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Decreased PBRM1 expression predicts unfavorable prognosis in patients with clear cell renal cell carcinoma
Urologic Oncology: Seminars and Original Investigations ] (2015) ∎∎∎–∎∎∎
Decreased PBRM1 expression predicts unfavorable prognosis in patients with clear cell renal cell carcinoma Soo Jeong Nam, M.D.a,b, Cheol Lee, M.D.a, Jeong Hwan Park, M.D.a, Kyung Chul Moon, M.D., Ph.D.a,c,* a
Department of Pathology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Korea b Department of Pathology, National Cancer Center, Goyang-si, Korea c Kidney Research Institute, Medical Research Center, Seoul National University College of Medicine, Seoul, Korea Received 6 October 2014; received in revised form 14 January 2015; accepted 14 January 2015
Abstract Aims: PBRM1 is one of the histone and chromatin regulators. A mutation in PBRM1 was recently identified in clear cell renal cell carcinoma (ccRCC). The aim of this study was to determine the clinicopathologic and prognostic significance of PBRM1 expression in ccRCC. Methods and results: Immunohistochemistry was performed for PBRM1 in 657 ccRCC cases. The number of positive cells was determined using image analyzer after virtual microscope scanning. There was a strong correlation between decreased PBRM1 expression and old age, increased tumor size, higher Fuhrman grade, higher pT stage, and higher stage (all P o 0.001). Patients with decreased PBRM1 expression showed significantly worse cancer-specific survival (CSS) and progression-free survival (PFS) (both P o 0.001). In multivariate analysis, PBRM1 expression was an independent predictor of shorter PFS (P ¼ 0.007). In lower-stage group (stages I and II), decreased expression of PBRM1 exhibited significantly worse CSS and PFS (both P o 0.001) but not in higher-stage group (stages III and IV). In multivariate analysis of lower-stage group, decreased expression of PBRM1 was significantly associated with both poor CSS and PFS (P ¼ 0.038 and 0.003, respectively). Conclusions: Decreased expression of PBRM1 predicts unfavorable clinical outcome in patients with ccRCC. r 2015 Elsevier Inc. All rights reserved.
Keywords: Kidney; PBRM1; Carcinoma; Renal cell; Immunohistochemistry; Prognosis
1. Introduction Cancer of the kidney accounts for 4% of the total human cancer burden [1], with approximately 271,000 new cases diagnosed each year [2]. Renal cell carcinoma (RCC) represents more than 90% of all malignancies of the kidney, and 70% of RCC cases are clear cell type [3]. Clear cell RCC (ccRCC) is characterized by inactivation of the von Hippel-Lindau (VHL) gene [4]. Inactivation of This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MOE) (2013R1A1A2007877). * Corresponding author. Tel.: þ82-2-2072-1767; fax: þ82-2-743-5530. E-mail address: [email protected] (K.C. Moon). http://dx.doi.org/10.1016/j.urolonc.2015.01.010 1078-1439/r 2015 Elsevier Inc. All rights reserved.
the VHL gene, located on chromosome 3p25, is considered a frequent and early event in renal carcinogenesis. Somatic inactivation of the VHL gene may occur by allelic deletion, mutation, or epigenetic silencing in nearly 90% of sporadic ccRCCs [5,6]. Recent studies have identified mutations in a number of genes that act as histone and chromatin regulators, including BAP1, PBRM1, SETD2, KDM6A, and JARID1c, in cases of ccRCC. The most common of these is PBRM1, which is found in 41% of ccRCCs [7]. The PBRM1 gene codes for the BAF180 subunit of the SWItch/sucrose nonfermentable (SWI/SNF) adenosine triphosphate (ATP)–dependent chromatin-remodeling complex that modifies chromatin structure and modulates transcription. SWI/SNF complexes are organized around
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an ATPase subunit that provides energy for mobilization of nucleosomes by transfer or replacement of histones [8]. These complexes are thought to have a widespread role in tumor suppression. Inactivating mutations in several SWI/ SNF subunits have been identified at a high frequency in a variety of cancers [9,10]. Very recently, several studies had focused on pathologic correlations of PBRM1. However, the clinicopathologic implications of PBRM1 protein expression remain unclear. Therefore, we evaluated the clinicopathologic features of patients according to PBRM1 expression and determined the prognostic significance of PBRM1. 2. Materials and methods 2.1. Patients We retrieved 657 ccRCC samples from patients who underwent radical or partial nephrectomy between January 1, 1995, and December 30, 2005, at the Seoul National University Hospital. Each ccRCC sample was reviewed regarding the carcinoma type, the stage of the tumor, and the nuclear grade according to the current World Health Organization criteria. Additionally, we retrospectively graded the tumors as grades I to IV according to the Fuhrman nuclear grading system. Clinical and pathologic information was obtained from electronic medical records and pathology reports. Recurrence or metastasis of ccRCC was determined according to the clinical and radiographic findings. Disease-related deaths were identified by reviewing the medical records. This study was approved by the Institutional Review Board of Seoul National University Hospital (H-1309-076-521). The declaration of Helsinki was followed.
blood vessels, and inflammatory cells as little as possible from virtual microscopic images. The PBRM1 expression was estimated based on the proportion of PBRM1-positive cells in all tumor cells using the nuclear v9 algorithm of ImageScope software (Aperio Technologies Inc., Vista, CA). We regarded PBRM1 to be positive when the tumor cell showed strong nuclear staining intensity according to v9 algorithm. The percentages of PBRM1-positive cells of each triple TMA slide were averaged and then used for further statistical analysis. 2.4. Statistical analysis All statistical analyses were performed with SPSS 21 (IBM Corp., NY). Student t test and one-way analysis of variance tests were performed to assess the differences in the percentage of PBRM1 expression according to the clinicopathologic variables. The cancer-specific survival (CSS) was measured from the time of primary radical or partial nephrectomy to cancer-related death and was censored at final follow-up visit alive or death from unrelated cause. The progression-free survival (PFS) was calculated from the time of nephrectomy to recurrence or metastasis of the ccRCC and was censored at final follow-up visit without evidence of ccRCC. For univariate survival analysis of CSS or PFS, we fitted a Kaplan-Meier model after dichotomizing the cases into 2 groups with 50% of nuclear expression as the cutoff. In addition, multivariate survival analyses were performed using the Cox proportional hazard model. We applied the percentage of PBRM1 expression as continuous variables to the Cox proportional hazard model. For all analyses, 2-sided P o 0.05 was considered statistically significant. 3. Results
2.2. Tissue microarray and immunohistochemistry
3.1. Quantitative analysis of PBRM1 expression in ccRCC
A set of 3 tissue microarray (TMA) blocks consisting of a representative tumor core section (2 mm in diameter) from each formalin-fixed paraffin-embedded block (SuperBioChips Laboratories, Seoul, Korea) was created for immunohistochemical (IHC) analysis. IHC staining was performed using the BenchMark XT Slide Preparation System (Ventana Medical Systems, Inc., Tucson, AZ) and a polyclonal rabbit antihuman PB1/BAF180 antibody (1:100, Bethyl Laboratories, Inc., Montgomery, TX).
IHC staining for PBRM1 exhibited a distinct nuclear expression (Fig. 1A and B). Nuclear PBRM1 was generally preserved in nonneoplastic tissue including renal parenchyma and tumor-infiltrating lymphocytes. In the ccRCC tumor cells, PBRM1 expression was observed at various levels throughout the 657 ccRCC cases. The automatically enumerated results of PBRM1 expression showed a range from a minimum of 0% to a maximum of 100%, with a median value of 40.16%.
2.3. Quantitative analysis of PBRM1 expression by image analysis
3.2. Relationships between clinicopathologic features and PBRM1 expression in patients with ccRCC
All immunostained slides were evaluated by virtual microscope scanning under high-power magnification (200) using ScanScope CS2 eSlide (Aperio Technologies, Vista, CA). For enumeration of PBRM1 expression, representative fields were selected including stromal tissues,
The clinicopathologic characteristics of patients with ccRCC and their associations with PBRM1 expression are summarized in Table 1. There was a strong correlation between loss of PBRM1 expression and old age (older than 55 y; P o 0.001), increased tumor size (more than 5 cm;
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Fig. 1. Immunohistochemical findings of PBRM1 expression. PBRM1 immunostaining showed a distinct nuclear positivity. Images were captured by virtual microscopy and submitted to an image analyzer. A series of representative images of different tumor stages were demonstrated. Respective images illustrated average counts of stages I (A; 63.65), II (B; 50.94), III (C; 44.15), and IV (D; 46.14). (Color version of figure is available online.)
P o 0.001), higher Fuhrman grade (P o 0.001), higher pT stage (P o 0.001), and higher stage (P o 0.001).
3.3. PBRM1 expression and the survival of patients with ccRCC The cases were separated into 2 groups according to the quantity of PBRM1 expression, with 50% positive cells as the cutoff. Survival analysis was performed using the Kaplan-Meier method with a log-rank test. Patients with decreased PBRM1 expression showed significantly worse CSS and PFS (both P o 0.001) (Fig. 2A and B) (Table 2). Furthermore, this prognostic pattern of PBRM1 expression
was maintained in a grade-dependent manner when analyzed according to the 4 groups (Fig. 2C and D). 3.4. Independent prognostic implications of PBRM1 expression in patients with ccRCC To determine the prognostic implications of PBRM1 expression, we performed multivariate survival analysis using the Cox proportional hazard model. We found that the clinical variables, including Fuhrman grade and tumor stage, were associated with CSS and PFS using a log-rank test (Table 2). In multivariate Cox regression analysis integrating other clinical risk factors, including Fuhrman grade and stage, PBRM1 expression had no independent
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Table 1 Clinicopathologic characteristics according to the PBRM1 expression in ccRCC Variables Age, y r55 455 Sex Female Male Tumor size, cm r5 45 Fuhrman grade 1 2 3 4 pT stagea 1 2 3 4 Stagea I II III IV
n (%)
PBRM1 expression (mean ⫾ SD)
313 (47.6) 344 (52.4)
63.36 ⫾ 30.06 54.34 ⫾ 30.14
P value o0.001
n.s. 172 (26.2) 485 (73.8)
60.88 ⫾ 30.25 57.84 ⫾ 30.46
372 (56.6) 285 (43.4)
65.76 ⫾ 29.67 49.35 ⫾ 28.87
51 298 242 66
69.92 ⫾ 28.26 62.71 ⫾ 30.43 54.08 ⫾ 29.43 48.22 ⫾ 30.25
431 93 124 8
64.21 ⫾ 29.70 50.58 ⫾ 28.51 46.83 ⫾ 28.88 30.38 ⫾ 27.76
424 79 87 66
64.63 ⫾ 29.51 50.47 ⫾ 28.49 44.00 ⫾ 29.12 48.60 ⫾ 29.01
expression of PBRM1 was significantly associated with both poor CSS and PFS (P ¼ 0.038 and 0.003, respectively) (Table 4).
o0.001 o0.001**
o0.001**
o0.001**
n.s. ¼ not significant; SD ¼ standard deviation. These variables contain missing values that lacked the information about the variables. ** These results are figured out using one-way analysis of variance (ANOVA) test. a
prognostic significance in patients with ccRCC regarding CSS. However, in PFS, PBRM1 expression was independently associated with a worse prognosis (P ¼ 0.007) (Table 3). 3.5. PBRM1 expression and the survival of patients with ccRCC according to stage We performed an additional survival analysis in patients with ccRCC who were divided into 2 groups according to the stage. In the lower-stage group, which included patients with ccRCC at stage I or II, decreased expression of PBRM1 exhibited significantly worse CSS and PFS (both P o 0.001) (Fig. 3A and B). In contrast, in the higher-stage group, which consisted of patients at stage III or IV, there was no statistically significant association between PBRM1 expression and CSS or PFS (Fig. 3C and D). 3.6. Independent prognostic implication of PBRM1 expression in the low-stage group of patients with ccRCC Multivariate survival analysis was also performed using the Cox proportional hazard model. In contrast to the result analyzed in patients with higher-stage ccRCC, decreased
4. Discussion Inactivation of the VHL gene is observed in most ccRCC cases. However, despite the central role of the VHL mutation, VHL loss alone is insufficient for the induction of ccRCC tumorigenesis. Therefore, it is presumed that additional genetic alterations are required to cause ccRCC [11]. Recent studies have identified recurrent mutations in histone-modifying and chromatin-remodeling genes, including PBRM1 [7], BAP1 [12], SETD2, KDM6A, and JARID1c [13]. All of these genes function in chromatin biology and have potential widespread roles as tumor suppressors. PBRM1, SETD2, and BAP1 are near the 3p21 locus, adjacent to the 3p25 locus, which is where VHL resides. The characteristic single-copy loss of 3p has been observed in 91% of ccRCC cases [7,14]. Therefore, simultaneous impairment of these 3 tumor suppressors and the VHL gene may be the key driver for ccRCC tumorigenesis based on loss of heterozygosity studies [15]. It has been suggested that the development of ccRCC is initiated by a focal mutation in VHL, followed by a 3p deletion. Loss of 3p may eliminate VHL gene function and would leave cells with just 1 copy of several tumor suppressor genes. Mutation of the remaining alleles of such tumor suppressor genes may initiate tumorigenesis [16]. Inactivation of PBRM1 is the second most common mutation in ccRCC development. Varela et al. conducted a functional study of ccRCC cell lines and reported that knockdown of PBRM1 significantly promoted colony formation and cell migration. In addition, they found that PBRM1 activity had a tumor-suppressive role, regulating pathways associated with chromosomal instability and cellular proliferation in ccRCC [7]. Previous studies revealed that PBRM1 is a critical transcriptional regulator of p21 in breast cancer cell lines [17]. In addition, PBRM1 is implicated in regulating the induction of senescence by TP53 transcriptional activity–mediated replicative and oncogenic stress [18]. The PBRM1 mutation acts as a direct effecter because it affects the expression of proteins. Therefore, we analyzed the protein expression of PBRM1 using IHC staining methods in 657 ccRCC cases. To perform an unbiased analysis of PBRM1 expression and to obtain objective and reproducible data, we used virtual microscopy and automatic enumeration by an image analyzer. We observed various patterns of PBRM1 protein expression. Some cases showed diffuse and even expression, and other cases displayed a complete loss of PBRM1. However, many cases exhibited PBRM1-positive and PBRM1negative nuclei mixed to varying degrees. The degree of mixing was continuous, and so the automatic enumerated values of PBRM1 expression showed no division into
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Fig. 2. Kaplan-Meier survival plot for PBRM1 expression. Patients with ccRCC were classified into 2 or 4 groups according to the expression of PBRM1. CSS and PFS of patients with ccRCC were evaluated according to the expression of PBRM1. Kaplan-Meier curves for CSS (A and C) and PFS (B and D) are illustrated, with the P values from the log-rank test. Table 2 Univariate analysis of cancer-specific survival and progression-free survival on clinicopathologic parameters and PBRM1 expression Prognostic factor Cancer-specific survival Mean ⫾ SE
Progression-free survival
P value Mean ⫾ SE
P value
177.194 ⫾ 2.496 o0.001 160.717 ⫾ 3.563 o0.001 126.211 ⫾ 4.221 90.691 ⫾ 4.389
Fuhrman grade
1–2 3–4
Stage
I–II 177.563 ⫾ 1.773 o0.001 154.717 ⫾ 3.483 o0.001 III–IV 85.870 ⫾ 6.675 50.648 ⫾ 5.945
PBRM1
Low High
145.507 ⫾ 3.823 o0.001 109.533 ⫾ 4.233 o0.001 171.751 ⫾ 2.973 156.208 ⫾ 4.469
SE ¼ standard error; SWI/SNF ¼ SWItch/sucrose nonfermentable.
distinct subgroups, such as a negative vs. a positive group. Therefore, we analyzed PBRM1 expression as a continuous variable or a categorized variable divided into 2 groups based on the 50% expression cutoff value. There was a strong correlation between loss of PBRM1 expression and advanced age, increased tumor size, higher Fuhrman grade, higher pT stage, and higher stage. Overall, patients with decreased PBRM1 expression showed a relatively worse survival. Furthermore, poor prognostic trend of decreased PBRM1 expression was maintained in a grade-dependent manner when analyzed according to the 4 groups. In particular, in the low tumor stage group, decreased expression of PBRM1 was independently associated with worse CSS and PFS. There
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Table 3 Multivariate analysis of cancer-specific survival and progression-free survival on clinicopathologic parameters and PBRM1 expression Prognostic factor
Cancer-specific survival HR
Fuhrman grade 1 and 2 vs. 3 and 4 Stage I and II vs. III and IV PBRM1 High vs. low
Progression-free survival
95% CI
P value
HR
95% CI
P value
3.924
2.303–6.686
o0.001
2.804
1.993–3.943
o0.001
10.199
6.448–16.131
o0.001
6.142
4.507–8.369
o0.001
1.216
0.764–1.935
0.409
1.633
1.143–2.332
0.007
HR ¼ hazard ratio; SD ¼ standard deviation.
Fig. 3. Kaplan-Meier survival plot for PBRM1 expression according to stage. Patients with ccRCC with low or high stage were classified into 2 groups according to the PBRM1 expression. Kaplan-Meier curves for CSSS (A and C) and PFS (B and D) are illustrated, with P values from the log-rank test.
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Table 4 Multivariate analysis of cancer-specific survival and progression-free survival on clinicopathologic parameters and PBRM1 expression in low-stage group Prognostic factor
Cancer-specific survival HR
95% CI
Fuhrman grade 1 and 2 vs. 3 and 4
2.174
0.957–4.938
Stage I vs. II
5.405
2.446–11.946
PBRM1 High vs. low
3.172
1.066–9.435
Progression-free survival P value
HR
95% CI
P value
0.064
2.152
1.357–3.413
0.001
o0.001
2.796
1.755–4.453
o0.001
0.038
2.214
1.299–3.771
0.003
HR ¼ hazard ratio; SD ¼ standard deviation.
was no statistically significant association between PBRM1 expression and prognosis in the higher-stage group. In patients with ccRCC, several previous studies have investigated the clinical and prognostic value of PBRM1. Pawlowski et al. [19] reported that a loss of PBRM1 was correlated with advanced tumor stage, low differentiation grade, and worse patient outcome. da Costa et al. [20] found that a loss of PBRM1 was associated with tumor stage, clinical stage, pN stage, tumor size, and markedly poor prognosis events in ccRCC. Hakimi et al. reported that tumors with mutations in PBRM1 are more likely to present with a higher disease stage. However, there was no statistically significant association between PBRM1 and survival [21]. In contrast, Gossage et al. [22] reported a trend of patients with PBRM1 mutations to have longer PFS than patients without PBRM1 mutations. Furthermore, Kapur et al. reported that overall survival was significantly shorter for patients with BAP1-mutant tumors than for patients with PBRM1-mutant tumors. However, there was no statistical association between survival and PBRM1 mutation [16]. Although the influence of PBRM1 expression on survival differed between previous studies and our present study, our study has some additional merits over previous studies. The cohort included more patients, and automatic cell counting was applied rather than manual evaluation. In addition, the clinical significance of PBRM1 expression was consistent between the previous studies and our study. However, potential limitation of our study is that we studied only protein expression without genetic test to analyze indirectly the mutation status of PBRM1. Although our result about PBRM1 protein expression did not reflect mutation status exactly, we revealed that altered PBRM1 expression had a strong influence on prognosis. The results of our study suggest that loss of PBRM1 may play an important role in the acquisition of tumor aggressiveness. Moreover, inactivation of PBRM1 may arise in the initiation step of tumorigenesis or the tumor growth step and may be an ongoing event during tumor progression. However, loss of PBRM1 expression is an effective prognostic variable in only the early stages. We speculated that tumor aggressiveness caused by PBRM1 mutation may be functioned early in the process of tumorigenesis.
5. Conclusions Our study revealed a strong correlation between the decrease of PBRM1 expression and higher Fuhrman grade or advanced stage. Overall, patients with decreased PBRM1 expression showed an independently worse prognosis, especially in the low-stage group. Therefore, PBRM1 may have a clinical significance for the management of patients with low-grade ccRCC.
References [1] Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin 2013;63:11–30. [2] Ferlay J, Shin HR, Bray F, et al. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010 2893–917. [3] Baldewijns MM, van Vlodrop IJ, Schouten LJ, et al. Genetics and epigenetics of renal cell cancer. Biochim Biophys Acta 2008;1785:133–55. [4] Latif F, Tory K, Gnarra J, et al. Identification of the von HippelLindau disease tumor suppressor gene. Science (New York, NY) 1993;260:1317–20. [5] Nickerson ML, Jaeger E, Shi Y, et al. Improved identification of von Hippel-Lindau gene alterations in clear cell renal tumors. Clin Cancer Res 2008;14:4726–34. [6] Moore LE, Nickerson ML, Brennan P, et al. Von Hippel-Lindau (VHL) inactivation in sporadic clear cell renal cancer: associations with germline VHL polymorphisms and etiologic risk factors. PLoS Genet 2011;7:e1002312. [7] Varela I, Tarpey P, Raine K, et al. Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma. Nature 2011;469:539–42. [8] Reisman D, Glaros S, Thompson EA. The SWI/SNF complex and cancer. Oncogene 2009;28:1653–68. [9] Wilson BG, Roberts CW. SWI/SNF nucleosome remodellers and cancer. Nat Rev Cancer 2011;11:481–92. [10] Shain AH, Pollack JR. The spectrum of SWI/SNF mutations, ubiquitous in human cancers. PloS One 2013;8:e55119. [11] Kaelin WG Jr. The von Hippel-Lindau tumor suppressor gene and kidney cancer. Clin Cancer Res 2004;10:6290s–5s. [12] Pena-Llopis S, Vega-Rubin-de-Celis S, Liao A, et al. BAP1 loss defines a new class of renal cell carcinoma. Nat Genet 2012; 44:751–9. [13] Dalgliesh GL, Furge K, Greenman C, et al. Systematic sequencing of renal carcinoma reveals inactivation of histone modifying genes. Nature 2010;463:360–3.
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[14] Toma MI, Grosser M, Herr A, et al. Loss of heterozygosity and copy number abnormality in clear cell renal cell carcinoma discovered by high-density affymetrix 10K single nucleotide polymorphism mapping array. Neoplasia (New York, NY) 2008;10:634–42. [15] Clifford SC, Prowse AH, Affara NA, et al. Inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene and allelic losses at chromosome arm 3p in primary renal cell carcinoma: evidence for a VHL-independent pathway in clear cell renal tumourigenesis. Genes Chromosomes Cancer 1998;22:200–9. [16] Kapur P, Pena-Llopis S, Christie A, et al. Effects on survival of BAP1 and PBRM1 mutations in sporadic clear-cell renal-cell carcinoma: a retrospective analysis with independent validation. Lancet Oncol 2013;14:159–67. [17] Xia W, Nagase S, Montia AG, et al. BAF180 is a critical regulator of p21 induction and a tumor suppressor mutated in breast cancer. Cancer Res 2008;68:1667–74.
[18] Burrows AE, Smogorzewska A, Elledge SJ. Polybromo-associated BRG1-associated factor components BRD7 and BAF180 are critical regulators of p53 required for induction of replicative senescence. Proc Natl Acad Sci U S A 2010;107:14280–5. [19] Pawlowski R, Muhl SM, Sulser T, et al. Loss of PBRM1 expression is associated with renal cell carcinoma progression. Int J Cancer 2013; 132:E11–7. [20] da Costa WH, Rezende M, Rocha RM, et al. PBRM1, a SWI/SNF complex subunit is a prognostic marker in clear cell renal cell carcinoma. BJU Int 2014;113:E157–163. [21] Hakimi AA, Chen YB, Wren J, et al. Clinical and pathologic impact of select chromatin-modulating tumor suppressors in clear cell renal cell carcinoma. Eur Urol 2013;63:848–54. [22] Gossage L, Murtaza M, Slatter AF, et al. Clinical and pathological impact of VHL, PBRM1, BAP1, SETD2, KDM6A, and JARID1c in clear cell renal cell carcinoma. Genes Chromosomes Cancer 2014;53:38–51.
Decreased PBRM1 expression predicts unfavorable prognosis in patients with clear cell renal cell carcinoma Soo Jeong Nam, M.D.a,b, Cheol Lee, M.D.a, Jeong Hwan Park, M.D.a, Kyung Chul Moon, M.D., Ph.D.a,c,* a
Department of Pathology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Korea b Department of Pathology, National Cancer Center, Goyang-si, Korea c Kidney Research Institute, Medical Research Center, Seoul National University College of Medicine, Seoul, Korea Received 6 October 2014; received in revised form 14 January 2015; accepted 14 January 2015
Abstract Aims: PBRM1 is one of the histone and chromatin regulators. A mutation in PBRM1 was recently identified in clear cell renal cell carcinoma (ccRCC). The aim of this study was to determine the clinicopathologic and prognostic significance of PBRM1 expression in ccRCC. Methods and results: Immunohistochemistry was performed for PBRM1 in 657 ccRCC cases. The number of positive cells was determined using image analyzer after virtual microscope scanning. There was a strong correlation between decreased PBRM1 expression and old age, increased tumor size, higher Fuhrman grade, higher pT stage, and higher stage (all P o 0.001). Patients with decreased PBRM1 expression showed significantly worse cancer-specific survival (CSS) and progression-free survival (PFS) (both P o 0.001). In multivariate analysis, PBRM1 expression was an independent predictor of shorter PFS (P ¼ 0.007). In lower-stage group (stages I and II), decreased expression of PBRM1 exhibited significantly worse CSS and PFS (both P o 0.001) but not in higher-stage group (stages III and IV). In multivariate analysis of lower-stage group, decreased expression of PBRM1 was significantly associated with both poor CSS and PFS (P ¼ 0.038 and 0.003, respectively). Conclusions: Decreased expression of PBRM1 predicts unfavorable clinical outcome in patients with ccRCC. r 2015 Elsevier Inc. All rights reserved.
Keywords: Kidney; PBRM1; Carcinoma; Renal cell; Immunohistochemistry; Prognosis
1. Introduction Cancer of the kidney accounts for 4% of the total human cancer burden [1], with approximately 271,000 new cases diagnosed each year [2]. Renal cell carcinoma (RCC) represents more than 90% of all malignancies of the kidney, and 70% of RCC cases are clear cell type [3]. Clear cell RCC (ccRCC) is characterized by inactivation of the von Hippel-Lindau (VHL) gene [4]. Inactivation of This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MOE) (2013R1A1A2007877). * Corresponding author. Tel.: þ82-2-2072-1767; fax: þ82-2-743-5530. E-mail address: [email protected] (K.C. Moon). http://dx.doi.org/10.1016/j.urolonc.2015.01.010 1078-1439/r 2015 Elsevier Inc. All rights reserved.
the VHL gene, located on chromosome 3p25, is considered a frequent and early event in renal carcinogenesis. Somatic inactivation of the VHL gene may occur by allelic deletion, mutation, or epigenetic silencing in nearly 90% of sporadic ccRCCs [5,6]. Recent studies have identified mutations in a number of genes that act as histone and chromatin regulators, including BAP1, PBRM1, SETD2, KDM6A, and JARID1c, in cases of ccRCC. The most common of these is PBRM1, which is found in 41% of ccRCCs [7]. The PBRM1 gene codes for the BAF180 subunit of the SWItch/sucrose nonfermentable (SWI/SNF) adenosine triphosphate (ATP)–dependent chromatin-remodeling complex that modifies chromatin structure and modulates transcription. SWI/SNF complexes are organized around
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an ATPase subunit that provides energy for mobilization of nucleosomes by transfer or replacement of histones [8]. These complexes are thought to have a widespread role in tumor suppression. Inactivating mutations in several SWI/ SNF subunits have been identified at a high frequency in a variety of cancers [9,10]. Very recently, several studies had focused on pathologic correlations of PBRM1. However, the clinicopathologic implications of PBRM1 protein expression remain unclear. Therefore, we evaluated the clinicopathologic features of patients according to PBRM1 expression and determined the prognostic significance of PBRM1. 2. Materials and methods 2.1. Patients We retrieved 657 ccRCC samples from patients who underwent radical or partial nephrectomy between January 1, 1995, and December 30, 2005, at the Seoul National University Hospital. Each ccRCC sample was reviewed regarding the carcinoma type, the stage of the tumor, and the nuclear grade according to the current World Health Organization criteria. Additionally, we retrospectively graded the tumors as grades I to IV according to the Fuhrman nuclear grading system. Clinical and pathologic information was obtained from electronic medical records and pathology reports. Recurrence or metastasis of ccRCC was determined according to the clinical and radiographic findings. Disease-related deaths were identified by reviewing the medical records. This study was approved by the Institutional Review Board of Seoul National University Hospital (H-1309-076-521). The declaration of Helsinki was followed.
blood vessels, and inflammatory cells as little as possible from virtual microscopic images. The PBRM1 expression was estimated based on the proportion of PBRM1-positive cells in all tumor cells using the nuclear v9 algorithm of ImageScope software (Aperio Technologies Inc., Vista, CA). We regarded PBRM1 to be positive when the tumor cell showed strong nuclear staining intensity according to v9 algorithm. The percentages of PBRM1-positive cells of each triple TMA slide were averaged and then used for further statistical analysis. 2.4. Statistical analysis All statistical analyses were performed with SPSS 21 (IBM Corp., NY). Student t test and one-way analysis of variance tests were performed to assess the differences in the percentage of PBRM1 expression according to the clinicopathologic variables. The cancer-specific survival (CSS) was measured from the time of primary radical or partial nephrectomy to cancer-related death and was censored at final follow-up visit alive or death from unrelated cause. The progression-free survival (PFS) was calculated from the time of nephrectomy to recurrence or metastasis of the ccRCC and was censored at final follow-up visit without evidence of ccRCC. For univariate survival analysis of CSS or PFS, we fitted a Kaplan-Meier model after dichotomizing the cases into 2 groups with 50% of nuclear expression as the cutoff. In addition, multivariate survival analyses were performed using the Cox proportional hazard model. We applied the percentage of PBRM1 expression as continuous variables to the Cox proportional hazard model. For all analyses, 2-sided P o 0.05 was considered statistically significant. 3. Results
2.2. Tissue microarray and immunohistochemistry
3.1. Quantitative analysis of PBRM1 expression in ccRCC
A set of 3 tissue microarray (TMA) blocks consisting of a representative tumor core section (2 mm in diameter) from each formalin-fixed paraffin-embedded block (SuperBioChips Laboratories, Seoul, Korea) was created for immunohistochemical (IHC) analysis. IHC staining was performed using the BenchMark XT Slide Preparation System (Ventana Medical Systems, Inc., Tucson, AZ) and a polyclonal rabbit antihuman PB1/BAF180 antibody (1:100, Bethyl Laboratories, Inc., Montgomery, TX).
IHC staining for PBRM1 exhibited a distinct nuclear expression (Fig. 1A and B). Nuclear PBRM1 was generally preserved in nonneoplastic tissue including renal parenchyma and tumor-infiltrating lymphocytes. In the ccRCC tumor cells, PBRM1 expression was observed at various levels throughout the 657 ccRCC cases. The automatically enumerated results of PBRM1 expression showed a range from a minimum of 0% to a maximum of 100%, with a median value of 40.16%.
2.3. Quantitative analysis of PBRM1 expression by image analysis
3.2. Relationships between clinicopathologic features and PBRM1 expression in patients with ccRCC
All immunostained slides were evaluated by virtual microscope scanning under high-power magnification (200) using ScanScope CS2 eSlide (Aperio Technologies, Vista, CA). For enumeration of PBRM1 expression, representative fields were selected including stromal tissues,
The clinicopathologic characteristics of patients with ccRCC and their associations with PBRM1 expression are summarized in Table 1. There was a strong correlation between loss of PBRM1 expression and old age (older than 55 y; P o 0.001), increased tumor size (more than 5 cm;
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Fig. 1. Immunohistochemical findings of PBRM1 expression. PBRM1 immunostaining showed a distinct nuclear positivity. Images were captured by virtual microscopy and submitted to an image analyzer. A series of representative images of different tumor stages were demonstrated. Respective images illustrated average counts of stages I (A; 63.65), II (B; 50.94), III (C; 44.15), and IV (D; 46.14). (Color version of figure is available online.)
P o 0.001), higher Fuhrman grade (P o 0.001), higher pT stage (P o 0.001), and higher stage (P o 0.001).
3.3. PBRM1 expression and the survival of patients with ccRCC The cases were separated into 2 groups according to the quantity of PBRM1 expression, with 50% positive cells as the cutoff. Survival analysis was performed using the Kaplan-Meier method with a log-rank test. Patients with decreased PBRM1 expression showed significantly worse CSS and PFS (both P o 0.001) (Fig. 2A and B) (Table 2). Furthermore, this prognostic pattern of PBRM1 expression
was maintained in a grade-dependent manner when analyzed according to the 4 groups (Fig. 2C and D). 3.4. Independent prognostic implications of PBRM1 expression in patients with ccRCC To determine the prognostic implications of PBRM1 expression, we performed multivariate survival analysis using the Cox proportional hazard model. We found that the clinical variables, including Fuhrman grade and tumor stage, were associated with CSS and PFS using a log-rank test (Table 2). In multivariate Cox regression analysis integrating other clinical risk factors, including Fuhrman grade and stage, PBRM1 expression had no independent
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Table 1 Clinicopathologic characteristics according to the PBRM1 expression in ccRCC Variables Age, y r55 455 Sex Female Male Tumor size, cm r5 45 Fuhrman grade 1 2 3 4 pT stagea 1 2 3 4 Stagea I II III IV
n (%)
PBRM1 expression (mean ⫾ SD)
313 (47.6) 344 (52.4)
63.36 ⫾ 30.06 54.34 ⫾ 30.14
P value o0.001
n.s. 172 (26.2) 485 (73.8)
60.88 ⫾ 30.25 57.84 ⫾ 30.46
372 (56.6) 285 (43.4)
65.76 ⫾ 29.67 49.35 ⫾ 28.87
51 298 242 66
69.92 ⫾ 28.26 62.71 ⫾ 30.43 54.08 ⫾ 29.43 48.22 ⫾ 30.25
431 93 124 8
64.21 ⫾ 29.70 50.58 ⫾ 28.51 46.83 ⫾ 28.88 30.38 ⫾ 27.76
424 79 87 66
64.63 ⫾ 29.51 50.47 ⫾ 28.49 44.00 ⫾ 29.12 48.60 ⫾ 29.01
expression of PBRM1 was significantly associated with both poor CSS and PFS (P ¼ 0.038 and 0.003, respectively) (Table 4).
o0.001 o0.001**
o0.001**
o0.001**
n.s. ¼ not significant; SD ¼ standard deviation. These variables contain missing values that lacked the information about the variables. ** These results are figured out using one-way analysis of variance (ANOVA) test. a
prognostic significance in patients with ccRCC regarding CSS. However, in PFS, PBRM1 expression was independently associated with a worse prognosis (P ¼ 0.007) (Table 3). 3.5. PBRM1 expression and the survival of patients with ccRCC according to stage We performed an additional survival analysis in patients with ccRCC who were divided into 2 groups according to the stage. In the lower-stage group, which included patients with ccRCC at stage I or II, decreased expression of PBRM1 exhibited significantly worse CSS and PFS (both P o 0.001) (Fig. 3A and B). In contrast, in the higher-stage group, which consisted of patients at stage III or IV, there was no statistically significant association between PBRM1 expression and CSS or PFS (Fig. 3C and D). 3.6. Independent prognostic implication of PBRM1 expression in the low-stage group of patients with ccRCC Multivariate survival analysis was also performed using the Cox proportional hazard model. In contrast to the result analyzed in patients with higher-stage ccRCC, decreased
4. Discussion Inactivation of the VHL gene is observed in most ccRCC cases. However, despite the central role of the VHL mutation, VHL loss alone is insufficient for the induction of ccRCC tumorigenesis. Therefore, it is presumed that additional genetic alterations are required to cause ccRCC [11]. Recent studies have identified recurrent mutations in histone-modifying and chromatin-remodeling genes, including PBRM1 [7], BAP1 [12], SETD2, KDM6A, and JARID1c [13]. All of these genes function in chromatin biology and have potential widespread roles as tumor suppressors. PBRM1, SETD2, and BAP1 are near the 3p21 locus, adjacent to the 3p25 locus, which is where VHL resides. The characteristic single-copy loss of 3p has been observed in 91% of ccRCC cases [7,14]. Therefore, simultaneous impairment of these 3 tumor suppressors and the VHL gene may be the key driver for ccRCC tumorigenesis based on loss of heterozygosity studies [15]. It has been suggested that the development of ccRCC is initiated by a focal mutation in VHL, followed by a 3p deletion. Loss of 3p may eliminate VHL gene function and would leave cells with just 1 copy of several tumor suppressor genes. Mutation of the remaining alleles of such tumor suppressor genes may initiate tumorigenesis [16]. Inactivation of PBRM1 is the second most common mutation in ccRCC development. Varela et al. conducted a functional study of ccRCC cell lines and reported that knockdown of PBRM1 significantly promoted colony formation and cell migration. In addition, they found that PBRM1 activity had a tumor-suppressive role, regulating pathways associated with chromosomal instability and cellular proliferation in ccRCC [7]. Previous studies revealed that PBRM1 is a critical transcriptional regulator of p21 in breast cancer cell lines [17]. In addition, PBRM1 is implicated in regulating the induction of senescence by TP53 transcriptional activity–mediated replicative and oncogenic stress [18]. The PBRM1 mutation acts as a direct effecter because it affects the expression of proteins. Therefore, we analyzed the protein expression of PBRM1 using IHC staining methods in 657 ccRCC cases. To perform an unbiased analysis of PBRM1 expression and to obtain objective and reproducible data, we used virtual microscopy and automatic enumeration by an image analyzer. We observed various patterns of PBRM1 protein expression. Some cases showed diffuse and even expression, and other cases displayed a complete loss of PBRM1. However, many cases exhibited PBRM1-positive and PBRM1negative nuclei mixed to varying degrees. The degree of mixing was continuous, and so the automatic enumerated values of PBRM1 expression showed no division into
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Fig. 2. Kaplan-Meier survival plot for PBRM1 expression. Patients with ccRCC were classified into 2 or 4 groups according to the expression of PBRM1. CSS and PFS of patients with ccRCC were evaluated according to the expression of PBRM1. Kaplan-Meier curves for CSS (A and C) and PFS (B and D) are illustrated, with the P values from the log-rank test. Table 2 Univariate analysis of cancer-specific survival and progression-free survival on clinicopathologic parameters and PBRM1 expression Prognostic factor Cancer-specific survival Mean ⫾ SE
Progression-free survival
P value Mean ⫾ SE
P value
177.194 ⫾ 2.496 o0.001 160.717 ⫾ 3.563 o0.001 126.211 ⫾ 4.221 90.691 ⫾ 4.389
Fuhrman grade
1–2 3–4
Stage
I–II 177.563 ⫾ 1.773 o0.001 154.717 ⫾ 3.483 o0.001 III–IV 85.870 ⫾ 6.675 50.648 ⫾ 5.945
PBRM1
Low High
145.507 ⫾ 3.823 o0.001 109.533 ⫾ 4.233 o0.001 171.751 ⫾ 2.973 156.208 ⫾ 4.469
SE ¼ standard error; SWI/SNF ¼ SWItch/sucrose nonfermentable.
distinct subgroups, such as a negative vs. a positive group. Therefore, we analyzed PBRM1 expression as a continuous variable or a categorized variable divided into 2 groups based on the 50% expression cutoff value. There was a strong correlation between loss of PBRM1 expression and advanced age, increased tumor size, higher Fuhrman grade, higher pT stage, and higher stage. Overall, patients with decreased PBRM1 expression showed a relatively worse survival. Furthermore, poor prognostic trend of decreased PBRM1 expression was maintained in a grade-dependent manner when analyzed according to the 4 groups. In particular, in the low tumor stage group, decreased expression of PBRM1 was independently associated with worse CSS and PFS. There
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Table 3 Multivariate analysis of cancer-specific survival and progression-free survival on clinicopathologic parameters and PBRM1 expression Prognostic factor
Cancer-specific survival HR
Fuhrman grade 1 and 2 vs. 3 and 4 Stage I and II vs. III and IV PBRM1 High vs. low
Progression-free survival
95% CI
P value
HR
95% CI
P value
3.924
2.303–6.686
o0.001
2.804
1.993–3.943
o0.001
10.199
6.448–16.131
o0.001
6.142
4.507–8.369
o0.001
1.216
0.764–1.935
0.409
1.633
1.143–2.332
0.007
HR ¼ hazard ratio; SD ¼ standard deviation.
Fig. 3. Kaplan-Meier survival plot for PBRM1 expression according to stage. Patients with ccRCC with low or high stage were classified into 2 groups according to the PBRM1 expression. Kaplan-Meier curves for CSSS (A and C) and PFS (B and D) are illustrated, with P values from the log-rank test.
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Table 4 Multivariate analysis of cancer-specific survival and progression-free survival on clinicopathologic parameters and PBRM1 expression in low-stage group Prognostic factor
Cancer-specific survival HR
95% CI
Fuhrman grade 1 and 2 vs. 3 and 4
2.174
0.957–4.938
Stage I vs. II
5.405
2.446–11.946
PBRM1 High vs. low
3.172
1.066–9.435
Progression-free survival P value
HR
95% CI
P value
0.064
2.152
1.357–3.413
0.001
o0.001
2.796
1.755–4.453
o0.001
0.038
2.214
1.299–3.771
0.003
HR ¼ hazard ratio; SD ¼ standard deviation.
was no statistically significant association between PBRM1 expression and prognosis in the higher-stage group. In patients with ccRCC, several previous studies have investigated the clinical and prognostic value of PBRM1. Pawlowski et al. [19] reported that a loss of PBRM1 was correlated with advanced tumor stage, low differentiation grade, and worse patient outcome. da Costa et al. [20] found that a loss of PBRM1 was associated with tumor stage, clinical stage, pN stage, tumor size, and markedly poor prognosis events in ccRCC. Hakimi et al. reported that tumors with mutations in PBRM1 are more likely to present with a higher disease stage. However, there was no statistically significant association between PBRM1 and survival [21]. In contrast, Gossage et al. [22] reported a trend of patients with PBRM1 mutations to have longer PFS than patients without PBRM1 mutations. Furthermore, Kapur et al. reported that overall survival was significantly shorter for patients with BAP1-mutant tumors than for patients with PBRM1-mutant tumors. However, there was no statistical association between survival and PBRM1 mutation [16]. Although the influence of PBRM1 expression on survival differed between previous studies and our present study, our study has some additional merits over previous studies. The cohort included more patients, and automatic cell counting was applied rather than manual evaluation. In addition, the clinical significance of PBRM1 expression was consistent between the previous studies and our study. However, potential limitation of our study is that we studied only protein expression without genetic test to analyze indirectly the mutation status of PBRM1. Although our result about PBRM1 protein expression did not reflect mutation status exactly, we revealed that altered PBRM1 expression had a strong influence on prognosis. The results of our study suggest that loss of PBRM1 may play an important role in the acquisition of tumor aggressiveness. Moreover, inactivation of PBRM1 may arise in the initiation step of tumorigenesis or the tumor growth step and may be an ongoing event during tumor progression. However, loss of PBRM1 expression is an effective prognostic variable in only the early stages. We speculated that tumor aggressiveness caused by PBRM1 mutation may be functioned early in the process of tumorigenesis.
5. Conclusions Our study revealed a strong correlation between the decrease of PBRM1 expression and higher Fuhrman grade or advanced stage. Overall, patients with decreased PBRM1 expression showed an independently worse prognosis, especially in the low-stage group. Therefore, PBRM1 may have a clinical significance for the management of patients with low-grade ccRCC.
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