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Loss of nuclear BAP1 protein expression is a marker of poor prognosis in patients with clear cell renal cell carcinoma
Urologic Oncology: Seminars and Original Investigations ] (2016) ∎∎∎–∎∎∎
Original article
Loss of nuclear BAP1 protein expression is a marker of poor prognosis in patients with clear cell renal cell carcinoma Daniele Minardi, M.D.a,1,*, Guendalina Lucarini, Ph.D.b,1, Giulio Milanese, M.D.a, Roberto Di Primio, M.D.b, Rodolfo Montironi, M.D.c, Giovanni Muzzonigro, M.D.a a
Dipartimento di Scienze Cliniche e Specialistiche—Sezione di Urologia, Università Politecnica delle Marche—Azienda Ospedaliero-Universitaria Ospedali Riuniti, Ancona, Italy b Dipartimento di Scienze Cliniche e Molecolari—Sezione di Istologia, Università Politecnica delle Marche, Ancona, Italy c Dipartimento di Scienze Biomediche e Sanità Pubblica—Sezione di Patologia e Istopatologia, Università Politecnica delle Marche—Azienda OspedalieroUniversitaria Ospedali Riuniti, Ancona, Italy Received 20 November 2015; received in revised form 24 February 2016; accepted 11 March 2016
Abstract Introduction: BRCA1-associated protein 1 (BAP1) is a gene situated on chromosome 3p in a region that is deleted in more than 90% of renal cell carcinomas (RCCs). In the present study, we studied BAP1 immunohistochemical expression in a large series of conventional clear cell RCCs (ccRCCs) treated with radical nephrectomy; we assessed the prognostic value of their expression in terms of patients' survival at long-term follow-up. Materials and methods: A total of 154 consecutive patients with ccRCC were selected from a prospective database and considered for the study purpose; all patients were treated with radical nephrectomy and lymphadenectomy at our Institute of Urology between 1983 and 1985. The features considered in this study were tumor size, grade and stage, vascular and capsular invasion, incidence of metastasis, and patient-specific survival; all these parameters were correlated with immunohistochemical cytoplasmic and nuclear expression of BAP1 in tumoral tissue. Results: Median follow-up was 196.18 months and median survival was 125.34 months. Nuclear BAP1 expression showed a high frequency of loss in tumoral cells; nuclear BAP1-negative tumors had higher tumor size, higher Fuhrman grade, and higher stage, a greater amount of vascular and capsular invasion and a higher incidence of metastases. In multivariate analysis, pathological stage and nuclear BAP1 expression resulted independent prognostic factors. Conclusion: We have demonstrated that nuclear BAP1 expression is a marker of prognosis in ccRCC, having an influence on cancerspecific survival. The clinical importance for BAP1 will be realized with the identification and application of targeted therapies and with individualized approaches in the adjuvant setting or in the metastatic setting or in both the settings. r 2016 Elsevier Inc. All rights reserved.
Keywords: BAP1 protein; Clear cell renal cell carcinoma; Immunohistochemistry; Cancer-specific survival
1. Introduction Renal cell carcinoma (RCC) represents the most common form of cancer originating from the renal parenchyma [1]; 1
Minardi D. and Lucarini G. contributed equally to the article. Corresponding author. Tel.: þ39-071-596-5667; fax: þ39-071-5963367. E-mail address: [email protected] (D. Minardi). *
http://dx.doi.org/10.1016/j.urolonc.2016.03.006 1078-1439/r 2016 Elsevier Inc. All rights reserved.
because of the increased use of abdominal imaging, most RCCs are detected at an early stage, can be surgically removed, and patients can be cured. In spite of this, there is a minority of patients who die of metastatic renal cancer; however, at present no markers exist to identify this group. The RCCs' classification has been recently updated to include morphology characteristics, growth pattern, cell of origin, and immunohistochemical staining [1,2]; in addition, investigators are exploring the molecular pattern of RCC
2
D. Minardi et al. / Urologic Oncology: Seminars and Original Investigations ] (2016) 1–8
subtypes and have reported that loss-of-function mutations are common events in clear cell renal cell carcinoma tumors (ccRCC) [3,4]. The recent discoveries about the molecular genetics of renal cancer have offered the opportunity to provide a therapeutic target. Von Hippel-Lindau (VHL) mutations were found to regulate hypoxia-inducible factor and vascular endothelial growth factor, leading to the development of vascular endothelial growth factor inhibitors. A second pathway has been implicated in renal cancer, the mammalian target of rapamycin complex 1 that is a major regulator of cell growth, and this has led to the development of mammalian target of rapamycin inhibitors. Studies involving the molecular pathogenesis of ccRCC focused on the loss of VHL gene located on chromosome 3p, an area frequently deleted in ccRCC. Among genes mutated in ccRCC, BRCA1-associated protein 1 (BAP1) has recently been identified; BAP1 is on chromosome 3p in a region that is deleted in more than 90% of ccRCCs [3–5]. Mutations in BAP1 occur in 5% to 15% of sporadic ccRCC tumors, and in some familial cases of ccRCC [6,7,8–14]. The BAP1 functions as a deubiquitinating (DUB) enzyme that regulates multiple cellular pathways related to tumorigenesis, an association between BAP1 mutations; and prognosis in patients with ccRCC has been investigated [12,14]. In the present study, we have studied BAP1 immunohistochemical expression in a large series of conventional ccRCCs treated with radical nephrectomy; we assessed the prognostic value of their expression in terms of patients' survival at long-term follow-up.
2. Materials and methods 2.1. Patients A total of 154 consecutive patients with clear cell RCC without sarcomatoid features were selected from a prospective database and considered for the study purpose; all patients were treated with radical nephrectomy and lymphadenectomy at our Institute of Urology between 1983 and 1985. Preoperative imaging consisted of ultrasound in 100% of cases, chest and abdominal computed tomography (CT) in 95% of cases (or chest and abdominal magnetic resonance [MR] in 5% of cases). For the study purpose, only patients without lymph nodes' involvement or distant metastases at the time of diagnosis were included. Followup examination at 3-month intervals in year 1 after surgery included chest and abdominal CT or MR; thereafter chest and abdominal CT or MR were performed at 6-month intervals, and bone scan was performed when clinically required. At year 6 after surgery and thereafter, chest X-ray, abdominal ultrasound and general examination were performed at 6-month intervals; and at year 10 after surgery, these tests were performed yearly.
Disease recurrence was defined as evidence of measurable disease on imaging, including CT, MR imaging, bone scan or ultrasound, and cytological/histological evaluation of suspected lesions. We have studied cancer-specific survival that is the percentage of people who have not died of kidney cancer during the long time of follow-up. 2.2. Histologic features Archived materials containing histological sections from the 154 patients were retrieved from the Institute of Pathological Anatomy and used for the study purpose. Tumor grade was based on the Fuhrman scheme. Fuhrman grade is on a scale of I to IV, where grade I carries the best prognosis and grade IV the worst [15]; tumor staging was based on Union for International Cancer Control classification [16]. The features considered in this study were tumor size, grade and stage, vascular and capsular invasion, and incidence of metastasis; furthermore, we have considered patient-specific survival; all these parameters were correlated with immunohistochemical cytoplasmic and nuclear expression of BAP1 in tumoral tissue. 2.3. Immunohistochemistry Immunohistochemistry (IHC) analysis was performed on 6-mm thick paraffin-embedded histological sections. After deparaffinization and rehydration, sections were treated with microwave for heat-induced epitope retrieval in EDTA Buffer (1 mM EDTA, 0.05% Tween 20, pH ¼ 9.0). Then the sections were incubated with the monoclonal antibody anti-BAP1 (clone C-4; 1:200 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, CA) and then they were immunostained using the streptavidin-biotin peroxidase technique (Envision peroxidase kit, Dako Cytomation, Milan, Italy). After incubation with 0.05% 3,30 diaminobenzidine (SigmaAldrich, Milan, Italy) in 0.05 M Tris buffer, pH ¼ 7.6 with 0.01% hydrogen peroxide, sections were counterstained with Mayer's hematoxylin (BioOptica, Milan, Italy), dehydrated in ethanol and coverslipped with Eukitt mounting medium (Electron Microscopy Sciences, PA). Positive controls for antibody against BAP1 were represented by internal positive staining in background stromal cells and intratumoral lymphocytes. For negative controls, primary antibody was replaced with nonimmune serum. All counts were performed separately by 2 investigators (G.L. and R.M.), blinded to the patient outcome. The reactivity for BAP1 was evaluated in nontumoral tissue, and in the nucleus and cytoplasm of the tumoral cells at least 10 fields/sample at 400 magnification and quantified as a percentage of the total counted cells. Images were captured with a Nikon DS-Vi1 digital camera (Nikon Instruments, EuropeBV, Kingston, Surrey, England) connected to a computer. The fields were randomly selected evaluating the most-positive, moderately positive, and
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less-positive areas. The area of each field (0.07 mm2) was standardized using NIS Elements BR 3.22 imaging software (Nikon Instruments). To evaluate intraobserver and interobserver variability, the counts were made 3-times by each examiner on each slide. Only the mean value was calculated for each case and used for statistical analysis. The k 4 0.80 showed a substantial agreement between the 2 observers and among different observations of the same observer. 2.4. Statistical analysis All data were represented as the means ⫾ standard deviation. Pearson's correlation was used to test the relationship between marker expressions and between marker expression and clinic-pathological features. Cox regression analysis was used to test variables on multivariate analysis. Survival parameters were compared using Kaplan-Meier curves and log-rank test. Patients without evidence of disease (local recurrence or metastasis) during follow-up were considered to have a good prognosis; and patients with local recurrence or distant metastasis during follow-up were considered to have a poor prognosis. The P o 0.05 was considered of statistical significance. 3. Results 3.1. Patients Of the patients, 98 were men and 56 were women. Mean age was 63.12 ⫾ 7.1 years (range: 35–84 y). A total of 71 tumors were in the right kidney and 83 tumors were in the left kidney. Mean diameter was 6.7 ⫾ 3.3 cm (range: 2.5– 17 cm). Pathological stage and grade are shown in Table 1. Median follow-up was 196.18 months (range: 5–274 mo) and median survival was 125.34 months (range: 5–274 mo). In all, 89 patients did not have evidence of metastases; whereas, 65 patients (42.2%) developed distant metastases in the follow-up: (1) 7 patients with pT1 disease developed multiple lung and bone metastases at 24.5 months after surgery (range: 10–48 mo) and died at 32.1 months (range: 18–59 mo); (2) 4 patients with pT2 disease developed disseminated metastases at 11.0 months (range: 10–12 mo) and died at 12.7 months (range: 10–16 mo); (3) 50 patients Table 1 Pathological stage and grade of RCC in our patients Fuhrman grade
Stage pT1 pT2 pT3 pT4 Total
Total
G1
G2
G3
G4
6 6 7
27 3 18
19
48
24 12 20 2 58
1 2 24 2 29
3
with pT3 disease had metastases after a mean time of 23 ⫾ 16.5 months (range: 15–32 mo) and died at 29.5 ⫾ 25.8 months (range: 17–36 mo) (overall, the patients had respectively multiple bone and liver metastases in 3 cases, multiple lung and bone metastases in 9 patients, multiple liver metastases in 15 patients, and multiple pulmonary metastases in 23 patients); and (4) the 4 patients with pT4 disease showed disseminated metastases at 3.2 months (range: 2–6 mo) after surgery and died at 5.1 months (range: 4–8 mo). None of the patients had local relapse. All the patients received adjuvant systemic therapy with interferon at the time of metastases discovery and thereafter. 3.2. BAP1 associations with clinic and pathological characteristics We found that BAP1 protein was expressed both as nuclear and cytoplasmic staining pattern (Fig. 1); however, nuclear BAP1 expression showed a high frequency of loss in tumoral cells compared to nontumoral tissue; in fact, nuclear staining was not present in 48% of our cases, whereas 8.7% of them did not show cytoplasm staining for BAP1. We found a significant correlation between the absence of nuclear BAP1 and the pathological features known to be associated with an adverse outcome; in particular, nuclear BAP1-negative tumors had higher tumor size (P o 0.0001; r ¼ 0.709), higher Fuhrman grade (P o 0.0001; r ¼ 0.677), and higher stage (P o 0.05; r ¼ 0.567); moreover, renal tumors that showed loss of nuclear BAP1 had a greater amount of vascular (P o 0.05; r ¼ 0.473) and capsular invasion (P o 0.005; r ¼ 0.461) and a higher incidence of metastases (P o 0.05; r ¼ 0.437) (Fig. 2). On the contrary, the cytoplasmic BAP1 expression did not show any correlation with the above considered features (Fig. 3). To evaluate whether loss of BAP1 expression correlates with prognosis, Kaplan-Meier survival curves were considered using overall or disease-specific 5-year survival; our data showed that nuclear BAP1 staining was correlated with both overall and disease-specific 5-year survival (log-rank test; P o 0.001) (Fig. 4A), whereas no correlation was observed between cytoplasmic BAP1 expression and overall and disease-specific 5-year survival (log-rank test; P o 0.343) (Fig. 4B). In multivariate analysis, pathological stage and nuclear BAP1 expression resulted independent prognostic factors (Table 2). 4. Discussion
58 23 69 4 154
Nowadays, most patients diagnosed with ccRCC present with small, organ-confined tumors with a “low risk” of progression and cancer-specific death, and which can be cured by surgery; however, there is a minor group of these
4
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Fig. 1. BAP1 immunohistochemical expression in ccRCC. (A) BAP1-negative tumor: all tumor nuclei and cytoplasms were uniformly negatively stained for BAP1, whereas lymphocytes serve as positive controls. (B) High magnification of BAP1-negative tumor: note that both nuclei and cytoplasms resulted negative. (C) Nuclear BAP1-positive tumor. (D) High magnification of BAP1-positive nuclei (↑), cytoplasm was not stained for BAP1. (E) Diffusely weak staining for BAP1 in tumor cytoplasms, whereas nuclei were uniformly negative. (F) High magnification of cytoplasm BAP1-positive tumor (↑). (G) Diffusely strong BAP1 staining in tumor cytoplasms, whereas nuclei were uniformly negative. (H) High magnification of BAP1-positive cytoplasm (↑). (Immunoperossidase, bars: 20 mm). (Color version of figure is available online.)
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Fig. 2. Correlation between nuclear BAP1 expression and clinic and pathological characteristics (Pearson's correlation test). Tumor size values were divided in 3 scores—1: 2.5–4 cm; 2: 4.10–7.00 cm; and 3: 47 cm. Vascular and capsular invasion were scored as 1 when absent and as 2 when present. Metastases status was scored as 1 when metastases were present and as 2 when metastases were absent.
patients (5%–10%) who are diagnosed with small ccRCC but develop metastatic disease and die of ccRCC. Therefore, it would be important to develop reliable tests that identify those patients with low-risk ccRCC who will experience progressive disease. The use of molecular genetics to identify these patients could be able to provide not only a useful prognostic marker but also a potential therapeutic target, helping us to identify potential new targets for anticancer drugs to be used in the adjuvant setting. Recent advances have illustrated that renal cancer genomes are very complex [17]. The loss of the VHL gene
that is located on chromosome 3p, an area frequently deleted in ccRCC, has been involved in the molecular pathogenesis of ccRCC [6,7]; recurrent mutations of additional genes located on chromosome 3p, including BAP1, PBRM, and SETD2, have also been identified [8–10]. Deletions in 3p are detected in almost 100% of small cell lung cancers, more than 90% of NSCLC cell lines, and 480% of breast carcinomas [17]. The 3p loss may eliminate VHL gene function and would leave cells with just a single copy of BAP1 and PBRM1. Mutation of the remaining BAP1 or PBRM1 allele may initiate tumorigenesis [18].
Fig. 3. Correlation between cytoplasmic BAP1 expression and clinic and pathological characteristics (Pearson's correlation test). Tumor size values were divided in 3 scores—1: 2.5–4 cm; 2: 4.10–7.00 cm; and 3: 47 cm. Vascular and capsular invasion were scored as 1 when absent and as 2 when present. Metastases status was scored as 1 when metastases were present and as 2 when metastases were absent.
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Fig. 4. Kaplan-Meier curves for disease-free survival in relation to nuclear BAP1 expression (A) and to cytoplasmic BAP1 expression (B) divided into groups of negative and positive expression. Overall survival was negatively correlated with negative BAP1 nuclear expression (P o 0.001). The log-rank test was used to calculate significance. Cum survival ¼ cumulative survival. (Color version of figure is available online.)
The BAP1 associates with several proteins involved in chromatin modification and transcription. The inhibition of cell cycle progression by BAP1 is closely related to the nuclear localization of BAP1, where it might interact with several transcription factors. In fact, BAP1 binds and deubiquitinates the transcriptional regulator host cell factor 1 that interacts with histone-modifying complexes [19]. You et al. [20] revealed BAP1-interacting partners, including the transcription factor Yin Yang 1, a zinc finger protein that possesses dual functionality by either activating or repressing gene expression depending on its association with specific transcription coactivators or corepressors at specific target gene promoters. Kapur et al. [8] observed that BAP1-negative tumors are more likely to be PDL1positive and have higher expression of some markers reported and validated to be associated with greater risk of RCC-specific death as survivin and Ki-67. However, the exact mechanism that control the nuclear BAP1 localization is still not fully understood, as well as the clinical implications of nuclear BAP1 loss remain to be explored. As BAP1 loss was associated with high tumor grade and correlated with metastasis development in uveal melanoma; an analysis of 176 tumors examined showed that BAP1 loss correlated with high Fuhrman nuclear grade and Table 2 Multivariate analysis of death-specific predictive factors
Sex Age Pathological stage Tumor size Furhman grade Vascular invasion Capsular invasion Nuclear BAP1 Cytoplasmic BAP1
P
Relative risk
95% CI
0.161 0.136 0.001 0.161 0.252 0.930 0.790 0.008 0.121
4.206 1.174 3.591 1.885 1.517 1.045 0.771 1.922 1.320
0.8–17.6 0.9–1.4 1.2–8.2 0.7–4.5 0.7–3.1 0.4–3.2 0.2–3.1 1.2–2.9 0.8–2.1
poor prognosis. From a therapeutic standpoint, RCC is considered radioresistant and BAP1-deficient tumors may be more sensitive [21]. Nuclear BAP1 loss confers increased susceptibility for the development of other tumors, including epithelioid atypical Spitz tumors, cutaneous melanoma, and mesothelioma [21]. It has been demonstrated that BAP1 inhibits tumor growth in vitro and in animal models [6,22]. The BAP1 functions as a DUB enzyme that regulates multiple cellular pathways related to tumorigenesis [4,13,17]. It has been observed that the BAP1 mutation rates increase with increasing of stage, suggesting that it may be implicated in ccRCC progression; and an association between BAP1 mutation and high Fuhrman grade was observed; furthermore, more than 50% of BAP1-mutated tumors exhibited coagulative necrosis that is a predictor of poor outcome. Kaplan-Meier analyses showed that BAP1 mutation was associated with a significantly worse overall survival (OS) [7,8,23]. The BAP1 is a nuclear-localized DUB enzyme, which contains a functional classic Nuclear Localization Signal; localization in the nucleus is required for BAP1mediated tumor suppression [6,24,25]; it has been supposed that BAP1 has a nuclear substrate that supports a mechanism, whereby BAP1 must enter the nucleus and its DUB activity is involved in functions that lead to tumor suppression. These functions may include DNA damage repair, regulation of apoptosis, or senescence or cell cycle regulation or all of these [25]. We have observed that in ccRCC loss of nuclear BAP1 expression correlates with tumor size, grade and stage, vascular and capsular invasion, and OS, whereas cytoplasmic expression does not seem to affect the patients' outcome. Similar findings were reported by Kapur et al. [8], which found in a multi-institutional cohort with ccRCC that the loss of nuclear BAP1 expression was associated with adverse clinicopathological variables, including high Fuhrman grade, advanced pT stage, worse disease-free
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survival and OS than patients with BAP1-positive tumors. Differently from us, Kapur et al. did not distinguish the nuclear and cytoplasmic BAP1 localization and considered mainly the nuclear BAP1 staining. Moreover, their study suggested that even if it has been previously shown that there is a good correlation between BAP1 mutations and loss of BAP1 protein, IHC assay has the ability to identify tumors that lack a BAP1 mutation but do not express BAP1 due to epigenetic silencing. Therefore, IHC may be superior to sequencing as a screening test. They also observed that BAP1-negative tumors are more likely to be PDL1-positive and have higher expression of survivin and Ki-67, so BAP1 could be combined with other known biomarkers to create more robust, multibiomarker panels for predicting ccRCC outcome. Other authors have reported different findings of BAP1 expression. Xin-Ke-Zhang et al. [26] have recently observed that in gliomas, high cytoplasmic expression of BAP1 was a significant independent biomarker for adverse OS, whereas nuclear expression of BAP1 was not correlated with the clinicopathological parameters. Another recent study in uveal melanoma showed that the wild-type BAP1 exhibits a typical nuclear staining, whereas mutants of BAP1 had negative nuclear expression and stronger cytoplasmic staining [27]. These findings could suggest that the expression of BAP1 protein could be mediated by multiple factors including the tumor site; however, BAP1 mutations associated with protein expression are very complex and it is difficult to explain correctly the complexity of BAP1 expression. Our data support that, in addition to pathological stage, nuclear BAP1 expression is the most important independent prognostic factor for cancer-specific death in ccRCC and could be able to identify the most aggressive forms of this tumor (even among those with low-risk tumors). Therefore, we strongly suggest for the use of BAP1 staining to obtain better informations about the postsurgical management for patients with clinically localized ccRCC; these finding can be possible useful targets for personalized therapies [22]. There are several limitations of our study. Although the number of patients is large, we believe that a further validation may be necessary to confirm that nuclear BAP1 loss is an independent marker of prognosis in the low-risk group of patients. Our patients exhibit limited racial/ethnic diversity (100% White). Further studies would be necessary to determine the prevalence of BAP1 mutations in metastatic lesions.
5. Conclusions We have demonstrated that BAP1 expression is a marker of prognosis in ccRCC, having an influence on cancerspecific survival. In particular, we confirmed that in ccRCC nuclear BAP1 expression is a prognostic marker rather than cytoplasmic expression even if the mechanism of varying
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expression remained poorly defined. The clinical importance for BAP1 would be realized with the identification and application of targeted therapies and with individualized approaches in the adjuvant setting or in the metastatic setting or in both the settings. References [1] Ho TH, Kapur P, Joseph RW, Serie DJ, Eckel-Passow JE, Parasramka M, et al. Loss of PBRM1 and BAP1 expression is less common in non–clear cell renal cell carcinoma than in clear cell renal cell carcinoma. Urol Oncol 2015;33:e9. [2] Lopez-Beltran A, Carrasco JC, Cheng L, Scarpelli M, Kirkali Z, Montironi R. 2009 Update on the classification of renal epithelial tumors in adults. Int J Urol 2009;16:432–43. [3] Varela I, Tarpey P, Raine K, Huang D, Ong CK, Stephens P, et al. Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma. Nature 2011;469:539–42. [4] Dalgliesh GL, Furge K, Greenman C, Chen L, Bignell G, Butler A, et al. Systematic sequencing of renal carcinoma reveals inactivation of histone modifying genes. Nature 2010;463:360–3. [5] Brugarolas J. PBRM1 and BAP1 as novel targets for renal cell carcinoma. Cancer J 2013;19:324–32. [6] Ventii KH, Devi NS, Friedrich KL, Chernova TA, Tighiouart M, Van Meir EG, et al. BRCA1-associated protein-1 is a tumor suppressor that requires deubiquitinating activity and nuclear localization. Cancer Res 2008;68:6953–62. [7] Kapur P, Pena-Llopis S, Christie A, Zhrebker L, Pavía-Jiménez A, Rathmell WK, 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. [8] Kapur P, Christie A, Raman JD, Then MT, Nuhn P, Buchner A, et al. BAP1 immunohistochemistry predicts outcomes in a multiinstitutional cohort with clear cell renal cell carcinoma. J Urol 2014;191:603–10. [9] Popova T, Hebert L, Jacquemin V, Gad S, Caux-Moncoutier V, Dubois-d'Enghien C, et al. Germline BAP1 mutations predispose to renal cell carcinomas. Am J Hum Genet 2013;92:974–80. [10] Farley MN, Schmidt LS, Mester JL, Pena-Llopis S, Pavia-Jimenez A, Christie A, et al. A novel germline BAP1 mutation predisposes to familial clear cell renal cell carcinoma. Mol Cancer Res 2013;11:1061–71. [11] Pena-Llopis S, Vega-Rubin-de-Celis S, Liao A, Leng N, Pavía-Jiménez A, Wang S,M, et al. BAP1 loss defines a new class of renal cell carcinoma. Nat Genet 2012;44:751–9. [12] Guo G, Gui Y, Gao S, Tang A, Hu X, Huang Y, et al. Frequent mutations of genes encoding ubiquitin-mediated proteolysis pathway components in clear cell renal cell carcinoma. Nat Genet 2011;44: 17–9. [13] Pawlowski R, Muhl SM, Sulser T, Krek W, Moch H, Schraml P. Loss of PBRM1 expression is associated with renal cell carcinoma progression. N Engl J Med 2013;132:E11–7. [14] Gerlinger M, Rowan AJ, Horswell S, et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med 2012;366:883–92. [15] Fuhrman SA, Lasky LC, Limas C. Prognostic significance of morphologic parameters in renal cell carcinoma. Am J Surg Pathol 1982;6:655–63. [16] Sobin LH, Witteking CH. TNM classification of malignant tumours, 6th ed. New York: Wiley-Liss, 2002. [17] Angeloni D. Molecular analysis of deletions in human chromosome 3p21 and the role of resident cancer genes in disease. Brief Funct Genomic Proteomic 2007;6:19–39. [18] Kapur P, Peña-Llopis S, Christie A, Zhrebker L, Pavía-Jiménez A, Rathmell WK, et al. Effects on survival of BAP1 and PBRM1
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[23] Joseph RW, Kapur P, Serie DJ, Eckel-Passow JE, Parasramka M, Ho TH, et al. Loss of BAP1 protein expression is an independent marker of poor prognosis in patients with low-risk clear cell renal cell carcinoma. Cancer 2014;120:1059–67. [24] Wang SS, Gu YF, Wolff N, Stefanius K, Christie A, Dey A, et al. Bap1 is essential for kidney function and cooperates with Vhl in renal tumorigenesis. Proc Natl Acad Sci U S A 2014;111:16538–43. [25] Murali R, Wiesner T, Scolyer RA. Tumours associated with BAP1 mutations pathology 2013;45:116–26. [26] Zhang XK, Xi SY, Sai K, Chen HD, Zhong ZH, Wu QL, et al. Cytoplasmic expression of BAP1 as an independent prognostic biomarker for patients with gliomas. Int J Clin Exp Pathol 2015;8:5035–43. [27] Koopmans AE, Verdijk RM, Brouwer RW, van den Bosch TP, van den Berg MM, Vaarwater J, et al. Clinical significance of immunohistochemistry for detection of BAP1 mutations in uveal melanoma. Mod Pathol 2014;27:1321–30.
Original article
Loss of nuclear BAP1 protein expression is a marker of poor prognosis in patients with clear cell renal cell carcinoma Daniele Minardi, M.D.a,1,*, Guendalina Lucarini, Ph.D.b,1, Giulio Milanese, M.D.a, Roberto Di Primio, M.D.b, Rodolfo Montironi, M.D.c, Giovanni Muzzonigro, M.D.a a
Dipartimento di Scienze Cliniche e Specialistiche—Sezione di Urologia, Università Politecnica delle Marche—Azienda Ospedaliero-Universitaria Ospedali Riuniti, Ancona, Italy b Dipartimento di Scienze Cliniche e Molecolari—Sezione di Istologia, Università Politecnica delle Marche, Ancona, Italy c Dipartimento di Scienze Biomediche e Sanità Pubblica—Sezione di Patologia e Istopatologia, Università Politecnica delle Marche—Azienda OspedalieroUniversitaria Ospedali Riuniti, Ancona, Italy Received 20 November 2015; received in revised form 24 February 2016; accepted 11 March 2016
Abstract Introduction: BRCA1-associated protein 1 (BAP1) is a gene situated on chromosome 3p in a region that is deleted in more than 90% of renal cell carcinomas (RCCs). In the present study, we studied BAP1 immunohistochemical expression in a large series of conventional clear cell RCCs (ccRCCs) treated with radical nephrectomy; we assessed the prognostic value of their expression in terms of patients' survival at long-term follow-up. Materials and methods: A total of 154 consecutive patients with ccRCC were selected from a prospective database and considered for the study purpose; all patients were treated with radical nephrectomy and lymphadenectomy at our Institute of Urology between 1983 and 1985. The features considered in this study were tumor size, grade and stage, vascular and capsular invasion, incidence of metastasis, and patient-specific survival; all these parameters were correlated with immunohistochemical cytoplasmic and nuclear expression of BAP1 in tumoral tissue. Results: Median follow-up was 196.18 months and median survival was 125.34 months. Nuclear BAP1 expression showed a high frequency of loss in tumoral cells; nuclear BAP1-negative tumors had higher tumor size, higher Fuhrman grade, and higher stage, a greater amount of vascular and capsular invasion and a higher incidence of metastases. In multivariate analysis, pathological stage and nuclear BAP1 expression resulted independent prognostic factors. Conclusion: We have demonstrated that nuclear BAP1 expression is a marker of prognosis in ccRCC, having an influence on cancerspecific survival. The clinical importance for BAP1 will be realized with the identification and application of targeted therapies and with individualized approaches in the adjuvant setting or in the metastatic setting or in both the settings. r 2016 Elsevier Inc. All rights reserved.
Keywords: BAP1 protein; Clear cell renal cell carcinoma; Immunohistochemistry; Cancer-specific survival
1. Introduction Renal cell carcinoma (RCC) represents the most common form of cancer originating from the renal parenchyma [1]; 1
Minardi D. and Lucarini G. contributed equally to the article. Corresponding author. Tel.: þ39-071-596-5667; fax: þ39-071-5963367. E-mail address: [email protected] (D. Minardi). *
http://dx.doi.org/10.1016/j.urolonc.2016.03.006 1078-1439/r 2016 Elsevier Inc. All rights reserved.
because of the increased use of abdominal imaging, most RCCs are detected at an early stage, can be surgically removed, and patients can be cured. In spite of this, there is a minority of patients who die of metastatic renal cancer; however, at present no markers exist to identify this group. The RCCs' classification has been recently updated to include morphology characteristics, growth pattern, cell of origin, and immunohistochemical staining [1,2]; in addition, investigators are exploring the molecular pattern of RCC
2
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subtypes and have reported that loss-of-function mutations are common events in clear cell renal cell carcinoma tumors (ccRCC) [3,4]. The recent discoveries about the molecular genetics of renal cancer have offered the opportunity to provide a therapeutic target. Von Hippel-Lindau (VHL) mutations were found to regulate hypoxia-inducible factor and vascular endothelial growth factor, leading to the development of vascular endothelial growth factor inhibitors. A second pathway has been implicated in renal cancer, the mammalian target of rapamycin complex 1 that is a major regulator of cell growth, and this has led to the development of mammalian target of rapamycin inhibitors. Studies involving the molecular pathogenesis of ccRCC focused on the loss of VHL gene located on chromosome 3p, an area frequently deleted in ccRCC. Among genes mutated in ccRCC, BRCA1-associated protein 1 (BAP1) has recently been identified; BAP1 is on chromosome 3p in a region that is deleted in more than 90% of ccRCCs [3–5]. Mutations in BAP1 occur in 5% to 15% of sporadic ccRCC tumors, and in some familial cases of ccRCC [6,7,8–14]. The BAP1 functions as a deubiquitinating (DUB) enzyme that regulates multiple cellular pathways related to tumorigenesis, an association between BAP1 mutations; and prognosis in patients with ccRCC has been investigated [12,14]. In the present study, we have studied BAP1 immunohistochemical expression in a large series of conventional ccRCCs treated with radical nephrectomy; we assessed the prognostic value of their expression in terms of patients' survival at long-term follow-up.
2. Materials and methods 2.1. Patients A total of 154 consecutive patients with clear cell RCC without sarcomatoid features were selected from a prospective database and considered for the study purpose; all patients were treated with radical nephrectomy and lymphadenectomy at our Institute of Urology between 1983 and 1985. Preoperative imaging consisted of ultrasound in 100% of cases, chest and abdominal computed tomography (CT) in 95% of cases (or chest and abdominal magnetic resonance [MR] in 5% of cases). For the study purpose, only patients without lymph nodes' involvement or distant metastases at the time of diagnosis were included. Followup examination at 3-month intervals in year 1 after surgery included chest and abdominal CT or MR; thereafter chest and abdominal CT or MR were performed at 6-month intervals, and bone scan was performed when clinically required. At year 6 after surgery and thereafter, chest X-ray, abdominal ultrasound and general examination were performed at 6-month intervals; and at year 10 after surgery, these tests were performed yearly.
Disease recurrence was defined as evidence of measurable disease on imaging, including CT, MR imaging, bone scan or ultrasound, and cytological/histological evaluation of suspected lesions. We have studied cancer-specific survival that is the percentage of people who have not died of kidney cancer during the long time of follow-up. 2.2. Histologic features Archived materials containing histological sections from the 154 patients were retrieved from the Institute of Pathological Anatomy and used for the study purpose. Tumor grade was based on the Fuhrman scheme. Fuhrman grade is on a scale of I to IV, where grade I carries the best prognosis and grade IV the worst [15]; tumor staging was based on Union for International Cancer Control classification [16]. The features considered in this study were tumor size, grade and stage, vascular and capsular invasion, and incidence of metastasis; furthermore, we have considered patient-specific survival; all these parameters were correlated with immunohistochemical cytoplasmic and nuclear expression of BAP1 in tumoral tissue. 2.3. Immunohistochemistry Immunohistochemistry (IHC) analysis was performed on 6-mm thick paraffin-embedded histological sections. After deparaffinization and rehydration, sections were treated with microwave for heat-induced epitope retrieval in EDTA Buffer (1 mM EDTA, 0.05% Tween 20, pH ¼ 9.0). Then the sections were incubated with the monoclonal antibody anti-BAP1 (clone C-4; 1:200 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, CA) and then they were immunostained using the streptavidin-biotin peroxidase technique (Envision peroxidase kit, Dako Cytomation, Milan, Italy). After incubation with 0.05% 3,30 diaminobenzidine (SigmaAldrich, Milan, Italy) in 0.05 M Tris buffer, pH ¼ 7.6 with 0.01% hydrogen peroxide, sections were counterstained with Mayer's hematoxylin (BioOptica, Milan, Italy), dehydrated in ethanol and coverslipped with Eukitt mounting medium (Electron Microscopy Sciences, PA). Positive controls for antibody against BAP1 were represented by internal positive staining in background stromal cells and intratumoral lymphocytes. For negative controls, primary antibody was replaced with nonimmune serum. All counts were performed separately by 2 investigators (G.L. and R.M.), blinded to the patient outcome. The reactivity for BAP1 was evaluated in nontumoral tissue, and in the nucleus and cytoplasm of the tumoral cells at least 10 fields/sample at 400 magnification and quantified as a percentage of the total counted cells. Images were captured with a Nikon DS-Vi1 digital camera (Nikon Instruments, EuropeBV, Kingston, Surrey, England) connected to a computer. The fields were randomly selected evaluating the most-positive, moderately positive, and
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less-positive areas. The area of each field (0.07 mm2) was standardized using NIS Elements BR 3.22 imaging software (Nikon Instruments). To evaluate intraobserver and interobserver variability, the counts were made 3-times by each examiner on each slide. Only the mean value was calculated for each case and used for statistical analysis. The k 4 0.80 showed a substantial agreement between the 2 observers and among different observations of the same observer. 2.4. Statistical analysis All data were represented as the means ⫾ standard deviation. Pearson's correlation was used to test the relationship between marker expressions and between marker expression and clinic-pathological features. Cox regression analysis was used to test variables on multivariate analysis. Survival parameters were compared using Kaplan-Meier curves and log-rank test. Patients without evidence of disease (local recurrence or metastasis) during follow-up were considered to have a good prognosis; and patients with local recurrence or distant metastasis during follow-up were considered to have a poor prognosis. The P o 0.05 was considered of statistical significance. 3. Results 3.1. Patients Of the patients, 98 were men and 56 were women. Mean age was 63.12 ⫾ 7.1 years (range: 35–84 y). A total of 71 tumors were in the right kidney and 83 tumors were in the left kidney. Mean diameter was 6.7 ⫾ 3.3 cm (range: 2.5– 17 cm). Pathological stage and grade are shown in Table 1. Median follow-up was 196.18 months (range: 5–274 mo) and median survival was 125.34 months (range: 5–274 mo). In all, 89 patients did not have evidence of metastases; whereas, 65 patients (42.2%) developed distant metastases in the follow-up: (1) 7 patients with pT1 disease developed multiple lung and bone metastases at 24.5 months after surgery (range: 10–48 mo) and died at 32.1 months (range: 18–59 mo); (2) 4 patients with pT2 disease developed disseminated metastases at 11.0 months (range: 10–12 mo) and died at 12.7 months (range: 10–16 mo); (3) 50 patients Table 1 Pathological stage and grade of RCC in our patients Fuhrman grade
Stage pT1 pT2 pT3 pT4 Total
Total
G1
G2
G3
G4
6 6 7
27 3 18
19
48
24 12 20 2 58
1 2 24 2 29
3
with pT3 disease had metastases after a mean time of 23 ⫾ 16.5 months (range: 15–32 mo) and died at 29.5 ⫾ 25.8 months (range: 17–36 mo) (overall, the patients had respectively multiple bone and liver metastases in 3 cases, multiple lung and bone metastases in 9 patients, multiple liver metastases in 15 patients, and multiple pulmonary metastases in 23 patients); and (4) the 4 patients with pT4 disease showed disseminated metastases at 3.2 months (range: 2–6 mo) after surgery and died at 5.1 months (range: 4–8 mo). None of the patients had local relapse. All the patients received adjuvant systemic therapy with interferon at the time of metastases discovery and thereafter. 3.2. BAP1 associations with clinic and pathological characteristics We found that BAP1 protein was expressed both as nuclear and cytoplasmic staining pattern (Fig. 1); however, nuclear BAP1 expression showed a high frequency of loss in tumoral cells compared to nontumoral tissue; in fact, nuclear staining was not present in 48% of our cases, whereas 8.7% of them did not show cytoplasm staining for BAP1. We found a significant correlation between the absence of nuclear BAP1 and the pathological features known to be associated with an adverse outcome; in particular, nuclear BAP1-negative tumors had higher tumor size (P o 0.0001; r ¼ 0.709), higher Fuhrman grade (P o 0.0001; r ¼ 0.677), and higher stage (P o 0.05; r ¼ 0.567); moreover, renal tumors that showed loss of nuclear BAP1 had a greater amount of vascular (P o 0.05; r ¼ 0.473) and capsular invasion (P o 0.005; r ¼ 0.461) and a higher incidence of metastases (P o 0.05; r ¼ 0.437) (Fig. 2). On the contrary, the cytoplasmic BAP1 expression did not show any correlation with the above considered features (Fig. 3). To evaluate whether loss of BAP1 expression correlates with prognosis, Kaplan-Meier survival curves were considered using overall or disease-specific 5-year survival; our data showed that nuclear BAP1 staining was correlated with both overall and disease-specific 5-year survival (log-rank test; P o 0.001) (Fig. 4A), whereas no correlation was observed between cytoplasmic BAP1 expression and overall and disease-specific 5-year survival (log-rank test; P o 0.343) (Fig. 4B). In multivariate analysis, pathological stage and nuclear BAP1 expression resulted independent prognostic factors (Table 2). 4. Discussion
58 23 69 4 154
Nowadays, most patients diagnosed with ccRCC present with small, organ-confined tumors with a “low risk” of progression and cancer-specific death, and which can be cured by surgery; however, there is a minor group of these
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Fig. 1. BAP1 immunohistochemical expression in ccRCC. (A) BAP1-negative tumor: all tumor nuclei and cytoplasms were uniformly negatively stained for BAP1, whereas lymphocytes serve as positive controls. (B) High magnification of BAP1-negative tumor: note that both nuclei and cytoplasms resulted negative. (C) Nuclear BAP1-positive tumor. (D) High magnification of BAP1-positive nuclei (↑), cytoplasm was not stained for BAP1. (E) Diffusely weak staining for BAP1 in tumor cytoplasms, whereas nuclei were uniformly negative. (F) High magnification of cytoplasm BAP1-positive tumor (↑). (G) Diffusely strong BAP1 staining in tumor cytoplasms, whereas nuclei were uniformly negative. (H) High magnification of BAP1-positive cytoplasm (↑). (Immunoperossidase, bars: 20 mm). (Color version of figure is available online.)
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Fig. 2. Correlation between nuclear BAP1 expression and clinic and pathological characteristics (Pearson's correlation test). Tumor size values were divided in 3 scores—1: 2.5–4 cm; 2: 4.10–7.00 cm; and 3: 47 cm. Vascular and capsular invasion were scored as 1 when absent and as 2 when present. Metastases status was scored as 1 when metastases were present and as 2 when metastases were absent.
patients (5%–10%) who are diagnosed with small ccRCC but develop metastatic disease and die of ccRCC. Therefore, it would be important to develop reliable tests that identify those patients with low-risk ccRCC who will experience progressive disease. The use of molecular genetics to identify these patients could be able to provide not only a useful prognostic marker but also a potential therapeutic target, helping us to identify potential new targets for anticancer drugs to be used in the adjuvant setting. Recent advances have illustrated that renal cancer genomes are very complex [17]. The loss of the VHL gene
that is located on chromosome 3p, an area frequently deleted in ccRCC, has been involved in the molecular pathogenesis of ccRCC [6,7]; recurrent mutations of additional genes located on chromosome 3p, including BAP1, PBRM, and SETD2, have also been identified [8–10]. Deletions in 3p are detected in almost 100% of small cell lung cancers, more than 90% of NSCLC cell lines, and 480% of breast carcinomas [17]. The 3p loss may eliminate VHL gene function and would leave cells with just a single copy of BAP1 and PBRM1. Mutation of the remaining BAP1 or PBRM1 allele may initiate tumorigenesis [18].
Fig. 3. Correlation between cytoplasmic BAP1 expression and clinic and pathological characteristics (Pearson's correlation test). Tumor size values were divided in 3 scores—1: 2.5–4 cm; 2: 4.10–7.00 cm; and 3: 47 cm. Vascular and capsular invasion were scored as 1 when absent and as 2 when present. Metastases status was scored as 1 when metastases were present and as 2 when metastases were absent.
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Fig. 4. Kaplan-Meier curves for disease-free survival in relation to nuclear BAP1 expression (A) and to cytoplasmic BAP1 expression (B) divided into groups of negative and positive expression. Overall survival was negatively correlated with negative BAP1 nuclear expression (P o 0.001). The log-rank test was used to calculate significance. Cum survival ¼ cumulative survival. (Color version of figure is available online.)
The BAP1 associates with several proteins involved in chromatin modification and transcription. The inhibition of cell cycle progression by BAP1 is closely related to the nuclear localization of BAP1, where it might interact with several transcription factors. In fact, BAP1 binds and deubiquitinates the transcriptional regulator host cell factor 1 that interacts with histone-modifying complexes [19]. You et al. [20] revealed BAP1-interacting partners, including the transcription factor Yin Yang 1, a zinc finger protein that possesses dual functionality by either activating or repressing gene expression depending on its association with specific transcription coactivators or corepressors at specific target gene promoters. Kapur et al. [8] observed that BAP1-negative tumors are more likely to be PDL1positive and have higher expression of some markers reported and validated to be associated with greater risk of RCC-specific death as survivin and Ki-67. However, the exact mechanism that control the nuclear BAP1 localization is still not fully understood, as well as the clinical implications of nuclear BAP1 loss remain to be explored. As BAP1 loss was associated with high tumor grade and correlated with metastasis development in uveal melanoma; an analysis of 176 tumors examined showed that BAP1 loss correlated with high Fuhrman nuclear grade and Table 2 Multivariate analysis of death-specific predictive factors
Sex Age Pathological stage Tumor size Furhman grade Vascular invasion Capsular invasion Nuclear BAP1 Cytoplasmic BAP1
P
Relative risk
95% CI
0.161 0.136 0.001 0.161 0.252 0.930 0.790 0.008 0.121
4.206 1.174 3.591 1.885 1.517 1.045 0.771 1.922 1.320
0.8–17.6 0.9–1.4 1.2–8.2 0.7–4.5 0.7–3.1 0.4–3.2 0.2–3.1 1.2–2.9 0.8–2.1
poor prognosis. From a therapeutic standpoint, RCC is considered radioresistant and BAP1-deficient tumors may be more sensitive [21]. Nuclear BAP1 loss confers increased susceptibility for the development of other tumors, including epithelioid atypical Spitz tumors, cutaneous melanoma, and mesothelioma [21]. It has been demonstrated that BAP1 inhibits tumor growth in vitro and in animal models [6,22]. The BAP1 functions as a DUB enzyme that regulates multiple cellular pathways related to tumorigenesis [4,13,17]. It has been observed that the BAP1 mutation rates increase with increasing of stage, suggesting that it may be implicated in ccRCC progression; and an association between BAP1 mutation and high Fuhrman grade was observed; furthermore, more than 50% of BAP1-mutated tumors exhibited coagulative necrosis that is a predictor of poor outcome. Kaplan-Meier analyses showed that BAP1 mutation was associated with a significantly worse overall survival (OS) [7,8,23]. The BAP1 is a nuclear-localized DUB enzyme, which contains a functional classic Nuclear Localization Signal; localization in the nucleus is required for BAP1mediated tumor suppression [6,24,25]; it has been supposed that BAP1 has a nuclear substrate that supports a mechanism, whereby BAP1 must enter the nucleus and its DUB activity is involved in functions that lead to tumor suppression. These functions may include DNA damage repair, regulation of apoptosis, or senescence or cell cycle regulation or all of these [25]. We have observed that in ccRCC loss of nuclear BAP1 expression correlates with tumor size, grade and stage, vascular and capsular invasion, and OS, whereas cytoplasmic expression does not seem to affect the patients' outcome. Similar findings were reported by Kapur et al. [8], which found in a multi-institutional cohort with ccRCC that the loss of nuclear BAP1 expression was associated with adverse clinicopathological variables, including high Fuhrman grade, advanced pT stage, worse disease-free
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survival and OS than patients with BAP1-positive tumors. Differently from us, Kapur et al. did not distinguish the nuclear and cytoplasmic BAP1 localization and considered mainly the nuclear BAP1 staining. Moreover, their study suggested that even if it has been previously shown that there is a good correlation between BAP1 mutations and loss of BAP1 protein, IHC assay has the ability to identify tumors that lack a BAP1 mutation but do not express BAP1 due to epigenetic silencing. Therefore, IHC may be superior to sequencing as a screening test. They also observed that BAP1-negative tumors are more likely to be PDL1-positive and have higher expression of survivin and Ki-67, so BAP1 could be combined with other known biomarkers to create more robust, multibiomarker panels for predicting ccRCC outcome. Other authors have reported different findings of BAP1 expression. Xin-Ke-Zhang et al. [26] have recently observed that in gliomas, high cytoplasmic expression of BAP1 was a significant independent biomarker for adverse OS, whereas nuclear expression of BAP1 was not correlated with the clinicopathological parameters. Another recent study in uveal melanoma showed that the wild-type BAP1 exhibits a typical nuclear staining, whereas mutants of BAP1 had negative nuclear expression and stronger cytoplasmic staining [27]. These findings could suggest that the expression of BAP1 protein could be mediated by multiple factors including the tumor site; however, BAP1 mutations associated with protein expression are very complex and it is difficult to explain correctly the complexity of BAP1 expression. Our data support that, in addition to pathological stage, nuclear BAP1 expression is the most important independent prognostic factor for cancer-specific death in ccRCC and could be able to identify the most aggressive forms of this tumor (even among those with low-risk tumors). Therefore, we strongly suggest for the use of BAP1 staining to obtain better informations about the postsurgical management for patients with clinically localized ccRCC; these finding can be possible useful targets for personalized therapies [22]. There are several limitations of our study. Although the number of patients is large, we believe that a further validation may be necessary to confirm that nuclear BAP1 loss is an independent marker of prognosis in the low-risk group of patients. Our patients exhibit limited racial/ethnic diversity (100% White). Further studies would be necessary to determine the prevalence of BAP1 mutations in metastatic lesions.
5. Conclusions We have demonstrated that BAP1 expression is a marker of prognosis in ccRCC, having an influence on cancerspecific survival. In particular, we confirmed that in ccRCC nuclear BAP1 expression is a prognostic marker rather than cytoplasmic expression even if the mechanism of varying
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expression remained poorly defined. The clinical importance for BAP1 would be realized with the identification and application of targeted therapies and with individualized approaches in the adjuvant setting or in the metastatic setting or in both the settings. References [1] Ho TH, Kapur P, Joseph RW, Serie DJ, Eckel-Passow JE, Parasramka M, et al. Loss of PBRM1 and BAP1 expression is less common in non–clear cell renal cell carcinoma than in clear cell renal cell carcinoma. Urol Oncol 2015;33:e9. [2] Lopez-Beltran A, Carrasco JC, Cheng L, Scarpelli M, Kirkali Z, Montironi R. 2009 Update on the classification of renal epithelial tumors in adults. Int J Urol 2009;16:432–43. [3] Varela I, Tarpey P, Raine K, Huang D, Ong CK, Stephens P, et al. Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma. Nature 2011;469:539–42. [4] Dalgliesh GL, Furge K, Greenman C, Chen L, Bignell G, Butler A, et al. Systematic sequencing of renal carcinoma reveals inactivation of histone modifying genes. Nature 2010;463:360–3. [5] Brugarolas J. PBRM1 and BAP1 as novel targets for renal cell carcinoma. Cancer J 2013;19:324–32. [6] Ventii KH, Devi NS, Friedrich KL, Chernova TA, Tighiouart M, Van Meir EG, et al. BRCA1-associated protein-1 is a tumor suppressor that requires deubiquitinating activity and nuclear localization. Cancer Res 2008;68:6953–62. [7] Kapur P, Pena-Llopis S, Christie A, Zhrebker L, Pavía-Jiménez A, Rathmell WK, 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. [8] Kapur P, Christie A, Raman JD, Then MT, Nuhn P, Buchner A, et al. BAP1 immunohistochemistry predicts outcomes in a multiinstitutional cohort with clear cell renal cell carcinoma. J Urol 2014;191:603–10. [9] Popova T, Hebert L, Jacquemin V, Gad S, Caux-Moncoutier V, Dubois-d'Enghien C, et al. Germline BAP1 mutations predispose to renal cell carcinomas. Am J Hum Genet 2013;92:974–80. [10] Farley MN, Schmidt LS, Mester JL, Pena-Llopis S, Pavia-Jimenez A, Christie A, et al. A novel germline BAP1 mutation predisposes to familial clear cell renal cell carcinoma. Mol Cancer Res 2013;11:1061–71. [11] Pena-Llopis S, Vega-Rubin-de-Celis S, Liao A, Leng N, Pavía-Jiménez A, Wang S,M, et al. BAP1 loss defines a new class of renal cell carcinoma. Nat Genet 2012;44:751–9. [12] Guo G, Gui Y, Gao S, Tang A, Hu X, Huang Y, et al. Frequent mutations of genes encoding ubiquitin-mediated proteolysis pathway components in clear cell renal cell carcinoma. Nat Genet 2011;44: 17–9. [13] Pawlowski R, Muhl SM, Sulser T, Krek W, Moch H, Schraml P. Loss of PBRM1 expression is associated with renal cell carcinoma progression. N Engl J Med 2013;132:E11–7. [14] Gerlinger M, Rowan AJ, Horswell S, et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med 2012;366:883–92. [15] Fuhrman SA, Lasky LC, Limas C. Prognostic significance of morphologic parameters in renal cell carcinoma. Am J Surg Pathol 1982;6:655–63. [16] Sobin LH, Witteking CH. TNM classification of malignant tumours, 6th ed. New York: Wiley-Liss, 2002. [17] Angeloni D. Molecular analysis of deletions in human chromosome 3p21 and the role of resident cancer genes in disease. Brief Funct Genomic Proteomic 2007;6:19–39. [18] Kapur P, Peña-Llopis S, Christie A, Zhrebker L, Pavía-Jiménez A, Rathmell WK, et al. Effects on survival of BAP1 and PBRM1
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