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Keywords:

  • kidney;
  • renal cell carcinoma;
  • prognosis;
  • molecular marker;
  • PBRM1

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. References

Objective

  • To analyse the immunohistochemical and mRNA expression of SWI/SNF (SWItch/Sucrose NonFermentable) complex subunit polybromo-1 (PBRM1) in clear cell renal cell carcinoma (ccRCC) and its impact on clinical outcomes.

Patients and Methods

  • In all, 213 consecutive patients treated surgically for renal cell carcinoma (RCC) between 1992 and 2009 were selected.
  • A single pathologist reviewed all cases to effect a uniform reclassification and determined the most representative tumour areas for construction of a tissue microarray.
  • In addition, mRNA expression of PBRM1 was analysed by reverse transcriptase-polymerase chain reaction.

Results

  • Of the 112-immunostained ccRCC specimens, 34 (30.4%) were PBRM1-negative, and 78 (69.6%) were PBRM1-positive.
  • The protein expression of PBRM1 was associated with tumour stage (P < 0.001), clinical stage (P < 0.001), pN stage (P = 0.035) and tumour size (P = 0.002).
  • PBRM1 mRNA expression was associated with clinical stage (P = 0.023), perinephric fat invasion (P = 0.008) and lymphovascular invasion (P = 0.042).
  • PBRM1 significantly influenced tumour recurrence and tumour-related death. Disease-specific survival rates for patients whose specimens showed positive- and negative-PBRM1 expression were 89.7% and 70.6%, respectively (P = 0.017).
  • Recurrence-free survival rates in patients with positive- and negative-expression of PBRM1 were 87.3% and 66.7%, respectively (P = 0.048).

Conclusions

  • PBRM1-negative expression is a markedly poor prognosis event in ccRCC.
  • We encourage PBRM1 study by other groups in order to validate our findings and confirm its possible role as a useful marker in the management of patients with ccRCC.

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. References

RCC accounts for 90% of renal neoplasms, which correspond to 3% of all adult malignancies [1]. RCC is increasing at a rate of ≈2% per year [2]. Clear cell RCC (ccRCC) is the most common subtype of RCC, accounting for 75% of the total. In most familial syndromes and also in sporadic tumours it is associated with loss of von Hippel-Lindau (VHL) gene function [3]. However, there is evidence that mutation of the VHL gene alone is not sufficient for the development of ccRCC, which suggests the need of one or more additional genetic events in the development of the disease [4].

Genes known to be important in various malignancies and frequently mutated in other epithelial tumours, e.g. Ras (rat sarcoma), BRAF (v-raf murine sarcoma viral oncogene homolog B1), TP53 (tumour protein 53), RB (retinoblastoma), PTEN (phosphatase and tensin homolog), EGFR (endothelial growth factor receptor) and ERBB2 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2), are not commonly detected and play a limited prognostic role in ccRCC [5]. However, a recent study described the presence of mutations in the gene polybromo-1 (PBRM1, chromosome 3p21) in 41% of cases of ccRCC [6]. It corresponds to the most frequently mutated gene found in ccRCC in addition to VHL. The study reported that 13 of 14 patients with ccRCC with no VHL mutations had mutations in PBRM1, confirming its importance in the development of the disease [6].

PBRM1 encodes BAF180 protein, which is a subunit of the ATP-dependent complex of chromatin remodelling called SWI/SNF (SWItch/Sucrose NonFermentable). This complex plays a role in the mobilisation of nucleosomes by promoting insertion or removal of histones from the chromatin [7]. Thus, the SWI/SNF is involved primarily in mechanisms of gene regulation in multiple cellular processes, e.g. tissue differentiation and carcinogenesis. It presents characteristics of a tumour suppressor and mutations in its subunits have been described in several cancers, e.g. breast and lung [7, 8].

Despite being a gene frequently altered in patients with ccRCC there is scarce information about its possible prognostic role. Therefore, we evaluated the gene and protein expression of PBRM1 in ccRCC, investigating its association with pathological features and its impact on clinical outcomes.

Patients and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. References

In all, 213 consecutive cases involving radical or partial nephrectomy for RCC between 1992 and 2009 were selected from the medical records of our institution. Abdominal CT was used as the standard imaging method for diagnostic confirmation. In suspected cases of systemic metastases, chest CT and bone scans were performed. Patients were evaluated quarterly during the first 2 years and every 6 months thereafter. A single pathologist (I.W.C.) reviewed all of the cases for uniform reclassification and determined the selection of the most representative tumour areas for the construction of the tissue microarray (TMA). For the purpose of this study, only cases of ccRCC were selected, resulting in 112 patients. Our internal Review Board approved the present study. Samples were provided by our institution biobank with patient's informed consent.

The following variables were included in the data bank: age, gender, Eastern Cooperative Oncology Group (ECOG) status, smoking, time since diagnosis, type of surgery, staging (TNM American Joint Committee on Cancer [AJCC]/Union Internationale Contre le Cancer [UICC] 2010), Fuhrman grade, histological subtype (WHO Classification/2004), lymphovascular invasion (LVI), perinephric fat invasion, lymph node involvement, presence of tumour necrosis, presence of metastases and analysis of PBRM1 mRNA expression and immunohistochemical expression patterns. During radical nephrectomy, retroperitoneal lymphadenectomy was restricted to the renal hilum and was performed for staging purposes only.

TMA Construction

Two cylinders measuring 1 mm in diameter taken from different parts of the tumour were used to build a TMA. Two cylinders from previously tested tumours, one showing positive expression of PBRM1 and one negative, were also included as internal controls for the TMA. Sequential 4-μm sections were obtained and stained with haematoxylin and eosin to confirm the diagnosis, and used for the immunohistochemical study. To minimise the possible impact of tumour heterogeneity in the results of immunohistochemical analysis, we chose to randomly select 20 cases that were analysed in six different areas of the tumour at least 5 mm apart from each other. For the selection of cases, the electronic tool Random Integer Generator® (http://www.random.org) was used. In addition, a further analysis in 10 additional whole-mount slides (seven tumoral tissue specimens and three non-neoplastic tissue specimens) was also performed.

Immunohistochemistry

The sections were mounted on positively charged glass slides and dried for 30 min at 37 °C. The sections were deparaffinized in xylene and rehydrated via a series of graded alcohols. Sections were then incubated with a primary rabbit polyclonal antibody against BAF180 (Sigma-Aldrich, St. Louis, MO, USA) at a 1:25 dilution for 60 min. All immunohistochemical procedures were performed automatically in the auto-stainer Link 48, DAKO®, using the Flex Plus visualization system according to the supplier's specifications. For control cases of immunohistochemical BAF180 expression, 37 specimens of non-neoplastic renal cortex at least 2.0 cm distant from the tumour boundary obtained from patients who underwent radical nephrectomy were included.

The same pathologist ‘blinded’ to the outcome of the cases, semi-quantitatively scored the nuclear staining intensity of PBRM1 in all specimens. For immunohistochemical score assessment, fields were chosen at random at ×400. For better interpretation, categories ‘negative-expression’ and ‘positive- expression’ groups were used based on the absence or presence of PBRM1 nuclear tissue staining, respectively.

Quantitative Real-time Reverse Transcriptase (qRT) PCR

In all, 44 patients had frozen tumoral tissue available in the tumour bank of our institution for qRT-PCR analysis. Total RNA was extracted from surgically removed tumours and 10 adjacent noncancerous frozen tissues using RNeasy Mini Kit (Qiagen, Austin, TX, USA). First strand cDNA was treated with TURBO DNA-free™ Kit (Life Technologies; Carlsbad, CA, USA) to remove contaminating DNA from RNA preparations. RT was performed using a 18-oligo(dT) primer and Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) using total RNA for 2 h at 42 °C. The primer sequence for PBRM1 was National Center for Biotechnology Information (NCBI, GenBank) Reference Sequence NM_018313.4 (Applied Biosystems). The qRT-PCR was performed by using Applied Biosystems 7500HT Fast Real-Time PCR System (Applied Biosystems). As a control, we used a pool of normal kidney tissue and β-actin was used to normalise the reactions. The values of CT (cycle threshold) in the corresponding reference group were accepted as normal. The relative comparative quantization method was applied to determine the gene expression levels. Five samples of normal kidney parenchyma were used for comparison of gene expression in non-neoplastic tissue and tumour tissue.

Statistical Analysis

To verify the association between PBRM1 immunohistochemical expression and the other variables, Pearson chi-square tests were used. Fisher's exact test was applied for those cases in which the expected frequencies were <5. The Mann–Whitney test and Kruskal–Wallis tests were used to compare means among different expression levels of PBRM1 groups.

Disease-specific survival (DSS) was defined as the interval between primary surgery and the last follow-up visit or disease-related death. Recurrence-free survival (RFS) was defined as the interval between primary surgery and the last follow-up visit without disease or evidence of recurrence. To analyse RFS, patients with metastatic disease were excluded. To study DSS and RFS, Kaplan–Meier curves and the log-rank test were used. Cox proportional hazards model was used to determine which variables influenced survival. The confidence interval was 5%.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. References

In all, 80 (71.4%) patients had radical nephrectomy, and 32 (28.6%) were treated with nephron-sparing procedures. Their mean (range) age was 55.5 (27–85) years. The median postoperative follow-up period was 40.8 months. There was metastatic disease at the initial clinical presentation in 14 (12.5%) patients; 53/112 (47.3%) presented with T1 stage disease. The entire cohort mean tumour size was 6.8 cm. At the end of the study, 18 (16.1%) patients had died from RCC (Table 1).

Table 1. Patients and pathological characteristics and the association between different variables and PBRM1 protein and gene expression
VariableNegative expression, n (%)Positive expression, n (%)PPBRM1 RT-PCRP
Gender:     
Male24 (70.6)42 (53.8)0.1434.8750.346
Female10 (29.4)36 (46.2) 6.048 
Smoking:     
No19 (55.9)55 (70.5)0.1926.0770.646
Yes15 (44.1)23 (29.5) 3.627 
ECOG status:     
021 (61.8)47 (60.3)0.8384.1570.399
1+213 (38.2)31 (39.7) 6.360 
Incidental tumour:     
Yes9 (26.5)45 (57.7)0.0047.3610.234
No25 (73.5)33 (42.3) 3.079 
Clinical stage:     
I or II13 (38.2)60 (76.9)<0.0016.8280.023
III or IV21 (61.8)18 (23.1) 3.917 
pT Stage:     
pT1a2 (5.9)20 (25.6)<0.0018.2500.286
pT1b2 (5.9)29 (37.2) 5.739 
pT2a7 (20.6)7 (9.0) 9.071 
pT2b3 (8.8)5 (6.4) 5.525 
pT3a14 (41.2)14 (17.9) 3.130 
pT3b1 (2.9)0 (0) NA 
pT45 (14.7)3 (3.8) 1.559 
Tumour size, cm8.796.020.002NANA
pN Stage:     
pN013 (68.4)43 (89.6)0.0355.5900.288
pN1 or N26 (31.6)5 (10.4) 1.407 
Metastasis:     
No27 (79.4)71 (91.0)0.1205.6490.980
Yes7 (20.6)7 (9.0) 4.214 
Fuhrman grade:     
Low grade21 (61.8)54 (70.1)0.5104.7160.552
High grade13 (38.2)23 (29.9) 4.893 
Perinephric fat invasion:     
No22 (64.7)66 (84.6)0.0255.7060.008
Yes12 (35.3)12 (15.4) 0.456 
Renal vein invasion:     
No26 (76.5)70 (89.7)0.0815.5900.223
Yes8 (23.5)8 (10.3) 3.276 
LVI:     
No27 (79.4)74 (94.9)0.0186.0270.042
Yes7 (20.6)4 (5.1) 1.484 
Necrosis:     
No14 (41.2)47 (60.3)0.1005.0610.464
Yes20 (58.8)31 (39.7) 5.721 

PBRM1-positive nuclear staining was seen in all non-neoplastic tissue sections (mainly in normal proximal tubules). In the evaluation of the 112 immunostained ccRCC specimens, 34 (30.4%) showed negative expression and 78 (69.6%) positive expression of PBRM1. Although also seen in the cytoplasm, the expression pattern was predominantly shown in the cell nucleus. An even distribution of the staining instead of the presence of hot spots was seen. When analysing the six tumour cores of each of the 20 randomly selected cases, there was a homogeneous PBRM1 immunohistochemical staining pattern. To confirm our findings in the TMA slide, we performed a further analysis in 10 additional whole-mount slides (seven tumoral tissue specimens and three non-neoplastic tissue specimens), which confirmed the PBRM1 expression pattern. Additionally, when divided into two groups according to storage time (≤10 years and >10 years), we noted no significant impact on staining intensity (P = 0.81) (Fig. 1).

figure

Figure 1. Photomicrographs of immunohistochemical expression of PBRM1. (A) Negative expression of PBRM1 in a whole mount slide. (B) Positive expression of PBRM1 in a whole mount slide. (C,D) Two different TMA slides 5 mm distant from each other showing similar expression of PBRM1.

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There was an association between PBRM1 protein expression and clinical stage (P < 0.001), pT stage (P < 0.001), tumour size (P = 0.002), pN stage (P = 0.035), perinephric fat invasion (P = 0.025) and LVI (P = 0.018) (Table 1).

The median (sd; range) gene expression level was 2.297 (8.111; 0.083–39.130). There was no significant difference in PBRM1 gene expression between non-tumoral tissue and RCC (P = 0.741). qRT-PCR analysis of PBRM1 in RCC specimens confirmed the immunohistochemical findings. There was an association between PBRM1 gene expression and clinical stage (P = 0.023), perinephric fat invasion (P = 0.008) and LVI (P = 0.042).

The 5-year RFS and DSS rates were 81.6% and 83.9%, respectively. Classical parameters, such as metastasis at presentation, lymph node involvement, clinical stage, tumour size and Fuhrman grade were associated with survival rates in univariate analysis. DSS rates in patients with positive- and negative-expression of PBRM1 were 89.7% and 70.6%, respectively (P = 0.017). RFS rates in patients with positive- and negative-expression of PBRM1 were 87.3% and 66.7%, respectively (P = 0.048) (Fig. 2).

figure

Figure 2. Survival analysis based on PBRM1 expression. (A) DSS with PBRM1 grouped into positive- vs negative-expression levels. (B) RFS with PBRM1 grouped into positive- vs negative-expression levels.

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Clinical stage, necrosis, Fuhrman grade, LVI and metastasis at presentation remained independent predictors of DSS and RFS in the multivariate analysis. Immunohistochemical low-expression levels of PBRM1 did not remain as independent predictors of DSS or RFS (Table 2).

Table 2. Cox regression analysis of DSS and RFS
Feature5-year RFS5-year DSS
UnivariateMultivariateUnivariateMultivariate
PHR95% CIPPHR95% CIP
  1. ECOG PS, ECOG performance status.

Clinical stage (III/IV vs I/II)<0.0011.0460.084–13.0920.972<0.0018.7371.708–44.6990.009
Incidental tumour (no vs yes)0.1371.4530.799–32430.230<0.0016.1561.143–33.1660.034
Fuhrman grade (III/IV vs I/II)0.0071.8401.046–7.3630.017<0.0015.1901.392–19.3500.014
LVI0.0064.7521.440–15.6790.011<0.00117.9392.664–25.7920.003
Necrosis0.8551.4320.788–1.9450.614<0.0013.0630.674–13.9080.147
MetastasisNANANANA<0.0016.7622.264–20.2000.001
ECOG PS (1/2 vs 0)0.6150.7650.601–2.4770.443<0.0011.2870.254–4.9360.882
Fat invasion<0.00111.7523.862–35.761<0.001<0.0010.6220.168–2.3120.479
PBRM1 (negative vs positive)0.0481.4450.399–5.2290.5750.0170.8570.261–2.8160.799

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. References

The present study found that established clinical and pathological parameters are the most important prognostic factors in the management of patients with ccRCC [9]. Some authors have incorporated molecular markers in to predictive models in an attempt to improve their accuracy [10]. However, we still lack markers that significantly impact clinical outcomes and might be used in clinical practice.

The importance of epigenetics in gene expression as well as in tumorigenesis and tumour progression is well recognised. Two distinct classes of complexes regulate chromatin structure: those that covalently modify histone tails and those that remodel nucleosomes in an ATP-dependent manner. Together, these classes dynamically regulate the structure of chromatin [11]. As a component of SWI/SNF complex and because of a significant proportion of ccRCC tumours have mutated PBRM1, it may be an interesting biomarker of the disease [5, 6].

PBRM1 activity regulates pathways associated with chromosomal instability and cellular proliferation. In breast cancer, it was shown to be a critical regulator of p21 [12]. There is also evidence that PBRM1 is implicated in regulating TP53-mediated replicative senescence [13]. The only functional study using small interfering RNA (siRNA)-mediated PBRM1 knock-down in vitro resulted in increased colony formation, cell proliferation and migration in ccRCC cell lines; suggesting a tumour suppressor role for PBRM1 [6]. A recent systematic review described SWI/SNF component genes mutations in 19% of human tumours (including ccRCC) compared with 26% mutation rate of TP53 gene. These authors found that 38.8% of SWI/SNF gene mutations lead to a functional consequence, a significantly higher proportion than seen on the complete exome analysis, which confirms their importance as driver alterations [14].

Loss of protein expression of PBRM1 has recently been associated with tumour progression in ccRCC [15]. However, there is evidence that the genetic loss of PBRM1 possibly through a ubiquitous mutation might be an early event in ccRCC tumorigenesis, which theoretically is present in all tumour cells [14, 16]. It is possible that a second mutation at the remaining allele results in a complete loss of PBRM1 expression, which might contribute in a decisive way to tumour development [14]. Such ubiquitous allelic imbalance events were also seen on other chromosome 3p genes, such as VHL and histone modifying genes SETD2 and JARID1C [5, 16]. Therefore, these genes become interesting candidate biomarkers and their signalling pathways potential therapeutic targets. It is curious to note that in the present study, 30.4% of patients had negative immunohistochemical expression. This rate is very close to PBRM1 gene mutation rates reported previously [6, 17].

The present study confirmed the role of PBRM1 as a possible biomarker in ccRCC. It's genetic and protein expression were associated with important prognostic parameters, e.g. clinical stage, tumour size and lymph node involvement. Such findings confirm previous studies that describe the loss of PBRM1 expression as a poor prognostic event [15, 17]. We found that 92.5% of pT1 tumours were PBRM1-positive, while only half of tumours with fat invasion or renal vein invasion (pT3a) expressed PBRM1. Other groups have reported similar findings in ccRCC tumours of <4.0 cm [15, 17]. In addition, 54.5% of patients given a radical nephrectomy with pN+ disease were PBRM1-negative. Possible molecular mechanisms that might explain such an aggressive phenotype in PBRM1-negative tumours are related to chromosomal instability, cytoskeleton malfunction and deregulation of cellular motility, which may contribute to invasion and metastasis [7]. If confirmed, such findings might have the potential to influence clinical decisions such as the inclusion of patients in active surveillance protocols.

PBRM1 expression status significantly impacted survival rates in univariate analysis. However, it did not remain as an independent predictor of either RFS or DSS. We think that, in part, our relatively small series and the few events might have contributed to such findings. Other classical prognostic factors, such as ECOG status and necrosis, did not remain as independent predictors of survival. There is only one study that confirmed PBRM1 expression pattern as an independent predictor of survival [15]. However, in that study the authors’ analysed tumours with different histological subtypes (including papillary and chromophobe tumours) and did not include N status or clinical stage in the multivariate analysis. We think that the missing data might have led to an analysis bias, because at least in our present cohort we observed a significant association between PBRM1-negative tumours and pN+ disease. Interestingly, Kapur et al. [18] described better survival rates for patients with PBRM1-mutated tumours when compared with BAP1-mutated tumours. However, there is growing evidence that although less frequent, mutation on BAP1 is a poor prognosis event irrespective of PBRM1 status [18-20].

Significant genetic heterogeneity has been reported in primary ccRCC and it is a major limitation of studies involving immunohistochemical analysis with TMA, as it is possible that different staining patterns occur in other parts of the tumour [16]. However, in the present study, we conducted a broader analysis with six samples of tumour tissue from 20 of our patients that showed homogeneous staining. We also confirmed that the expression pattern of PBRM1 was homogeneous in 10 additional whole-tissue slides. There is evidence that in this situation, immunohistochemical analysis with TMA is reliable [21-23]. The present study has other limitations that should be mentioned. This is a single-centre retrospective analysis. The immunohistochemical procedure itself may have had problems posed by inadequate technique of fixation in formalin material. However, the present study is the first one to address the prognostic importance of PBRM1 mRNA expression in ccRCC tumours, which was very similar to the protein expression found in our immunohistochemical studies.

It is known that molecular signatures are able to distinguish specific subtypes of ccRCC with different clinical behaviours [20]. In the present series, PBRM1 was associated with major prognostic factors in renal cancer and significantly influenced tumour recurrence and tumour-related death. We encourage PBRM1 studies by other groups to validate our present findings and confirm the possible role of PBRM1 as a useful marker in the management of patients with ccRCC.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. References
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Abbreviations
ccRCC

clear cell RCC

DSS

disease-specific survival

ECOG

Eastern Cooperative Oncology Group

LVI

lymphovascular invasion

PBRM1

polybromo-1

qRT-PCR

quantitative reverse transcriptase PCR

RFS

recurrence-free survival

SWI/SNF

SWItch/Sucrose NonFermentable

TMA

tissue microarray

VHL

von Hippel-Lindau