Gelsolin, NF-κB, and p53 expression in clear cell renal cell carcinoma: Impact on outcome

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Original Article

Gelsolin, NF-␬B, and p53 expression in clear cell renal cell carcinoma: Impact on outcome Duygu Kankaya a,∗ , Saba Kiremitci a , Ozden Tulunay a , Sumer Baltaci b a b

Department of Pathology, Medical School of Ankara University, Ankara, Turkey Department of Urology, Medical School of Ankara University, Ankara, Turkey

a r t i c l e

i n f o

Article history: Received 14 August 2014 Received in revised form 27 February 2015 Accepted 6 March 2015 Keywords: Clear cell renal cell carcinoma Gelsolin Nuclear factor kappa B p53 Prognosis

a b s t r a c t Objectives: To examine the prognostic significance of Gelsolin, NF-␬B, and p53 in clear cell renal cell carcinoma (CRCC), which has an unpredictable behavior and tendency for recurrence and metastasis. Materials and methods: Immunohistochemistry was performed on 100 consecutive cases of CRCC using antibodies against Gelsolin, NF-␬B, and p53. Tumors were grouped by nuclear grade (NG) as low NG (NG1, 2) or high NG (NG3, 4), and by pathological stage as localized (pT1, 2) or locally invasive (pT3, 4). Clinical stage was grouped as early stage (stage I, II) or late stage (stage III, IV). Evaluation was based on cytoplasmic (NF-␬BCyt ) and nuclear (NF-␬BNuc ) expression for NF-␬B, nuclear expression for p53, membranous and cytoplasmic expression for Gelsolin. Results: Gelsolin expression correlated with high NG (p = 0.001), metastasis (p = 0.003), late stage (p = 0.008), and cancer death (p = 0.001). NF-␬BCyt expression correlated with high NG (p = 0.002), perirenal invasion (p = 0.010), local invasion (p = 0.020), and late stage (p = 0.003). NF-␬BNuc expression failed to predict the prognosis of CRCC. p53 expression correlated with high NG (p = 0.045), lymphovascular invasion (p = 0.05), metastasis (p = 0.001), late stage (p = 0.028), and cancer death (p = 0.034). However, only Gelsolin was found to correlate with disease-specific survival, (p = 0.006), and neither NF-␬B nor p53 showed such relation. Conclusion: Expressions of Gelsolin, NF-␬BCyt , and p53 associated with aggressive behavior of CRCC, while Gelsolin expression specifically indicated poor disease-specific survival. The results of the present study served to determine biomarkers for predicting high-risk patients with CRCC, expected to show aggressive phenotype. © 2015 Elsevier GmbH. All rights reserved.

Introduction One-third of the patients diagnosed with renal cell carcinoma (RCC) present with metastatic lesions, or will develop these lesions during the course of the disease [1]. Classic cytotoxic chemotherapy is reported to have little antitumour activity in RCC [2], and metastatic RCC is currently one of the most treatment-resistant malignancies. Though, mechanisms responsible for tumor growth and the molecular basis underlying the dissemination of cancer cells in RCC are still unclear; clinical stage and histologic nuclear grade have so far been considered as the best predictors of tumor behavior. However, patients with tumors of similar grade and stage may show a wide variation in biological behavior and clinical

∗ Corresponding author at: Ankara University, School of Medicine, Department of Pathology, Sihhiye, Ankara 06100, Turkey. Tel.: +90 312 5958103; fax: +90 312 5958135. E-mail address: [email protected] (D. Kankaya).

outcome. Therefore, additional biomarkers for RCC are needed to provide further prognostic information and possibly offer an insight into the mechanisms of the disease. Tumor cell physiology includes rapid growth, avoiding apoptosis, being irresponsive to growth-inhibitory signals and having the capability of invasion and metastasis. Almost all of the genes involved in the regulation of these processes are under the control of nuclear factor kappa B (NF-␬B) transcription, a family of transcription factors. The role of NF-␬B in various tumors has been described previously [3]. Regarding RCC, cell culture studies indicated that NF␬B activity is under the control of Von Hippel Lindau (VHL) tumor suppressor gene, and its activity increases due to VHL inactivation, which is the most important genetic change in clear cell renal cell carcinoma (CRCC) [4,5]. It is very likely that increased activity of NF-␬B following VHL loss contributes to the tumorigenesis, progression and spread of CRCCs, and in keeping with that, increased expression of NF-␬B in human RCC tissues in comparison with normal renal parenchyma has been reported [6], and a higher expression rate has been detected in the CRCC subtype than

http://dx.doi.org/10.1016/j.prp.2015.03.002 0344-0338/© 2015 Elsevier GmbH. All rights reserved.

Please cite this article in press as: D. Kankaya, et al., Gelsolin, NF-␬B, and p53 expression in clear cell renal cell carcinoma: Impact on outcome, Pathol. – Res. Pract (2015), http://dx.doi.org/10.1016/j.prp.2015.03.002

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in other RCC subtypes [7]. However, most of the studies dealing with the role of NF-␬B in RCC have been arranged on RCC cell lines [8–10], and studies investigating the expression of NF-␬B protein on human RCC tissues regarding its role on prognosis are limited and mostly gave contradictory results [11,12]. Tumor suppressor gene, p53, provides genetic stability and reduces the risk of cancer through induction of growth arrest or apoptosis in the presence of damaged DNA [13,14]. The importance of the tumor suppressor function of p53 is also highlighted by the frequent inactivating mutations encountered in over 50% of human cancers [15]. The direct implication of the p53 pathway in RCC carcinogenesis and its impact on prognosis is not clear. Studies on RCC show a low p53 expression rate of 20–50%, indicating a rare existence of p53 mutation in primary RCC [16,17], and p53 mutation rate varies with the subtype of RCC, the lowest rate observed in CRCC subtype [17,18]. A large number of studies published in the literature, with respect to the predictive role of p53 expression in the prognosis of RCC, however, gave conflicting results. Some reported a significant correlation between p53 expression and survival in CRCC [16,17,19–22], whereas no such correlation was detected by others [23,24]. On the other hand, the prognostic importance of p53 was demonstrated for only advanced localized or metastasized CRCC cases, referring to its contribution in the late stage of carcinogenesis of CRCC [25,26]. Gelsolin, a member of the actin-binding protein family, is involved in the regulation of actin cytoskeleton dynamics and signaling pathways, as well as in apoptosis in normal human cells [27]. Gelsolin acts as a tumor suppressor with a potential prognostic impact in some neoplasias, but as a tumor activator in others [28,29]. There has also been controversy in the literature due to the prognostic role of Gelsolin in CRCC. One of the few studies investigating this issue reported that Gelsolin is an indicator of decreased survival [20], in contrast to another study that determined it as an indicator of increased survival [30]. This study was designed to investigate the utility of Gelsolin, NF-␬B, and p53 for the prediction of the biological behavior of CRCC. Materials and methods Patients The study consisted of 100 consecutive CRCC cases who underwent radical nephrectomy between 1995 and 2007 at our university. Hematoxylin and eosin-stained histological sections were reviewed for the purpose of the study, and histological grade, pathological stage, and clinical stage were determined according to the original Fuhrman grading system and TNM staging system (6th edition), respectively [31,32]. Clinical stage was determined according to “College of American Pathologists (CAP) Cancer Protocol 2005”, which was based on TNM, 6th edition. Lymphovascular invasion, invasion of renal vein, renal sinus, perirenal tissues, regional lymph node involvement, and presence of metastasis were noted. Clinicopathological parameters, including patient age, gender, multifocality, clinical stage and clinical follow-up findings, were retrieved from the records of the urology department. Tumors were grouped by nuclear grade (NG) as low-NG (NG1, NG2) or high NG (NG3, NG4), by pathological stage (pT) as localized (pT1, pT2) or locally invasive (pT3, pT4), by clinical stage as early stage (stage I, II) or late stage (stage III, IV). Immunohistochemistry Gelsolin (3G5, 1/150, Abcam), NF-␬B (P65C, 1/100, Zymed) and p53 (DO-7, 1/200, Neomarkers) expressions were investigated by immunohistochemistry. Four ␮m sections of representative blocks

of each tumor were mounted on poly-Lysine-coated slides. All steps of the immunohistochemical analysis, including deparaffinization, antigen retrieval and counterstaining, were performed on an automatic immunostainer (Benchmark XT Staining Module, Ventana Medical Systems) by using Streptavidin-biotin complex immunodetection system. CC1 (EDTA, ph:8), CC2 (citrate, ph:6) or protease solutions (Ventana) were used for antigen retrieval. Diaminobenzidine was used as chromogen and followed by hematoxylin counterstaining. Positive control tissues were used as recommended by the suppliers. Immunohistochemical evaluation Evaluation was based on cytoplasmic (NF-␬BCyt ) and nuclear (NF-␬BNuc ) expression for NF-␬B; nuclear expression for p53. Gelsolin frequently showed both cytoplasmic and membranous expression. Therefore, further evaluation of staining pattern was not performed (Fig. 1). For Gelsolin, the intensity of the immunostaining (scale 0–3) was multiplied by the percentage of cells with positive staining to give a score between 0 and 300. Due to similar staining intensities of NF-␬B and p53 among tumors, only the percentage of positive cells was considered in the evaluation. Cut-off value for Gelsolin was 30, which was the mean staining score. Cut-off values for NF-␬B and p53 were 5 and 1, respectively, which were determined according to the median of the percentage of positive cells and literature data [7,17]. Tumors with a staining score or a percentage of positive cells higher than the cut-off were assigned as positive. Statistical analysis The relationship between immunohistochemical expressions and clinicopathologic parameters was compared using the 2 test or Fisher’s exact test. Their effects on disease-free survival were investigated by using the Kaplan–Meier method and compared by the log rank test. Cox’s proportional hazards regression model was used for univariate and multivariate testing. A p value of ≤0.05 was considered statistically significant. Results The median age of 100 patients (M/F:2) was 58 years (33–77 years; 57 ± 10). Median tumor diameter was 7 cm (1.5–20 cm; 7.2 ± 3.49), and 9 of the tumors showed multifocality. Tumor characteristics are summarized in Table 1. It was found that tumors with larger size and high NG had an increased incidence of local invasion (58.7% vs 29.6%, p = 0.003; 60.9% vs 33.3, p = 0.006 respectively). Patients with older age (>57 years) and with tumors showing high NG presented with a higher frequency of metastases (34.7% vs 17.9%, p = 0.045; 71.1% vs 30.6%, p = 0.001 respectively). Mean and median values of antibodies are shown in Table 2. Tumors with Gelsolin expression showed more perirenal invasion (73.3% vs 34.3%, p = 0.001), metastasis (60% vs 28.6%, p = 0.003), late stage disease (73.3% vs 44.3%, p = 0.008), and cancer death (57.1% vs 20.9%, p = 0.001) in comparison with tumors without Gelsolin expression. NF-␬BCyt expression correlated with high NG (57.8% vs 25%, p = 0.002), perirenal invasion (51.6% vs 25%, p = 0.010), locally invasive tumor (54.7% vs 30.6%, p = 0.020), and late stage disease (64.1% vs 33.3%, p = 0.003), whereas no influence of NF␬BNuc expression was found on the prognosis of CRCC. Furthermore, p53 expression correlated with high nuclear grade (56% vs 36%, p = 0.045), lymphovascular invasion (22% vs 8%, p = 0.05), metastasis (56% vs 20%, p = 0.001), late stage disease (64% vs 42%, p = 0.028), and cancer death (42.2% vs 22%, p = 0.034). These results are summarized in Table 3.

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Fig. 1. NF-␬B expression, only nuclear (a) and nuclear-cytoplasmic (b) staining. Gelsolin expression, predominantly membranous (c) and cytoplasmic (d) p53 expression (e) (×400, ×200, ×200, ×400, ×400, respectively).

Kaplan–Meier curves and the log-rank test survival analysis indicated that NG (p = 0.001), lymphovascular invasion (p = 0.035), renal vein invasion (0.001), perirenal invasion (p = 0.008), pT (p = 0.004), regional lymph node involvement (p = 0.001), metastasis (p = 0.001), clinical stage (p = 0.001) together with increased expressions of Gelsolin (p = 0.006) were significant prognostic factors (Fig. 2). However, NF-␬BCyt , NF-␬BNuc or p53 expressions showed no correlation with disease-specific survival. According to the results of Univariate Cox regression analysis of these factors, clinical stage was found to be the most important predictor, with an 7.88-fold increased risk for death and with an 6.54-fold increased risk for metastasis (Table 4). Discussion The unpredictable behavior of CRCCs makes the management of these patients difficult. It has been well established that histological grade and clinical stage are significant prognostic parameters for CRCC. In the present study, NG, clinical stage and additionally lymphovascular invasion, renal vein invasion, perirenal invasion,

Fig. 2. Kaplan–Meier curves for gelsolin staining.

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4 Table 1 Tumor characteristics. Grade (n) (%) Low nuclear grade High nuclear grade pT (n) (%) Localized Locally invasive

1 2 3 4

10 (10) 44 (44) 26 (26) 20 (20)

pT1 pT2 pT3 pT4

37 (37) 17 (17) 44 (44) 2 (2)

Clinical stage (n) (%) Early stage

Stage I Stage II Late stage Stage III Stage IV Regional lymph node involvement (n) (%) N0 N1 N2 Lymphovascular invasion (n) (%) Renal vein invasion (n) (%) Perirenal invasion (n) (%) Renal sinus invasion (n) (%) Metastasis (n) (%) At presentation During follow-up Median follow up (months) Dead of cancer (n) (%)

apoptosis in normal human cells [27]. In the present study, Gelsolin expression indicated poor prognosis by correlating with aggressive clinicopathological parameters like perirenal invasion, metastasis, late stage, and it proved to be a significant predictor of diseasespecific survival in univariate analysis. Studies in the literature investigating the role of Gelsolin in tumorigenesis and its effect on prognosis are rare, with inconsistent results (Table 5). The mechanisms by which Gelsolin exerts its functions in cancer development are uncertain. Although several reports indicate that Gelsolin acts as a tumor suppressor [28,34], it may also act as a tumor activator in carcinogenesis, as suggested by in vivo and in vitro studies [29,35] which notate its dual role in carcinogenesis, and this could in part explain the contradictory findings in the literature. Studies on the role of Gelsolin in renal carcinogenesis and its effect on the prediction of survival of the patients are also limited and contradictory [20,22]. Klatte et al. [22] reported no correlation of Gelsolin with disease-free survival for localized CRCC, while Kim et al. indicated its significance as a predictor of disease-specific survival [20], which is in accordance with our results. In the present study, NF-␬BCyt expression correlated with high NG, perirenal invasion, local invasion or late stage, whereas no correlation was found between NF-␬BNuc expression and clinicopathological parameters. However, none of the expression patterns had any influence on the prediction of survival. NF-␬B is a family of closely related transcription factors that includes five genes, and plays a role in the regulation of many gene product expressions associated with inflammation, cell survival, proliferation, invasion, and angiogenesis [3,37]. NF-␬B signaling pathway activation, mediated by the stimulation of various signaling pathways, like cytokines, growth factors and tyrosine kinases, results in translocation of NF-␬B subunit in the nucleus [38], thus providing identification of NF-␬B activation by positive nuclear immunostaining of various NF-␬B subunits. The role of NF␬B in hematological and solid tumors has been described previously in many studies [3,39–41]. Reports on the association of NF-␬B and RCC published in the literature largely comprise the results of RCC cell line studies [8–10,42]. Cell culture studies indicated that NF␬B activity is under the control of VHL tumor suppressor gene, and its activity increases due to VHL inactivation, which is the most important genetic change in CRCCs [4,5]. It is very likely that increased activity of NF-␬B following VHL loss contributes to the progression and spread of CRCCs if NF-␬B is in fact elevated in CRCC tumors and not just in RCC cell lines. Then, targeting NF-␬B may provide

35 (35) 12 (12) 36 (36) 17 (17) 87 (87) 10 (10) 3 (3) 15 (15) 17 (17) 42 (42) 6 (6) 38 (38) 13 (13) 25 (25) 24 (1–144) 30 (30)

Table 2 Biomarker expressions in CRCC. Antibody

Mean ± SD

Gelsolina NF-␬BNuc b NF-␬BCyt b P53b

30.11 16.9 28.5 5.98

a b

± ± ± ±

4.9 21.8 31.49 16.3

Median (min–max)

Cut-off level

8.0 (0–300) 8.0 (0–95) 12.5 (0–100) 0.5 (0–100)

30 5 5 1

Values according to staining score. Values according to the percentage of positive cells.

pT, regional lymph node involvement, and metastasis proved to be significant prognostic factors that were found to correlate with disease-specific survival. Alterations in cell morphology and motility are very important in invasion and metastasis of cancer cells. Gelsolin, a member of the actin-binding protein family, is involved in the regulation of actin cytoskeleton dynamics and signaling pathways, as well as in Table 3 Correlation of biomarker expressions with clinicopathological parameters. Gelsolin N Multifocality (%)

7.2

NF-␬BNuc P

N

13

13

p = 0.446 High nuclear grade (%)

37.1

53.3

52.5

12.9

20

10

20.9

5.6 27.8

48.3

31.6

40

51.6

36

18.8

8

17.2

10

43.8

20

33.3

64.1

42

34.4

22

p = 0.003 31.6

p = 0.587

54.7

p = 0.114

p = 0.252 57.1

p = 0.001

36

p = 0.097 35

60

p = 0.008 Cancer death (%)

13.3

42.5

57.8

p = 0.161

p = 0.449 73.3

44.3

8.3

p = 0.903 60

6

p = 0.01 11.7

12.5

p = 0.003 Late stage (%)

25

p = 0.253 20

12

p = 0.02 35

20

p = 0.201 28.6

30.6

p = 0.082

p = 0.372

N

p = 0.002 40

52.5

p = 0.001 Lymphovascular invasion (%) Lymph node involvement (%) Metastasis (%)

25

p = 0.14 73.3

34.3

3

p53 P

p = 0.103 41.7

55

p = 0.161 Perirenal invasion (%)

7

p = 0.287 56.7

41.4

N

p = 0.318

p = 0.133 Local invasion (%)

NF-␬BCyt P

26.5 p = 0.287

P 2 p = 0.295 56 p = 0.045 52 p = 0.229 48 p = 0.224 22 p = 0.05 16 p = 0.372 56 p = 0.001 64 p = 0.028 42.2 p = 0.034

N: negative; P: positive.

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Table 4 Uni-variant Cox proportional hazards models for clinicopathological parameters and biomarker expressions. Variable

DF

SE

p value

HR

NG LVI RVI PRI pT RLN Metastasis Clinical stage Gelsolin NF-␬BNuc NF-␬BCyt p53

1 1 1 1 1 1 1 1 1 1 1 1

0.406 0.441 0.397 0.380 0.394 0.403 0.438 0.542 0.372 0.386 0.409 0.407

0.001 0.042 0.001 0.012 0.006 0.001 0.001 0.001 0.008 0.713 0.476 0.51

3.75 2.44 4.14 2.61 2.92 4.24 6.54 7.88 2.68 1.15 0.74 0.76

a new therapeutic option especially for advanced CRCC. Previous studies regarding the expression of NF-␬B in human RCC tissues are summarized in Table 6. Some reports demonstrated that NF-␬B (p65 and p50 subunits) showed increased activity in advanced stage RCCs, playing a role in the progression of the tumors [6]. Dordevic et al. [11] observed nuclear and cytoplasmic staining of NF-␬B/p65 in the majority of tumor cells of RCC. In their study, an association between NF-␬B and proliferation rate of tumor cells was significant for cytoplasmic staining but not for nuclear NF-␬B, similar to our findings. Furthermore, activation of NF-␬B has been held to account for chemotherapy and radiotherapy resistance in various malignancies, including RCC, by protecting cancer cells from death [3,43]. As signaling through the epidermal growth factor receptor (EGFR) has been shown to result in NF-␬B activation, inhibition of the EGFR sensitizes RCC cells to the cytotoxic agents by inhibition of NF-␬B activity [8]. In keeping with this view, a positive correlation between EGFR and NF-␬B was detected, as part of the present study not mentioned here. The elucidation of the interactions of NF␬B with EGFR may solve the problem of chemotherapy resistance, which is an important problem especially for the advanced stage RCCs, and lead to the development of new therapeutic modalities for these patients [44,45]. The p53 protein functions as a transcription factor at times of cellular or genotoxic stress by promoting cell cycle arrest and DNA repair or promoting apoptosis. p53 dysfunction by inactivating mutations has been detected in over 50% of human cancers [47]. The direct implication of the p53 pathway in RCC carcinogenesis is not clear. Not only the role but also the prognostic value of p53 in renal cell carcinoma is still inconsistent and controversial [48–52] (Table 7). Although some studies demonstrated that p53

95%CI 1.69–8.31 1.03–5.08 1.90–9.02 1.23–5.49 1.35–6.33 1.92–9.36 2.77–15.44 2.72–22.81 1.29–5.56 0.54–2.45 0.33–1.66 0.34–1.69

Table 5 Summary of previous studies related to the role of Gelsolin in CRCC. Study group

Results

Visapaa et al., 2003 [30]

257 CRCC

Kim et al., 2004 [20]

318 CRCC

Klatte et al., 2009 [36]

158 PRCC

Klatte et al., 2009 [22]

170 localized CRCC

Decreased Gelsolin expression, combined with increased Ki-67 expression indicating poor cancer specific survival (p = 0.0507) Increased Gelsolin staining correlating with decreased survival in univariate (p < 0.001) and multivariate (p = 0.030) analysis Inverse correlation between Gelsolin and tumor stage (p < 0.05), grade (p < 0.05) and metastasis (p < 0.01) No correlation of Gelsolin with survival (p = 0.312)

CRCC: clear cell renal cell carcinoma, PRCC: papillary renal cell carcinoma.

mutation is relatively rare [49–51] and its expression has no prognostic effect on RCC [49,50], some articles strongly suggest that loss of p53 function is a critical event in the evolution of RCC, and is associated with an aggressive biological behavior [48,52]. Studies on RCC also identified p53 expression as an independent prognostic indicator not only in metastatic lesions, but also in earlystage tumors [17,48,54,55]. These divergencies may be the result of studies with low patient numbers or study groups of tumors with different stage, grade, and histologic subtypes. In addition, technical differences, including the use of various antibodies and staining procedures, make data comparison difficult. In the present study, we found that p53 overexpression correlated with poor

Table 6 Summary of previous studies related to the role of NF␬B in CRCC.

Oya et al., 2003 [6] Sourbier et al., 2007 [33] Djordjevic et al., 2008 [11] Meteoglu, 2008 [12] Matusan-Ilijas et al., 2011 [46]

Method

Study group

Results

p50, p65 subunits Nuclear staining Anti-phospho-NF-␬B P65 Nuclear staining P65 Nuclear and cytoplasmic staining

42 CRCC, 3 non-CRCC 212CRCC, 43 non-CRCC)

P50 Nuclear and cytoplasmic staining P65 Nuclear

40 RCC

Correlation with tumor grade (p = 0.03), invasion (p = 0.005), and metastasis (p = 0.04) Higher expression of NF-␬B in CRCCsubtype (p = 0.002) Correlated with tumor dimension (p = 0.02). No correlation with survival NF-␬B cytoplasmic expression correlated with Ki-67 proliferation index (p = 0.039) Both of cytoplasmic (p = 0.018) and nuclear (p = 0.029) expression correlated with tumor stage Correlation with VEGF, EGFR, bcl-2, p53 (markers involved in angiogenesis and apoptosis) No correlation with tumor grade, stage Inverse correlation between NF-␬B expression and apopitotic index (p = 0.022)

22 CRCC, 9 non-CRCC

87 CRCC

RCC: renal cell carcinoma, CRCC: clear cell renal cell carcinoma.

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Table 7 Summary of previous studies related to the role of p53 in CRCC. Study group

Antibody clone

Cut-off (percentage of positive cells)

Results

50 RCC (old histologic classification) 134 CRCC, 50 non-CRCC

?

Positive staining (≥20%)

Significant correlation with DSS (p = 0.0027)

DO-7

Positive staining (≥1%)

Kim et al., 2004 [20]

318 CRCC

DO-7

–

Kramer et al., 2005 [23] Shvarts et al., 2005 [16]

104 CRCC, 15 non-CRCC 350 localized RCC

?

Positive staining (≥5%)

Lowest expression in CRCC subtype (11.9%) (p < 0.0001) Significant correlation with DSS only in the CRCC group (p = 0.0005) Significant predictor of DSS in univariate [HR (95% CI): 3.25 (1.83–5.76)], (p < 0.001) and multivariate analysis [HR (95% CI): 2.28 (1.19–4.35) (p = 0.014)] No correlation with DSS (p = 0.6210)

?

–

Kankuri et al., 2006 [26] Mombini et al., 2006 [18] Phuoc et al., 2007 [21] Klatte et al., 2009 [22] Baytekin et al., 2011 [24]

101 CRCC, 16 non-CRCC 34 CRCC, 32 non-CRCC 119 CRCC

DO-7 ?

170 localized CRCC

?

Positive staining (≥10%) Positive staining (≥10%) Positive staining (≥10%) ?

66 CRCC, 32 non-CRCC

Polyclonal

Positive staining (≥10%)

Erdem et al., 2013 [58]

24 CRCC, 26 non-CRCC

DO-7

Scoring: 0 (no positive tumor cells), 1 (<10%) 2 (10–50%) 3 (>50%)

Weber et al., 2014 [25]

145 CRCC

DO-1

Hejnold et al., 2014 [59]

470 RCC

DO-7, PAb1801

–

Hodorova et al., 2014 [60]

42 RCC (29 CRCC, 13 non-CRCC)

DO-7

Positive staining (≥10%)

Girgin et al., 2001 [19] Zigeuner et al., 2004 [53]

DO-7

Correlated significantly with nuclear grade (p = 0.023) Significant predictor of DSS in univariate (p < 0.05) and multivariate analysis (p = 0.003) No correlation with tumor stage or metastasis Correlated with DSS only in the metastatic group (p = 0.030) Higher expression in non-CRCC type (p < 0.001) and in higher grade tumors (p = 0.001)Significant predictor of DSS in univariate (p = 0.0029) and multivariate analysis (p = 0.0059) Significant predictor of DSS in univariate (<0.001) and multivariate analysis (p = 0.005) Inverse correlation with tumor stage (p = 0.014) and grade (p = 0.04) No correlation with survival (p > 0.05) Correlation with tumor diameter (p = 0.028), renal sinus invasion (p = 0.05) and grade (p = 0.026)

Significantly predictive for DSS for advanced localized (p T3-4, N0 M0) (p = 0.009) or metastasized CRCC (Any T, N1/M1) (p = 0.025) but not for organ confined disease Correlation with tumor diameter, grade, presence of sarcomatoid component, necrosis (p < 0.05) No correlation with stageHigher expression in non-CRCC type but not statistically significant No correlation with grade (p = 0.17)-

RCC: renal cell carcinoma, CRCC: clear cell renal cell carcinoma, DSS: disease specific survival, HR: hazard ratio, CI: confidence interval.

clinicopathological parameters as high NG, lymphovascular invasion, metastasis, late stage and cancer death. Some investigators [20,22,55] found that p53 overexpression had a significant impact on survival, being one of the independent predictors of diseasespecific survival in CRCC, whereas our survival analysis did not show any role of p53 on survival. Oda et al. [56], however, studied sarcomatoid transformation in RCCs. They observed that p53 was overexpressed with a high mutation rate in sarcomatoid component in contrast to a low mutation rate in usual carcinomatous areas, and this finding also confirms the correlation between p53 overexpression and tumor dedifferentiation. In normal cells, the expression level of p53 protein is generally below the detection level of immunohistochemical methods, but p53 mutations result in accumulation and overexpression of mutant p53 protein, and high-level expression of p53 is used as an indicator of p53 mutation. However, this interpretation is not reliable as high levels of p53 protein expression are not always accompanied by p53 mutations [57]. Moreover, in case of harboring p53 deletions or non-sense mutations in which mutant proteins are less stable to be detected immunohistochemically, tumors may show no p53 expression or low expression rates and are grouped together with cases expressing wild type p53, although these mutations define worse outcome and this grouping together (nonsense mutation, deletion and wild type 53) lead to misinterpretation of the prognostic role of p53 in CRCC. In the present study, half of the patients were completely p53-negative, and p53 expression

did not show a statistically significant correlation with survival. As no mutational analysis was performed in this study, we do not know whether some of these negative cases had nonsense mutation or deletion and/or whether this sort of misgrouping concealed the predictive role of p53 expression on survival of CRCC patients.

Conclusions Expressions of Gelsolin, NF-␬BCyt , and p53 associated with aggressive behavior of CRCC, and Gelsolin expression indicated poor disease-aspecific survival. The results of the present study served to determine biomarkers for predicting a high-risk group of patients with CRCC, expected to show aggressive phenotype.

Conflict of interest None declared.

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