PTRF independently predicts progression and survival in multiracial upper tract urothelial carcinoma following radical nephroureterectomy

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Laboratory-Bladder cancer

PTRF independently predicts progression and survival in multiracial upper tract urothelial carcinoma following radical nephroureterectomy Hsin-Chih Yeh, M.D., Ph.D.a,b,c,d, Vitaly Margulis, M.D.d, Nirmish Singla, M.D.d, Elizabeth Hernandez, B.S.d, Vandana Panwar, M.D.e, Solomon L. Woldu, M.D.d, Jose A. Karam, M.D.f, Christopher G. Wood, M.D.f, Alon Z. Weizer, M.D.g, Jay D. Raman, M.D.h, Mesut Remzi, M.D.i, Nathalie Rioux-Leclercq, M.D., Ph.D.j, Andrea Haitel, M.D.k, Marco Roscigno, M.D.l, Christian Bolenz, M.D.m, Karim Bensalah, M.D., Ph.D.n, Ching-Chia Li, M.D., Ph.D.b,c, Hung-Lung Ke, M.D., Ph.D.b,c, Wei-Ming Li, M.D., Ph.D.b,c, Hsiang-Ying Lee, M.D.a,b,c, Leonid M. Rapoport, M.D., Ph.D.o, Yair Lotan, M.D.d, Payal Kapur, M.D.e, Shahrokh F. Shariat, M.D.d,i,o, Jer-Tsong Hsieh, Ph.D.d,p,*, Wen-Jeng Wu, M.D., Ph.D.b,c,q,** a

Department of Urology, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung, Taiwan Department of Urology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan c Department of Urology, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan d Department of Urology, University of Texas Southwestern Medical Center, Dallas, TX e Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX f Department of Urology, MD Anderson Cancer Center, Houston, TX g Department of Urology, University of Michigan, Ann Arbor, MI h Division of Urology, Penn State Milton S. Hershey Medical Center, Hershey, PA i Department of Urology, Medical University of Vienna, Vienna, Austria j Department of Pathology, Centre Hospitalier Universitaire de Rennes, Rennes, France k Department of Pathology, Medical University of Vienna, Vienna, Austria l Department of Urology, Ospedali Riuniti of Bergamo, Bergamo, Italy m Department of Urology, University of Ulm, Ulm, Germany n Department of Urology, Centre Hospitalier Universitaire de Rennes, Rennes, France o Institute for Urology and Reproductive Health, Sechenov University, Moscow, Russia p Department of Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan q Institute of Medical Science and Technology, National Sun Yat-sen University, Kaohsiung, Taiwan b

Received 3 September 2019; received in revised form 22 November 2019; accepted 25 November 2019

Abstract Objectives: Polymerase I and transcript release factor (PTRF) has been implicated in cancer biology but its role in upper tract urothelial carcinoma (UTUC) is unknown. From a pilot transcriptome, we identified PTRF was significantly upregulated in high stage UTUC. Bladder cancer transcriptome from The Cancer Genome Atlas (TCGA) supported our finding and high PTRF level also predicted poor survival. We, therefore, investigated the correlation of PTRF with patients’ clinicopathologic characteristics and outcomes in a multiracial UTUC cohort. Materials and methods: By immunohistochemical staining, PTRF expression was determined using H-score. PTRF expression of 575 UTUCs from 8 institutions, including 118 Asians and 457 Caucasians, was compared with various clinicopathologic parameters. Human urothelial cancer cell lines were used to evaluate the level of PTRF protein and mRNA expression, and PTRF transcript level was assessed This work was supported by the Ministry of Science and Technology [grant number MOST 105-2628-B-037-004-MY2], Kaohsiung Medical University Hospital [grant number KMUH105-5R45] and Kaohsiung Municipal Ta-Tung Hospital [grant number kmtth-103-054]. *Corresponding author. Tel.: +1-214-648-3988; fax: +1-214-648-8786. https://doi.org/10.1016/j.urolonc.2019.11.010 1078-1439/Ó 2019 Elsevier Inc. All rights reserved.

**Corresponding author. Tel.: +886-7-3208212; fax: +886-7-3211033. E-mail addresses: [email protected] (J.-T. Hsieh), [email protected] (W.-J. Wu).

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in fresh samples from 12 cases of the cohort. The impact of PTRF expression on disease progression, cancer-specific death and overall mortality was also examined. Results: High PTRF expression was significantly associated with multifocality (P = 0.023), high pathologic tumor stage (P < 0.00001), nonurothelial differentiation (P = 0.035), lymphovascular invasion (P = 0.003) and lymph node metastasis (P = 0.031). PTRF mRNA expression was also markedly increased in advanced stage UTUC (P = 0.0003). High PTRF expressing patients had consistently worse outcomes than patients with low PTRF expression regardless of demographic variation (all P < 0.005). In multivariate analysis, high PTRF expression was an independent predictor for progression-free survival (hazard ratio [HR] 1.70, 95% confidence interval [CI] 1.07−2.69, P = 0.025), cancer-specific survival (HR 2.09, 95% CI 1.28−3.42, P = 0.003), and overall survival (HR 2.04, 95% CI 1.33−3.14, P = 0.001). Conclusions: Results indicate that PTRF is a predictive biomarker for progression and survival and an independent prognosticator of UTUC. Elevated PTRF could probably propel clinically aggressive disease and serve as a potential therapeutic target for UTUC. Ó 2019 Elsevier Inc. All rights reserved.

Keywords: Upper tract urothelial carcinoma; Polymerase I and transcript release factor; Immunohistochemical staining; Progression; Prognosis; Urothelial carcinoma of bladder

1. Introduction Urothelial carcinoma (UC) arises from epithelial lining of pelvicalyceal cavities, ureter, bladder, and urethra and is the sixth most common cancer in the United States [1]. Urothelial carcinoma of bladder (UCB) accounts for 90% to 95% of UCs, and upper tract urothelial carcinoma (UTUC) is substantially less common (5%−10%). However, the incidence of UTUC can be as high as 30% in endemic area, like Taiwan [2,3]. In addition, Taiwanese patients may have better survival in early stage disease and worse outcomes in advanced stage UTUC [2,4]. Moreover, it has been shown that UTUC is highly heterogeneous, and discrepancies in clinicopathologic characteristics and predictors of non-organ-confined disease were shown between Chinese and American UTUC [5,6], implying that differences in risk factors, presentation, and evaluation may exist. Molecular factors exhibiting universal prognostic significance could be valuable and may explain common biology underlying the diverse clinical behavior of UTUC. More importantly, integration of predictive molecular biomarkers may improve risk stratification and facilitate tailored therapy. Although many tissue-based biomarkers have been reported for UTUC [7,8], no systemic validation in multiple demographics has been reported. Thus, in this study, we applied transcriptomic analyses to explore potential markers for disease progression from Taiwanese UTUC patients and identified polymerase I and transcript release factor (PTRF) as a candidate. PTRF, a soluble protein with putative nuclear localization sequences, is first identified as a regulator of RNA polymerase I and involved in dissociation of transcription complexes [9]. The role of PTRF in malignancies appears to be controversial. For example, PTRF plays an oncogenic role in pancreatic cancer [10], glioma [11], breast cancer [12], and glioblastoma [13] by increasing proliferation, invasion, metastasis, or drug resistance. In contrast, PTRF is a suppressor for epithelial-mesenchymal transition or MMP9 expression in non-small-cell lung cancer [14],

colorectal cancer [15], and prostate cancer [16,17]. Apparently, it is important to understand the implication of PTRF in a tumor-specific manner [18]. So far, the role of PTRF in UTUC has not been reported. Consistent with a positive correlation between PTRF transcript expression and pathologic tumor (pT) stage in genome-wide expression profiling of a pilot set of Taiwanese UTUC patients, the immunostaining data of PTRF showed a significant correlation with disease characteristics. The clinical significance of PTRF in UTUC outcomes was validated in 2 separate cohorts of Asian and Caucasian patients and also in the combined cohort. Furthermore, data mining from The Cancer Genome Atlas (TCGA) dataset supported the oncogenic role of PTRF in UCB. Our study demonstrated that PTRF was an independent prognosticator to predict disease progression and survival for UTUC from different ethnicities. 2. Materials and Methods 2.1. Transcriptome profiling Snap frozen tumor samples from a set of 48 Asian Taiwanese UTUC patients were subjected to high-throughput gene expression profiling using a microarray of Illumina HumanHT-12 v3 Expression BeadChip. Next, we extracted TCGA dataset (version 2016_01_28) which profiled 408 UCB tumor specimens from FireBrowse database (http:// www.firebrowse.org). The normalized PTRF expression level was obtained from BLCA.rnaseqv2__illuminahiseq_ rnaseqv2__unc_edu__Level_3__RSEM_genes_normalized__data.data file, and clinicopathologic information of each patient was extracted from BLCA.clin.merged.picked file for analysis. 2.2. Patient collection Two independent populations of UTUC were included in this study. The Asian cohort comprised 118 Taiwanese

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patients receiving radical nephroureterectomy (RNU) for UTUC at Kaohsiung Medical University Hospital between 2000 and 2009. The study protocol was approved by the review board of our institution (KMUHIRB-E(I)-20180288). The Caucasian population consisting of 457 patients treated with RNU between 1990 and 2008 was derived from a multicenter international cohort, and institutional review board approval was also obtained at all sites. In total, 575 UTUC patients were included for analyzing PTRF expression. First, the correlation of PTRF with disease progression and survival was analyzed in individual ethnical populations. In turn, PTRF was systematically validated in the well-characterized cases of UTUC altogether. Clinicopathologic data were recorded for each patient. Lymph node dissection was performed based on surgeon discretion, typically in patients with enlarged nodes on preoperative imaging or intraoperative examination. Patients were regularly followed with cystoscopy, urine cytology, and imaging studies postoperatively following institutional guidelines. Follow-up visits were generally every 3 months in the first year with increasing interval, thereafter if no relapse but at least once annually. Disease progression was defined as tumor recurrence in regional operation site or distant organ. A total of 59 patients (10.3%) who had deeply invasive tumors (≥ pT2) and/or positive lymph nodes underwent cisplatin-based adjuvant chemotherapy.

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2.3. Immunohistochemical staining and H-scoring Formalin-fixed, paraffin-embedded pathologic specimens were sectioned on slides, deparaffinized, and rehydrated. After performing with antigen retrieval method, endogenous peroxidase activity was blocked. Slides were subsequently incubated with a rabbit monoclonal primary antibody (IgG) targeting PTRF (#69036, Cell Signaling Technology). The optimal concentration (1:1000, 525 mg/l) was determined by serial dilutions. For antibody validation, ACHN renal cancer xenograft was used as a positive control and PC3 prostate cancer xenograft as a negative control. The immunostaining intensity was defined as negative (0+, no staining at all), weak (1+, faint staining), moderate (2+, modest staining), and strong (3+, intense staining). Two independent genitourinary pathologists blinded to patient information determined immunohistochemical staining (IHC) results using H-score. H-score (0−300) was calculated by multiplying percentage of stained tumor cells (Pi: 0−100) Pand each intensity of cytoplasmic staining (i: 0−3+) as Pi £ i. A cutoff of H-score best discriminating disease outcomes was determined by testing all possible values within central 80% distribution. An H-score ≥115 was defined as the high expression accordingly.

Fig. 1. Transcriptomic analyses of PTRF expression in UTUC and UCB patients. Significant elevation of PTRF expression in higher stage (pT3˗4) vs. lower stage (pTa-2) of UTUC patients from a pilot set of 48 Asian Taiwanese cases (A). From TCGA dataset, significant elevation of PTRF expression in higher stage (pT3˗4) vs. lower stage (pTa-2) (B) and N+ vs. N0/Nx (C) of UCB patients. Kaplan-Meier analysis of OS according to PTRF expression in UCB patients (D).

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2.4. Quantitative real time RT-PCR

2.6. Statistical analysis

The mRNA expression of PTRF was analyzed in fresh samples from 12 UTUC cases of this cohort using quantitative real time RT-PCR (qRT-PCR). Total RNA was isolated from human tissues or UC cell lines, quantified and reversely transcribed. Gene expression levels were amplified and detected with a Power SYBR Green PCR Master Mix kit in a QuantStudio 3 Real-Time PCR Detection System (Applied Biosystems). Primers used to target PTRF gene expression were as follows: sense: 50 -CAAGAAGCTGGAGGTCAACGA-30 ; antisense: 50 -AGCGCCTCCGACTCTTTCAG-3’. The expression fold of PTRF relative to normal urothelium was calculated after normalization to the internal control GAPDH.

Student’s t test was used to evaluate PTRF transcript level in relation to tumor status. The study cohort was divided into high and low PTRF expression groups by cutoff of H-score. The association between PTRF expression and clinicopathologic factors was analyzed by chi-square test for categorical parameters and Student’s t test for continuous parameters. The Kaplan-Meier method was used to estimate the impact of PTRF on progression-free survival (PFS), cancer-specific survival (CSS), and overall survival (OS). Survival rate was calculated from the date of RNU to the date of progression, cancer-specific death, death of any cause, or last visit. Survival curve was compared using the log-rank test. Significant variable in univariate analysis was incorporated in the multivariate Cox proportional hazards model to identify independent predictive factors for PFS, CSS, and OS. Statistical analyses were performed with SPSS, version 22.0, and 2-tailed P < 0.05 was considered statistically significant.

2.5. Western blot assay Human UC cells were lysed in RIPA lysis buffer containing protease inhibitor and the cell lysates were subjected to Bradford assay for determining protein concentration. Equal amount of protein extracts was electrophoresed on Bolt 4% to 12% Bis-Tris Plus gels (Thermo Fisher Scientific) and transferred to nitrocellulose membrane using Trans-Blot Turbo Transfer system (BIORAD). Membranes were blocked with 5% skimmed milk in PBST buffer for 1 hour and then incubated with same primary antibody for IHC at 4˚C for 16 to 18 hours, followed by incubating with horseradish peroxidaseconjugated secondary antibodies at room temperature for 1 hour. Enhanced chemiluminescence was used to visualize targeted proteins.

3. Results 3.1. Oncogenic role of PTRF in UC patients from transcriptomic analyses From the microarray analysis, a significantly higher PTRF expression was found in the higher stage (pT3˗4) compared with the lower stage (pTa˗2) UTUC from 48 Asian Taiwanese patients (Fig. 1A, P = 0.001). In addition, PTRF transcript was significantly elevated in cases with higher stage disease and positive lymph node involvement compared to each counterpart from TCGA dataset

Fig. 2. Representative images of PTRF IHC in tumor cells with negative (A), weak (B), moderate (C) and strong staining (D), and normal urothelium with negative staining (E, F). No PTRF staining in normal urothelium adjacent to tumor cells (C, D). Magnification, 100£.

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containing 408 UCB patients (Fig. 1B and C, P = 0.00004 and 0.017, respectively). Survival analysis also revealed a significantly worse outcome for patients with high PTRF expression (Fig. 1D, P = 0.005). 3.2. Correlation of PTRF expression with clinicopathologic characteristics of UTUC patients Mean age of the enrolled patients was 68.7 years, and mean duration of follow-up was 40.4 months. According to the American Joint Committee on Cancer staging system the distribution of pT stage in this cohort was pTa, pT1, pT2, pT3, and pT4 in 82 (14.3%), 178 (31.0%), 116 (20.2%), 165 (28.7%), and 34 tumors (5.9%), respectively. In terms of lymph node status, 87 cases (15.1%) were N0, 443 (77.0%) were Nx and 45 (7.8%) were N1˗2. Histological grade was classified based on the 2004 WHO/International

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Society of Urologic Pathology classification; 133 (23.1%) were low grade and 442 (76.9%) were high grade. Lymphovascular invasion, carcinoma in situ, and nonurothelial differentiation were observed in 126 (21.9%), 83 (14.4%), and 60 patients (10.4%), respectively. Of the patients, 135 (23.5%) experienced progression, 116 (20.2%) died of UTUC, and 178 (31.0%) died of any cause. From IHC, PTRF antibody exhibited a clear cytoplasmic staining rather than nuclear staining in tumor cells (Fig. 2). There was no staining in normal urothelium (Fig. 2E and F), irrespective of the positive staining of adjacent tumor cells (Fig. 2C and D). Forty-six patients (8.0%) were categorized as high PTRF expression. As shown in Table 1, there was no significant difference in age, gender, location, history of UCB, grade, and carcinoma in situ between high and low PTRF group. However, elevated PTRF level was significantly associated with aggressive disease features,

Table 1 Analyses of PTRF expression with clinicopathologic factors of UTUC patients Variable

Age, years (mean, SD) Gender Tumor location History of UCB Type of RNU Focality Grade pT stage

Lymphovascular invasion pN stage

Carcinoma in situ Nonurothelial differentiation Adjuvant chemotherapy Progression Death of UTUC All-cause death

Category (Value for age)

68.7 (10.2) Male Female Renal pelvis Ureter No Yes Open Laparoscopy Unifocal Multifocal Low High pTa pT1 pT2 pT3 pT4 No Yes pN0 pNx pN+ No Yes No Yes No Yes No Yes No Yes No Yes

No. of patients (%)

575 (100.0) 313 (54.4) 262 (45.6) 410 (71.3) 165 (28.7) 395 (68.7) 180 (31.3) 429 (74.6) 146 (25.4) 451 (78.4) 124 (21.6) 133 (23.1) 442 (76.9) 82 (14.3) 178 (31.0) 116 (20.2) 165 (28.7) 34 (5.9) 449 (78.1) 126 (21.9) 87 (15.1) 443 (77.0) 45 (7.8) 492 (85.6) 83 (14.4) 515 (89.6) 60 (10.4) 516 (89.7) 59 (10.3) 440 (76.5) 135 (23.5) 459 (79.8) 116 (20.2) 397 (69.0) 178 (31.0)

PTRF expression Low

High

68.6 (10.3) 288 (54.4) 241 (45.6) 383 (71.2) 155 (28.8) 359 (67.9) 170 (32.1) 389 (73.5) 140 (26.5) 421 (79.6) 108 (20.4) 124 (23.4) 405 (76.6) 80 (15.1) 170 (32.1) 109 (20.6) 147 (27.8) 23 (4.3) 421 (79.6) 108 (20.4) 79 (14.9) 413 (78.1) 37 (7.0) 457 (86.4) 72 (13.6) 478 (90.4) 51 (9.6) 482 (91.1) 47 (8.9) 420 (79.4) 109 (20.6) 436 (82.4) 93 (17.6) 379 (71.6) 150 (28.4)

69.9 (8.9) 25 (54.3) 21 (45.7) 27 (73.0) 10 (27.0) 36 (78.3) 10 (21.7) 40 (87.0) 6 (13.0) 30 (65.2) 16 (34.8) 9 (19.6) 37 (80.4) 2 (4.3) 8 (17.4) 7 (15.2) 18 (39.1) 11 (23.9) 28 (60.9) 18 (39.1) 8 (17.4) 30 (65.2) 8 (17.4) 35 (76.1) 11 (23.9) 37 (80.4) 9 (19.6) 34 (73.9) 12 (26.1) 20 (43.5) 26 (56.5) 23 (50.0) 23 (50.0) 18 (39.1) 28 (60.9)

P value

0.409 0.990 0.817 0.145 0.045 0.023 0.55 <0.00001

0.003 0.031

0.057 0.035 0.0002 <0.00001 <0.00001 <0.00001

PTRF = polymerase I and transcript release factor; RNU = radical nephroureterectomy; SD = standard deviation; UCB = urothelial carcinoma of bladder; UTUC = upper tract urothelial carcinoma.

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including multifocality (P = 0.023), lymphovascular invasion (P = 0.003), nonurothelial differentiation (P = 0.035), and a need for adjuvant chemotherapy (P = 0.0002). High PTRF expression was also significantly correlated with increased pT stage (P < 0.00001) and lymph node metastasis (P = 0.031), implying its role in disease progression. In order to understand whether the elevated PTRF protein expression in tissues corresponded to its gene expression, we performed qRT-PCR using a panel of human UC cell lines. As shown in Fig. 3A, aggressive cell lines exhibited an increased PTRF mRNA level, which can be correlated with the level of PTRF protein expression detected by Western blot (Fig. 3B) using the same antibody from IHC. Furthermore, using total cellular RNA extracted from fresh frozen samples of 12 cases in the cohort, the level of PTRF mRNA expression was significantly elevated in patients with advanced stage (Fig. 3C, P = 0.0003). In addition, patients with PTRF immunostaining had significantly higher PTRF transcript than those without (Fig. 3D, P = 0.026). Taken together, these data indicated a good correlation between PTRF mRNA expression detected by qRT-PCR and its protein expression detected by Western blot or IHC.

3.3. Correlation of PTRF expression with survival of UTUC patients Survival was analyzed in 2 independent ethnical populations. High PTRF expression was associated with a significant decline in PFS, CSS, and OS by Kaplan-Meier analysis in both Asian Taiwanese and Caucasian cohorts (Fig. 4, all P < 0.005), supporting its overall impact on UTUC progression regardless of ethnicities. Indeed, patients with high PTRF expression had apparently worse PFS, CSS, and OS than those with low PTRF expression in the entire cohort (all P < 0.00001, Fig. 4). Table 2 summarized the prognostic value of each variable in univariate and multivariate analyses. Universally significant factors for PFS, CSS, and OS in univariate analysis were tumor focality (P = 0.00002, P < 0.00001, and P = 0.00006, respectively), grade (P = 0.00003, 0.0001, and 0.0001, respectively), pT stage (all P < 0.00001), lymphovascular invasion (all P < 0.00001), pN stage (all P < 0.00001), carcinoma in situ (P = 0.0005, 0.001, and 0.0001, respectively), adjuvant chemotherapy (all P < 0.00001), and PTRF expression (all P < 0.00001). In multivariate analysis, PTRF status was significantly associated with PFS

Fig. 3. The expression of PTRF mRNA (A) detected by pRT-PCR and PTRF protein (B) detected by Western blot in human UC cell lines. Significantly increased PTRF mRNA in pT3-4 UTUC (C) and patients with positive IHC (D).

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Fig. 4. Kaplan-Meier analysis revealed high PTRF expression was significantly associated with decreased PFS, CSS and OS in Asian Taiwanese (A−C), Caucasian (D−F) and combined cohort (G−I) of UTUC patients, respectively.

(P = 0.025) in addition to pT stage (P < 0.00001), lymph node metastasis (P = 0.005), and adjuvant chemotherapy (P = 0.049). Age (P = 0.008), pT stage (P < 0.00001), lymph node involvement (P = 0.048), and PTRF expression (P = 0.003) were significant multivariate-adjusted predictors for CSS. As for OS, significant factors were age (P < 0.00001), history of UCB (P = 0.006), grade (P = 0.032), pT stage (P < 0.00001), lymphovascular invasion (P = 0.032), pN stage (P = 0.049), and PTRF expression (P = 0.001) in multivariate analysis. Notably, high PTRF expression appeared to be an independent prognostic factor for PFS, CSS, and OS (hazard ratio [HR] 1.698, 95% confidence interval [CI] 1.070−2.693; HR 2.091, 95% CI 1.279 −3.416; HR 2.042, 95% CI 1.329−3.139, respectively). 4. Discussion The potential value of PTRF as a marker in UTUC was initially identified by transcriptomic analysis, which showed a significant elevation of PTRF transcript in higher stage disease. Subsequently, IHC results demonstrated a

significant correlation of PTRF expression with adverse pathologic features, supporting its oncogenic role in UTUC progression. The potential clinical utility of PTRF in predicting disease progression and patient survival was validated from 2 different ethnical populations. This is the first report regarding the clinical relevance of PTRF in UTUC. The level of PTRF expression was an independent prognosticator of UTUC and referred to a 1.70-, 2.09-, and 2.04fold increase in the risk of progression, cancer-specific death, and all-cause death after multivariate adjustment, respectively. PTRF, also known as cavin1, is one of the 4 cavin isoforms and has been shown to play an oncogenic role in many cancer types. Cavin proteins can oligomerize into an outer coat complex to remodel lipid raft, a specific compartment in the plasma membrane essential for drug resistance. Higher PTRF level leading to lipid raft abundance is found to promote drug resistance of breast cancer and glioblastoma, and the chemoresistance can be reversed by PTRF knockdown [12,13]. PTRF induced by EGFR/PI3K/AKT pathway is responsible for cell proliferation of glioma tumor-derived

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Table 2 Univariate and multivariate analyses of PFS, CSS, and OS in 575 UTUC patients treated with RNU Variable

PFS

CSS

Univariate analysis HR

95% CI

P value

0.999-1.034

HR

95% CI

OS

Univariate analysis P value

Multivariate analysis

Univariate analysis

Multivariate analysis

95% CI

P value

HR

95% CI

P value

HR

95% CI

0.062

1.027

1.008-1.046

0.006

1.030

1.008-1.052

0.008

1.043

1.027-1.059 <0.00001 1.048 1.030-1.065 <0.00001

0.680

1 1.020

0.708-1.468

1 1.065

0.793-1.431

1 1.011

0.673-1.520

1 0.910

0.649-1.277

1 1.251

0.858-1.824

1 1.412

1.043-1.911

1 0.627

0.384-1.026

1 0.698

0.474-1.029

1 2.476

1.701-3.603

1 1.918

1.396-2.634

0.766-1.505 0.908 0.670-1.427 0.254 0.864-1.738 0.149 0.470-1.122

0.957

0.244

0.063

<0.00001 1 1.405

95% CI

P value

0.674

0.587

0.026

1 1.560 1.138-2.139

0.006

0.00006 1 1.109 0.777-1.583

0.569

0.0001

0.032

0.069

0.00002 1 1.254

0.847-1.856

2.055-7.453

0.00003 1 1.572

0.801-3.083

1 4.088

1.993-8.383

1 1.966

0.923-4.186

1 2.615

1.606-4.258

<0.00001 0.648-38.175 1.461-83.183 2.881-158.452 7.943-511.653

1 4.247 14.538 28.993 183.971

<0.00001 0.548-32.901 1.963-107.655 4.012-209.526 24.860-1361.470

1 3.027 8.597 14.631 41.885

<0.00001 0.386-23.773 1.133-65.251 1.959-109.277 5.135-341.611

1 1.015 1.924 3.664 19.522

0.509-2.027 0.977-3.791 1.944-6.903 9.580-39.783

<0.00001 1 0.756 1.258 2.179 6.472

1 3.992

<0.00001 1 1.456

0.954-2.223

1 3.133

2.308-4.251

<0.00001 1 1.502 1.036-2.177

0.032

2.765-5.764

1 0.800 7.053

<0.00001 1 1.030 2.133

0.582-1.821 1.015-4.480

1 0.841 5.127

0.542-1.305 3.033-8.665

<0.00001 1 1.033 0.659-1.619 2.036 1.060-3.914

0.049

0.457-1.399 3.811-13.054

1 2.040

1.318-3.158

1 2.012

1.405-2.883

1 1.376

0.785-2.412

1 1.037

0.609-1.765

1 4.255

2.848-6.358

<0.00001 1 0.959

0.572-1.609

1 2.663

1 3.735

2.365-5.899

<0.00001 1 2.091

1.279-3.416

1 2.828

1 4.975 11.023 21.367 63.751

0.188

2.674-5.307

<0.00001 1 1.112

0.742-1.668

0.429-1.147 4.202-12.665

<0.00001 1 0.863 2.034

0.524-1.423 1.068-3.877

0.0005 1.372-3.092

1 1.248

0.606

0.005

0.338 0.793-1.963

0.328 0.769-2.194

4.231-8.765

<0.00001 1 1.585

1.003-2.506

2.420-5.704

<0.00001 1 1.698

1.070-2.693

0.049

0.025

0.0001

0.001

1 1.026

0.107

HR

1.526-3.101

<0.00001 0.814-46.685 2.244-122.236 5.224-271.254 32.713-1791.244

0.258

0.917

P value

0.929-2.125 0.080

0.082

0.048

0.916 0.636-1.657

0.265

0.875

0.003

0.0001

1 1.777 1.050-3.007

<0.00001 0.371-1.541 0.616-2.570 1.092-4.348 2.643-15.848

1 1.237 0.839-1.823

0.283

<0.00001 1 0.689 0.425-1.117

0.131

1.836-3.860

<0.00001 1 2.042 1.329-3.139

0.001

1.887-4.239

0.894

CI = confidence interval; CSS = cancer-specific survival; HR = hazard ratio; OS = overall survival; PFS = progression-free survival; PTRF = polymerase I and transcript release factor; RNU = radical nephroureterectomy; UCB = urothelial carcinoma of bladder.

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HR

H.-C. Yeh et al. / Urologic Oncology: Seminars and Original Investigations 00 (2019) 1−10

Age, years (continuous) 1.017 Gender Male 1 Female 1.074 Tumor location Renal pelvis 1 Ureter 0.978 History of UCB No 1 Yes 1.226 Type of RNU Open 1 Laparoscopy 0.726 Focality Unifocal 1 Multifocal 2.175 Grade Low 1 High 3.914 pT stage pTa 1 pT1 6.166 pT2 16.562 pT3 37.643 pT4 242.068 Lymphovascular invasion No 1 Yes 3.767 pN stage pN0 1 pNx 0.702 pN+ 7.295 Carcinoma in situ No 1 Yes 2.060 Nonurothelial differentiation No 1 Yes 1.299 Adjuvant chemotherapy No 1 Yes 6.089 PTRF expression Low 1 High 3.716

Multivariate analysis

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exosome that exhibits a paracrine induction manner from in vitro and in vivo [11]. Clinically, patients with high grade glioma and relapsed glioblastoma express significantly higher PTRF than low grade and primary tumor, respectively [11,13]. In pancreatic cancer cells, PTRF overexpression activates Rho kinases and MMP9, while PTRF depletion can diminish PI3K/AKT-elicited epithelial-mesenchymal transition and reduce in vitro invasion and in vivo liver metastasis [10]. Furthermore, PTRF knockdown in rhabdomyosarcoma is shown to reduce cell proliferation, migration, and anchorage-independent cell growth [19]. Node-positive UTUC is potentially life-threatening with dismal survival [4]. However, inaccuracy of clinical N staging is not uncommon [20], and micrometastases are difficult to detect. Molecular biomarkers are expected to make up the inadequacy of current diagnostic imaging and improve prediction of non-organ-confined disease. Since PTRF expression was significantly elevated in patients with lymph node metastasis, high pT stage, and lymphovascular invasion, its expression in specimens obtained from ureteroscopy-guided biopsy could improve the identification of patients with positive lymph nodes and/or micrometastases. Patients with high PTRF expression may benefit from perioperative adjunct therapy; extended lymphadenectomy might be considered but survival advantage of this procedure has not yet been proved. From this study, high PTRF expression can also distinguish UTUC patients at high risk of progression, and perhaps PTRF could help determine if closer follow-up is needed for early detection. While clinicopathologic characteristics and prognostic predictors of UTUC could vary among demographic distribution [5,6], the molecular biomarker will be more precise and useful for this application under the assumption of universal molecular pathogenesis of this disease. In this study, no difference in PTRF expression between Asian Taiwanese and Caucasian patients was noticed. Increased PTRF level was significantly correlated with high pT stage and well predicted PFS, CSS, and OS in both ethnicities, implying its general applicability. In addition, although UTUC and UCB have been demonstrated to exhibit variances in the prevalence of genomic alterations despite their similar histologic appearances [21], the significant association of PTRF expression with adverse pathologic features and survival was also found in UCB from TCGA cohort. Taken together, PTRF is likely to be a prognosticator for all UCs. There are a number of limitations in the present study. First, data are retrospectively collected and RNU is performed by several surgeons from multicenter. This is mainly due to rarity of the disease and in fact, international collaboration allows discovering a new biomarker and its potential applicability. In this study, we find abundance in PTRF is a feature of clinically aggressive UTUC in both Asian Taiwanese and Caucasian patients. Second, it is unclear what mechanism is responsible for higher PTRF expression leading to disease progression and poor outcome, but our findings warrant further supplementary

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investigation specifically to UC. Third, although inherent problems exist in IHC, the present study follows standard protocol of immunostaining and 2 dedicated genitourinary pathologists independently review all slides. In addition, we adopt H-score since this combined scoring system offers advantages due to a dynamic range and excellent correspondence to immunoblot [22]. Lastly, prospective studies are necessary to validate our findings. Nevertheless, this study is strengthened by the sizable and multiracial cohort derived from multiple institutions globally. Further exploration of PTRF in UTUC is needed to evaluate the value in decision making and its potential as a therapeutic target. 5. Conclusions This multicenter study finds that PTRF is associated with adverse clinicopathologic features and is an independent predictor of progression and survival of this disease. With further validation, high-risk UTUC patients identified by PTRF IHC should be considered for neoadjuvant or adjuvant systemic therapy and frequent disease surveillance. Acknowledgments We thanked Dr. Samarpita Sengupta for editorial assistance. Dislosures The authors declare they have no conflict of interest. References [1] Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin 2019;69:7–34. [2] Li CC, Chang TH, Wu WJ, et al. Significant predictive factors for prognosis of primary upper urinary tract cancer after radical nephroureterectomy in Taiwanese patients. Eur Urol 2008;54:1127–34. [3] Lai MN, Wang SM, Chen PC, Chen YY, Wang JD. Population-based case-control study of Chinese herbal products containing aristolochic acid and urinary tract cancer risk. J Natl Cancer Inst 2010;102:179–86. [4] Margulis V, Shariat SF, Matin SF, et al. Outcomes of radical nephroureterectomy: a series from the upper tract urothelial carcinoma collaboration. Cancer 2009;115:1224–33. [5] Singla N, Fang D, Su X, et al. A multi-institutional comparison of clinicopathological characteristics and oncologic outcomes of upper tract urothelial carcinoma in China and the United States. J Urol 2017;197:1208–13. [6] Singla N, Fang D, Su X, et al. Preoperative predictors of nonorganconfined disease in upper-tract urothelial carcinoma differ between China and the United States. Urol Oncol 2018;36:88 e11-88 e18. [7] Favaretto RL, Zequi SC, Oliveira RAR, et al. Tissue-based molecular markers in upper tract urothelial carcinoma and their prognostic implications. Int Braz J Urol 2018;44:22–37. [8] Mbeutcha A, Roupret M, Kamat AM, et al. Prognostic factors and predictive tools for upper tract urothelial carcinoma: a systematic review. World J Urol 2017;35:337–53. [9] Jansa P, Mason SW, Hoffmann-Rohrer U, Grummt I. Cloning and functional characterization of PTRF, a novel protein which induces

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