead to new therapeutic options for patients with such tumors (Wendel et al., 2006).

The aim of the present work was to study the expression of p21 and PTEN in RCC and to correlate their expression with the different clinicopathological features.

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Materials and methods

Specimens and clinical data

This study was carried out on 50 consecutive cases of RCC. Specimens were submitted to the Pathology Department, Faculty of Medicine, Alexandria University, during the period from July 2009 to November 2010. Specimens included radical nephrectomy (38 cases) and partial nephrectomy (12 cases). Ten cases had preaortic and/or para-aortic lymphadenectomy. The clinical and radiological data were collected from the archives of the Pathology and Urosurgery Departments, Faculty of Medicine, Alexandria University. The outcome was determined after a follow-up period from the date of diagnosis to the date of death or the last follow-up before study closure (minimum follow-up period: 12 months).

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Histopathological examination

The histopathology of all cases was reviewed on complete tissue sections to determine the histological type and grade of the tumor, presence/absence of invasion of the capsule, perinephric fat, renal sinus, Gerota’s fascia and renal vein, and also for the detection of lymph node involvement.

The histological type of RCC was determined according to the Heidelberg and UICC/AJCC classification (Kovacs et al., 1997). Tumor grading was performed according to the Fuhrman grading system (Fuhrman et al., 1982) and staging was carried out according to the 2009 TNM staging system (Sobin et al., 2009).

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Tissue microarray construction (Kononen et al., 1998)

H&E-stained sections of RCC were used for the selection of morphologically representative regions of each tumor for tissue microarray (TMA) study. Two tumor spots were chosen under microscopy for each case and the corresponding spots were marked on the tissue block. A manual tissue arrayer punch (Beecher Instruments Inc., Sun Prairie, Wisconsin, USA) was used to remove tissue cores 1 mm in diameter in the marked area on the donor block. These tissue cores were then transferred to corresponding receiver pores in the recipient paraffin block, arranged in a precisely spaced array pattern in order to eventually construct a TMA block according to a predetermined scheme. The block was heated at 40°C for 15 min and the surface was flattened. Sections from this block were cut using a microtome. An H&E-stained section of each TMA block was used to establish the adequacy of sampling by ensuring representative selection for the histological type and Fuhrman grade of RCC. Other sections were mounted on charged slides for immunohistochemical staining.

A hundred tumor spots representing the 50 cases of RCC studied were performed (two spots per case). In addition, four spots of normal kidney were used as control spots. Results were interpreted with reference to a map of the TMA, with labeled rows and columns and their corresponding case number.

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Immunohistochemical staining

Immunohistochemical staining was performed on 5 μm-thick sections cut from the tumor TMA block. The TMA paraffin sections were deparaffinized in xylene, rehydrated in descending grades of alcohol, and then immersed in 0.3% hydrogen peroxide in methanol for 20 min to inhibit endogenous peroxidase activity. Antigen retrieval was performed by placing the TMA slides in citrate buffer (0.01 mol/l, pH 6.0) in a 700 W microwave oven for 8 min. Slides were allowed to cool to room temperature, and then an ultra V block was applied for 3–5 min to block nonspecific background staining.

The following primary antibodies were applied: p21 WAF1 Ab-5 (Clone HZ52) mouse monoclonal antibody (Lab Vision, Fremont, California, USA; diluted 1 : 10) and PTEN Ab-4 (Clone 17.A) mouse monoclonal antibody (Lab Vision; diluted 1 : 100). The sections were incubated overnight at 4°C in a humidity chamber. The TMA slides were then incubated with biotinylated rabbit antipolyvalent secondary antibody for 20 min, and then in peroxidase-conjugated steptavidin for 20 min at room temperature. The sections were washed with PBS for 5 min after each step. The reaction product was developed using a diaminobenzidine tetrachloride mixture for 10 min.

Then, the TMA slides were rinsed with tap water for 5 min, counterstained with hematoxylin stain, dehydrated in ascending grades of alcohol, cleared in xylene, and mounted. The positive control used was a case of colorectal carcinoma for p21 and normal kidney tissue for PTEN. Sections where the primary antibody has been omitted served as negative controls.

In addition, for each histological subtype of RCC, two 5-μm-thick complete tissue sections cut from formalin-fixed, paraffin-embedded blocks of the tumor were immunostained as described, one with p21 and one with PTEN, to evaluate the distribution and consistency of the protein expression throughout a given tumor.

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Interpretation of immunohistochemical staining
p21

Assessment of p21 staining was performed under a magnification of ×400. The percentage of tumor cells showing nuclear staining was categorized semiquantitatively as follows (Røtterud et al., 2001): score 0: no staining; score 1: nuclear staining in more than 0–5% of tumor cells; score 2: nuclear staining in more than 5–50% of tumor cells; and score 3: nuclear staining in more than 50% of tumor cells. Cytoplasmic expression was scored on a scale of 0 to 100 reflecting a combined distribution and intensity within the core (Weiss et al., 2007).

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PTEN

Immunostaining of PTEN was assessed as follows (David, 2005): score 0: no appreciable staining in tumor cells; score 1: barely detectable staining in the cytoplasm, nucleus, or both compared with stromal elements; score 2: readily appreciable staining distinctly marking the cytoplasm, nucleus, or both; and score 3: strong staining in tumor cells completely obscuring the cytoplasm, nucleus, or both.

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Statistical analysis

Statistical analysis was carried out using the SPSS software package, version 17.0 (SPSS, Chicago, Illinois, USA). Continuous variables were expressed as mean±SD, whereas categorical variables were expressed as numbers and percentages. Statistical correlations between two categorical variables were assessed using the χ2 or the Fisher exact test. Statistical correlations between categorical and continuous variables were assessed using the Mann–Whitney U-test. For statistical purposes, on assessment of correlation of p21 and PTEN staining with patients’ outcome, cases were divided into two groups: negative/low expression (scores 0 and 1) versus moderate/high expression (scores 2 and 3). The level of significance was set at a P value of up to 0.05.

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Results

Clinicopathological data

This study included 50 cases of RCC. Patient ages ranged from 18 to 95 years (mean 50.64±15.19 years). Thirty-two patients (64%) were men and 18 (36%) were women. The size of the tumor ranged from 4 to 21 cm (mean 17.77±10.08 cm); in eight cases (16%) the tumor was up to 7 cm and in 42 cases (84%) it was greater than 7 cm in diameter. Multicentric tumor masses were seen in four cases (8%). Invasion of the renal capsule and perinephric fat was detected in 10 cases (20%), renal sinus invasion in five cases (10%), Gerota’s fascia invasion in one case (2%), adrenal gland invasion in one case (2%), and invasion of the collecting system in two cases (4%). Lymph node metastases were found in five out of the 10 patients (50%) who had undergone lymphadenectomy.

In the present study, five histological types of RCC were recognized (according to Heidelberg and UICC/AJCC classification): 32 cases (64%) were clear cell RCC (CCRCC); 12 cases (24%) were papillary RCC (PRCC); three cases (6%) were chromophobe RCC (chRCC); one case (2%) was collecting duct RCC (CDRCC); and two cases (4%) were RCC with sarcomatoid change (SRCC). Eleven cases (22%) were Fuhrman grade 1; 21 cases (42%) were grade 2; 14 cases (28%) were grade 3; and four cases (8%) were grade 4. According to the TNM staging system 2009, 17 cases (34%) were stage I; eight cases (16%) were stage II; 10 cases (20%) were stage III; and 15 cases (30%) were stage IV. Fifteen cases (30%) were metastatic. Venous invasion was found in seven cases (14%) (Table 1). In terms of the outcome, 31 patients (62%) showed no evidence of disease, 15 patients (30%) were alive with disease, and four patients (8%) died of their disease (Table 2).

Table 1
Table 1
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Table 2
Table 2
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Immunohistochemical staining of TMA

The tissue microarray technique was applied in this study. The total number of spots performed was 104; 100 spots represented the 50 studied cases of RCC (two spots per case) and four spots of normal kidney represented the control spots. A total of 96 tissue spots were informative for immunohistochemistry analysis including 61 CCRCC, 20 PRCC, five chRCC, two CDRCC, four SRCC, and four normal kidney tissue.

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Expression of p21

Immunostaining showed that p21 was localized in the cytoplasm of normal renal tubular epithelium (Fig. 1a), whereas in tumor cells, immunoreactivity was most often nuclear (Fig. 1b–f). Thirty-six out of the 50 studied cases (72%) expressed p21. Analysis of the RCC tissue microarray for nuclear p21 expression showed the following distribution: negative score 0, 14 cases (28%); score 1, 20 cases (40%); score 2, nine cases (18%); and score 3, seven cases (14%).

Fig. 1
Fig. 1
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p21 nuclear expression was found to increase with increasing tumor stage and this relation was statistically significant (P=0.022) (Table 1). In addition, a significant relation was found between p21 nuclear expression and the disease outcome of patients (P=0.050) (Table 2).

p21 expression scores were higher in metastatic RCC than in nonmetastatic RCC, where 60% of metastatic cases had moderate/high scores (scores 2 and 3) versus 20% of nonmetastatic cases. p21 expression scores were also higher in RCC with venous invasion than in RCC without venous invasion, where 85.72% of RCC with venous invasion had moderate/high scores (scores 2 and 3) versus 23.25% of cases without venous invasion, but these findings were not statistically significant (P=0.330 and 0.414, respectively). No significant correlation was found between p21 expression and each of the tumor histological subtype, tumor grade, tumor size, patient age, and patient sex (P=0.264, 0.543, 0.089, 0.123, and 0.981, respectively) (Table 1).

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Expression of PTEN

In normal renal tubular epithelium, PTEN immunostaining was always cytoplasmic (Fig. 2a). In tumor cells, immunoreactivity was most often cytoplasmic. Nuclear reactivity was also observed but less often (Fig. 2b–f). Forty out of the 50 studied cases (80%) expressed PTEN. Analysis of the RCC tissue microarray for cytoplasmic and nuclear PTEN expression indicated the following distribution: negative score 0, 10 cases (20%); score 1, 19 cases (38%); score 2, 12 cases (24%); and score 3, nine cases (18%).

Fig. 2
Fig. 2
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The PTEN expression score was significantly higher in PRCC than in CCRCC (P=0.003). In addition, PTEN expression was significantly higher in men than in women (P=0.017) (Table 1).

PTEN expression scores were lower in metastatic RCC than in nonmetastatic RCC, where 66.66% of metastatic cases showed a negative/low expression (scores 0 and 1) versus 54.29% of nonmetastatic cases. PTEN expression scores were also lower in RCC with venous invasion than in RCC without venous invasion, where 71.43% of RCC with venous invasion showed a negative/low expression (scores 0 and 1) versus 55.81% of cases without venous invasion. However, these findings were not significant (P=0.127 and 0.197, respectively). No significant correlation was found between PTEN expression and each of tumor size, tumor stage, and tumor grade as well as patient age and the disease outcome of patients (P=0.116, 0.675, 0.211, 0.226, and 0.530, respectively) (Tables 1 and 2).

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Relation between p21 and PTEN expression

Out of the 34 cases with a negative/low p21 expression (scores 0 and 1), 17 cases (50%) showed a negative/low expression of PTEN (scores 0 and 1) and 17 cases (50%) showed a moderate/high expression of PTEN (scores 2 and 3). Out of the 16 cases with a moderate/high p21 expression (scores 2 and 3), 12 cases (75%) showed a negative/low expression of PTEN (scores 0 and 1) and four cases (25%) showed a high expression of PTEN (scores 2 and 3). The relation between p21 and PTEN expression was not statistically significant (P=0.095) (Table 3).

Table 3
Table 3
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Discussion

RCC is characterized by resistance to DNA-damaging chemotherapy. The mechanism of this effect, although incompletely understood, appears to be related to activation of survival pathways that occurs in the setting of repair of DNA damage (Lin et al., 2007).

p21 is a cyclin kinase inhibitor that is induced by p53 in situations of DNA damage. It likely plays a role in the decision pathways leading to apoptosis or DNA repair (Weiss, 2003). p21 has been shown to have multiple and diverse effects in various cancer-derived and noncancer-derived cell lines. Such effects include cell cycle promotion through cyclin/cyclin-dependent kinase assembly, positive effect on cell proliferation, and, more recently, its pleotropic functions in apoptosis (Park et al., 2008).

In the present work, immunohistochemical analysis of p21 was carried out in 50 cases of RCC including different histological subtypes and in normal kidney tissue. In tumor cells, immunoreactivity was most often nuclear. Cytoplasmic staining was encountered less often. In normal renal tubular epithelium, p21 staining was cytoplasmic. Interestingly, the subcellular localization of p21 may determine the differences in the function of this protein; cytoplasmic p21 has been shown to be pro-proliferative in vascular smooth muscle cells (Dong et al., 2004), whereas proliferating cell nuclear antigen binding and cyclin/cyclin-dependent kinase interaction occur when p21 is in its usual intranuclear location (Rössig et al., 2001).

In the present study, the relation between p21 nuclear immunostaining and the histological subtype of RCC was not statistically significant. It has been reported previously that nuclear p21 expression was the highest in CDRCC compared with all other RCC subtypes (Lam et al., 2007). This finding could not be assessed in the current work, because only one case of CDRCC was included.

In the present work, a statistically significant relation was found between p21 expression and the stage of RCC, where higher p21 nuclear expression scores were detected in more advanced stages of RCC. This finding is in agreement with the findings reported by Weiss et al. (2007), who reported that immunohistochemical expression of p21 correlates with more aggressive disease. Moreover, Perroud et al. (2006) reported that elevated levels of p21 predict a poorer prognosis in patients who have metastatic CCRCC. Contradictory results have been reported on the prognostic value of p21 in RCC. Samlowski (2009) reported that high p21 expression was associated with better prognosis in patients with localized RCC, whereas it was an indicator of poor prognosis in patients with metastatic RCC. Meanwhile, Haitel et al. (2001) found no prognostic value for p21 expression in RCC.

The present study also showed that higher p21 expression (scores 2 and 3) was more frequent in metastatic RCC and in RCC with venous invasion in comparison with nonmetastatic RCC and RCC without venous invasion. Although not reaching statistical significance, these findings suggest that p21 expression is associated with more aggressive disease.

A statistically significant relation was found, in the current work, between p21 expression and patient outcome, where moderate/high scores of p21 nuclear immunostaining (scores 2 and 3) were found in 75% of cases who died of their disease as compared with 35.48% of cases with no evidence of disease. These results suggest that p21 may have a prognostic value in RCC. Such findings are in agreement with the findings reported by Lam et al. (2007), who suggested that p21 may be a useful prognostic marker for RCC and may help in selection of patients for specific therapeutic approaches.

In the present study, the relation between p21 nuclear immunostaining, on the one hand, and tumor size, age, and sex of RCC patients, on the other, was statistically insignificant. In addition, the relation between p21 nuclear expression and RCC grade was not significant. Weiss et al. (2007) reported that p21 nuclear positivity correlated with tumor grade in CCRCC, but was only significantly increased in grade 3 compared with grade 1. The paradox that p21 nuclear expression correlated with tumor stage but not with tumor grade or size in the present study may be explained by the fact that nuclear grade has been reported to be an important predictor of survival in CCRCC but not in papillary, chromophobe, or SRCC (Medeiros et al., 1997; De Peralta-Venturina et al., 2001; Minervini et al., 2002), which altogether represent 35% of our cases. In addition, 50% of our cases were stages III and IV, in which other factors (other than tumor size), namely, tumor extension beyond the kidney, lymph node metastasis, and distant metastasis contribute toward tumor stage.

It is well known that inactivation of apoptosis is essential for cancer development. Thus, a major strategy for chemotherapy involves channeling malignant cells into apoptotic pathways, and, consequently, a frequent cause of failure of chemotherapy results from malfunction of apoptosis. As p21 is increased in DNA damaged cells, likely in an attempt to repair the damage, its antiapoptotic property can be used to therapeutic advantage in chemotherapy-resistant cancer. Transient attenuation of this protein in combination with conventional DNA-damaging chemotherapy would sensitize kidney cancer cells to apoptosis by directing cancerous cells into the apoptotic, rather than the DNA repair pathway (Park et al., 2008). It has been suggested that p21 plays a key role in the mechanisms by which tumors deficient in PTEN resist chemotherapy (Lin et al., 2007).

PTEN is a ubiquitous regulator of the cellular PI3k signaling pathway. This pathway is characterized by the regulated activation of class I PI 3-kinase enzymes, producing phosphatidylinositol 3,4,5-trisphosphate (PIP3), which, in turn, mediates downstream signaling through a range of effector proteins, including the proto-oncogene product protein kinase B (PKB)/Akt. PTEN antagonizes PI3k signaling by dephosphorylating PIP3, thus inactivating downstream signaling and inhibiting cellular proliferation, survival, and growth (Leslie and Downes, 2004). The PTEN tumor suppressor gene is frequently mutated or deleted in a wide variety of cancers and these tumors are generally more aggressive and resistant to treatment than those with the wild-type PTEN (Lin et al., 2007). RCC is commonly associated with treatment failures (∼90% in metastatic cases), and these tumors frequently have PTEN abnormalities (Youssif et al., 2011).

In the present work, TMA sections representing the 50 studied cases of RCC were stained with the anti-PTEN antibody and compared with normal kidney control. The normal kidney showed diffuse strong cytoplasmic staining of tubular epithelial cells. In RCCs, PTEN staining was predominantly cytoplasmic in the majority of cases (only 10% of the cases showed predominantly nuclear staining) and varied in different cases. These findings are in agreement with those of Pantuck et al. (2007), who reported that the anti-PTEN antibody stained the cytoplasm of RCCs and that tumors showed a lower expression than normal renal tissues. Similarly, Perren et al. (1999) and Zhou et al. (2002) described tumors with reduced PTEN expression relative to the surrounding tissues in their studies on colorectal cancer and breast cancer, respectively. Immunohistochemical studies of a variety of tissues have shown that the protein is largely cytoplasmic, although in some cases, predominantly nuclear staining was found (Perren et al., 1999; Tachibana et al., 2002; Zhou et al., 2002). It has been suggested that nuclear compartmentalization of PTEN may be related to its tumor-suppressive activity; however, its nuclear function remains poorly defined (Song et al., 2011).

In the current work, 58% of the studied cases of RCC showed a negative/low PTEN expression (scores 0 and 1). Similarly, Sitaram et al. (2009) reported that reduced PTEN protein expression is encountered in a high frequency in RCC. Meanwhile, Figlin et al. (2005) reported PTEN loss in only 20% of RCCs. PTEN protein and gene expression have been described variously as reduced (Hara et al., 2005), absent (Brenner et al., 2002), mutated (Alimov et al., 1999), or deleted (Sükösd et al., 2001) in human RCCs. Brenner et al. (2002) reported that loss of PTEN occurs during renal carcinogenesis. There is substantial evidence now that loss of PTEN expression is far more frequent than mutation of the PTEN gene in most cancers; thus, future research must focus on processes regulating the transcription of the PTEN gene and/or turnover of the PTEN protein (Leslie and Downes, 2004).

In the present study, PRCC showed significantly higher PTEN expression than CCRCC, suggesting that PRCC may less likely benefit from mammalian target of rapamycin (mTOR) inhibitor therapy than CCRCC. Furthermore, two out of the three studied cases of chRCC showed high PTEN expression. These findings are in agreement with those of Pantuck et al. (2007), who reported higher PTEN expression in nonclear cell subtypes of RCC, and thus proposed that not all RCC tumor types are equally amenable to mTOR-targeted therapy.

In the current work, most of stage IV cases and all the Fuhrman grade 4 cases showed negative/low PTEN expression (scores 0 and 1). In addition, negative/low PTEN expression (scores 0 and 1) was more frequently observed in metastatic RCC and in RCC with venous invasion in comparison with nonmetastatic RCC and RCC without venous invasion. Although not reaching statistical significance, these results predict that patients with lower PTEN expression do worse than those with higher PTEN expression. These findings are in agreement with those of Leslie and Downes (2004), who reported that PTEN loss is associated with adverse prognosis in RCC. In addition, Cho et al. (2007) reported that lack of PTEN expression is a negative prognostic factor for disease-specific survival in patients with metastatic RCC. The lack of statistical significance in our results may be attributed to the limited number of cases included.

On correlation of PTEN expression with patient outcome, negative/low expression (scores 0 and 1) was detected in 75% of patients who died of their disease, 60% of patients who were alive with disease, and 54.84% of patients with no evidence of disease. This is in agreement with results obtained by Kim et al. (2005), who reported that low PTEN expression correlates with worse survival. PTEN as well as pAkt, p27, and pS6 have been suggested to serve as surrogate parameters for predicting prognosis in RCC (Pantuck et al., 2007).

One of the important pathways related to cancer is the mTOR pathway, which involves downstream signaling from PI3K/Akt that leads to phosphorylation of mTOR, thus activating its substrate, p70S6 kinase, in turn promoting mRNA translation, cell cycle progression, and angiogenesis. This pathway is of special interest in RCC because of frequent inactivation of PTEN in these tumors (Youssif et al., 2011). In PTEN-deficient cells, increased PIP3 levels result in constitutive activation of PKB/Akt, which itself lies upstream of mTOR, with subsequent stimulation of growth-promoting pathways. Thus, the mTOR inhibitors may be ideally suitable for use in RCC (Weiss and Lin, 2006) and patients with a highly activated mTOR pathway are expected to benefit most from this therapy (Pantuck et al., 2007). The therapeutic benefit from mTOR inhibitors has been variable among RCC patients who were selected only with respect to patient-related clinical factors, without considering tumor-related factors (Youssif et al., 2011). Immunohistochemical detection of PTEN expression in RCC, as performed in the present work, may be helpful in selection of patients who can benefit from mTOR inhibitor therapy.

In the present study, p21 expression was correlated inversely with PTEN expression, but the statistically significant level was not reached, possibly because of the limited number of cases studied. The inverse correlation between p21 and PTEN expression has also been reported by Yoo et al. (2006), who detected higher levels of p21 protein and transcripts in PTEN-deficient bladder tissue. Chen et al. (2005) observed a similar phenomenon in PTEN-deficient prostate tissue, which they interpreted as confirming the activation of a p21-related senescence pathway. The inverse correlation between p21 and PTEN expression may be explained by the fact that PTEN inactivation renders it unable to carry out its phosphatase function. PIP3 can no longer be deactivated; thus, it continues to propagate its signal downstream, with continuous activation of PKB/Akt. p21 is phophorylated by Akt. This phophorylation increases p21 stability (Zhou et al., 2001). Thus, PTEN inactivation augments p21 stability.

Using a PTEN knockdown cell line, Lin et al. (2007) showed that PTEN attenuation increases resistance to cisplatin-induced apoptosis, a finding that was associated with increased levels of p21. They concluded that augmentation of p21 may be an important mechanism through which PTEN-altered tumors acquire resistance to chemotherapy. They also reported that PTEN-deficient tumors are sensitive to mTOR inhibitors, which, in turn, sensitize tumors to DNA damage-induced apoptosis through inhibition of p21 translation.

Recent breakthroughs have led to greater knowledge of the molecular genetic and oncogenic pathways of RCC. This would allow for individualized application of systemic and targeted therapies on the basis of the molecular signature of the tumor to be targeted, thus ultimately improving patient outcome (Leppert et al., 2007). The findings of the present work suggest that p21 expression may serve as a poor prognostic indicator in RCC and that PRCC show higher PTEN expression, and thus may less likely benefit from mTOR inhibitor therapy as compared with CCRCC. Immunohistochemical detection of expression of p21 and PTEN may help in identification of the appropriate candidates for targeted therapy; however, larger studies on these two markers are required before such therapeutic modalities become established.

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Acknowledgements
Conflicts of interest

There are no conflicts of interest.

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References

Alimov A, Li C, Gizatullin R, Fredriksson V, Sundelin B, Klein G, et al..Somatic mutation and homozygous deletion of PTEN/MMAC1 gene at 10q23 in renal cell carcinoma.Anticancer Res1999;195B3841–3846.

Brenner W, Färber G, Herget T, Lehr H-A, Hengstler JG, Thüroff JW.Loss of tumor suppressor protein PTEN during renal carcinogenesis.Int J Cancer2002;99:53–57.

Bukowski RM, Negrier S, Elson P, Yang JC.Prognostic factors in patients with advanced renal cell carcinoma: development of an international kidney cancer working group.Clin Cancer Res2004;1018 II6310s–6314s.

Chen Z, Trotman LC, Shaffer D, Lin H-K, Dotan ZA, Niki M, et al..Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis.Nature2005;436:725–730.

Cho D, Signoretti S, Regan M, Mier JW, Atkins MB.The role of mammalian target of rapamycin inhibitors in the treatment of advanced renal cancer.Clin Cancer Res2007;13II758s–762s.

David OHayat MA.PTEN and cancer.Handbook of immunohistochemistry and in situ hybridization of human carcinomas2005.Burlington, MA, USA: Elsevier Academic Press93–101.

De Peralta-Venturina M, Moch H, Amin M, Tamboli P, Hailemariam S, Mihatsch M, et al..Sarcomatoid differentiation in renal cell carcinoma: a study of 101 cases.Am J Surg Pathol2001;25:275–284.

Dong Y, Chi SL, Borowsky AD, Fan Y, Weiss RH.Cytosolic p21Waf1/Cip1 increases cell cycle transit in vascular smooth muscle cells.Cell Signal2004;16:263–269.

Figlin RA, Seligson DB, Wu H, Thomas G, Leppert JT, O'Toole T, et al..Characterization of the mTOR pathway in renal cell carcinoma and its use in predicting patient selection for agents targeting this pathway.J Clin Oncol2005;23:4539.

Fuhrman SA, Lasky LC, Limas C.Prognostic significance of morphologic parameters in renal cell carcinoma.Am J Surg Pathol1982;6:655–663.

Haitel A, Wiener HG, Neudert B, Marberger M, Susani M.Expression of the cell cycle proteins P21, P27, and PRB in clear cell renal cell carcinoma and their prognostic significance.Urology2001;58:477–481.

Hara S, Oya M, Mizuno R, Horiguchi A, Marumo K, Murai M.Akt activation in renal cell carcinoma: contribution of a decreased PTEN expression and the induction of apoptosis by an Akt inhibitor.Ann Oncol2005;16:928–933.

Kim HL, Seligson D, Liu X, Janzen N, Bui MHT, Yu H, et al..Using tumor markers to predict the survival of patients with metastatic renal cell carcinoma.J Urol2005;173:1496–1501.

Kononen J, Bubendorf L, Kallioniemi A, Bärlund M, Schraml P, Leighton S, et al..Tissue microarrays for high-throughput molecular profiling of tumor specimens.Nat Med1998;4:844–847.

Kovacs G, Akhtar M, Beckwith BJ, Bugert P, Cooper CS, Delahunt B, et al..The Heidelberg classification of renal cell tumours.J Pathol1997;183:131–133.

Lam JS, Pantuck AJ, Belldegrun AS, Figlin RA.Protein expression profiles in renal cell carcinoma: staging, prognosis, and patient selection for clinical trials.Clin Cancer Res2007;13II703s–706s.

Lam JS, Klatte T, Breda A.Staging of renal cell carcinoma: current concepts.Indian J Urol2009;25:446–454.

Leppert JT, Pantuck AJ, Figlin RA, Belldegrun AS.The role of molecular markers in the staging of renal cell carcinoma.BJU Int2007;995B1208–1211.

Leslie NR, Downes CP.PTEN function: how normal cells control it and tumour cells lose it.Biochem J2004;382:1–11.

Lin P-Y, Fosmire SP, Park S-H, Park J-Y, Baksh S, Modiano JF, Weiss RH.Attenuation of PTEN increases p21 stability and cytosolic localization in kidney cancer cells: a potential mechanism of apoptosis resistance.Mol Cancer2007;6:16.

Medeiros LJ, Jones EC, Aizawa S, Aldape HC, Cheville JC, Goldstein NS, et al..Grading of renal cell carcinoma: Workgroup No. 2. Union Internationale Contre le Cancer and the American Joint Committee on Cancer (AJCC).Cancer1997;80:990–991.

Minervini A, Lilas L, Minervini R, Selli C.Prognostic value of nuclear grading in patients with intracapsular (pT1-pT2) renal cell carcinoma: long-term analysis in 213 patients.Cancer2002;94:2590–2595.

Pantuck AJ, Seligson DB, Klatte T, Yu H, Leppert JT, Moore L, et al..Prognostic relevance of the mTOR pathway in renal cell carcinoma: implications for molecular patient selection for targeted therapy.Cancer2007;109:2257–2267.

Park S-H, Park J-Y, Weiss RH.Antisense attenuation of p21 sensitizes kidney cancer to apoptosis in response to conventional DNA damaging chemotherapy associated with enhancement of phospho-p53.J Urol2008;180:352–360.

Perren A, Weng L-P, Boag AH, Ziebold U, Thakore K, Dahia PLM, et al..Immunohistochemical evidence of loss of PTEN expression in primary ductal adenocarcinomas of the breast.Am J Pathol1999;155:1253–1260.

Perroud B, Lee J, Valkova N, Dhirapong A, Lin P-Y, Fiehn O, et al..Pathway analysis of kidney cancer using proteomics and metabolic profiling.Mol Cancer2006;5:64.

Roninson IB.Oncogenic functions of tumour suppressor p21Waf1/Cip1/Sdi1: association with cell senescence and tumour-promoting activities of stromal fibroblasts.Cancer Lett2002;179:1–14.

Rössig L, Jadidi AS, Urbich C, Badorff C, Zeiher AM, Dimmeler S.Akt-dependent phosphorylation of p21Cip1 regulates PCNA binding and proliferation of endothelial cells.Mol Cell Biol2001;21:5644–5657.

Røtterud R, Berner A, Holm R, Skovlund E, Fosså SD.p53, p21 and mdm2 expression versus the response to radiotherapy in transitional cell carcinoma of the bladder.BJU Int2001;88:202–208.

Samlowski W.Targeted molecular therapy for renal cell carcinoma: impact on existing treatment paradigms.J Manag Care Med2009;13:40–45.

Sitaram RT, Cairney CJ, Grabowski P, Keith WN, Hallberg B, Ljungberg B, Roos G.The PTEN regulator DJ-1 is associated with hTERT expression in clear cell renal cell carcinoma.Int J Cancer2009;125:783–790.

Sobin LH, Gospodarowicz MK, Wittekind C.TNM classification of malignant tumours. UICC International Union against cancer2009:7th ed..Weinheim, Germany:Wiley-Blackwell;255–257.

Song MS, Carracedo A, Salmena L, Song SJ, Egia A, Malumbres M, Pandolfi PP.Nuclear PTEN regulates the APC-CDH1 tumor-suppressive complex in a phosphatase-independent manner.Cell2011;144:187–199.

Sükösd F, Digon B, Fischer J, Pietsch T, Kovacs G.Allelic loss at 10q23.3 but lack of mutation of PTEN/MMAC1 in chromophobe renal cell carcinoma.Cancer Genet Cytogenet2001;128:161–163.

Tachibana M, Shibakita M, Ohno S, Kinugasa S, Yoshimura H, Ueda S, et al..Expression and prognostic significance of PTEN product protein in patients with esophageal squamous cell carcinoma.Cancer2002;94:1955–1960.

Weiss RH.p21Waf1/Cip1 as a therapeutic target in breast and other cancers.Cancer Cell2003;4:425–429.

Weiss RH, Lin P-Y.Kidney cancer: identification of novel targets for therapy.Kidney Int2006;69:224–232.

Weiss RH, Borowsky AD, Seligson D, Lin P-Y, Dillard-Telm L, Belldegrun AS, et al..p21 is a prognostic marker for renal cell carcinoma: implications for novel therapeutic approaches.J Urol2007;177:63–69.

Wendel H-G, Malina A, Zhao Z, Zender L, Kogan SC, Cordon-Cardo C, et al..Determinants of sensitivity and resistance to rapamycin-chemotherapy drug combinations in vivo.Cancer Res2006;66:7639–7646.

Yoo LI, Liu DW, Le Vu S, Bronson RT, Wu H, Yuan J.Pten deficiency activates distinct downstream signaling pathways in a tissue-specific manner.Cancer Res2006;66:1929–1939.

Youssif TA, Fahmy MA, Koumakpayi IH, Ayala F, Al Marzooqi S, Chen G, et al..The mammalian target of rapamycin pathway is widely activated without PTEN deletion in renal cell carcinoma metastases.Cancer2011;117:290–300.

Zhou BP, Liao Y, Xia W, Spohn B, Lee M-H, Hung M-C.Cytoplasmic localization of p21 CIP1/WAF1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells.Nat Cell Biol2001;3:245–252.

Zhou X-P, Loukola A, Salovaara R, Nystrom-Lahti M, Peltomäki P, De la Chapelle A, et al..PTEN mutational spectra, expression levels, and subcellular localization in microsatellite stable and unstable colorectal cancers.Am J Pathol2002;161:439–447.

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