Prognostic Value of Leptin Receptor Overexpression in Upper Tract Urothelial Carcinomas in Taiwan

Accepted Manuscript Prognostic value of leptin receptor overexpression in upper tract urothelial carcinomas in Taiwan Yi-Chen Lee, Wen-Jeng Wu, Hui-Hui Lin, Wei-Ming Li, Chun-Nung Huang, Wei-Chi Hsu, Lin-Li Chang, Ching-Chia Li, Hsin-Chih Yeh, Chien-Feng Li, Hung-Lung Ke PII:

S1558-7673(17)30002-2

DOI:

10.1016/j.clgc.2017.01.002

Reference:

CLGC 769

To appear in:

Clinical Genitourinary Cancer

Received Date: 12 November 2016 Revised Date:

27 December 2016

Accepted Date: 3 January 2017

Please cite this article as: Lee Y-C, Wu W-J, Lin H-H, Li W-M, Huang C-N, Hsu W-C, Chang L-L, Li CC, Yeh H-C, Li C-F, Ke H-L, Prognostic value of leptin receptor overexpression in upper tract urothelial carcinomas in Taiwan, Clinical Genitourinary Cancer (2017), doi: 10.1016/j.clgc.2017.01.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Prognostic value of leptin receptor overexpression in upper tract urothelial carcinomas in Taiwan Yi-Chen Leea,b, Wen-Jeng Wub,c,d,e, Hui-Hui Linb,c,d, Wei-Ming Lib,c,d,f, Chun-Nung Huangb,c,d, Wei-Chi Hsub,c,d, Lin-Li Changb,g, Ching-Chia Lib,c,d,e, Hsin-Chih Yehb,c,d,e,

a

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Chien-Feng Lih,i, Hung-Lung Keb,c,d

Department of Anatomy, School of Medicine, College of Medicine, Kaohsiung

b

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Medical University, Kaohsiung, Taiwan

Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University,

c

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Kaohsiung, Taiwan

Department of Urology, Kaohsiung Medical University Hospital, Kaohsiung Medical

University, Kaohsiung, Taiwan d

Department of Urology, School of Medicine, College of Medicine, Kaohsiung

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Medical University, Kaohsiung, Taiwan e

Department of Urology, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung, Taiwan

f

Taiwan g

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Department of Urology, Ministry of Health and Welfare Pingtung Hospital, Pingtung,

h

i

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Department of Microbiology, Kaohsiung Medical University, Kaohsiung, Taiwan Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan

National Cancer Research Institute, National Health Research Institutes, Tainan,

Taiwan Correspondence to: Hung-Lung Ke, Department of Urology, School of Medicine, College of Medicine, Kaohsiung Medical University, No. 100, TzYou 1st Road, Kaohsiung City 807, Taiwan 1

ACCEPTED MANUSCRIPT Tel.: +886-7-3208212; Fax: +886-7-3211033

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E-mail address: [email protected]

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ACCEPTED MANUSCRIPT MicroAbstract Leptin receptor is involved in cancer development and progression. Leptin receptor expression

was

examined

in

upper

tract

urothelial

carcinoma

by

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immunohistochemistry. Leptin receptor expression could predict patient survival.

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ACCEPTED MANUSCRIPT Abstract Objectives: Leptin and its receptor (LEPR) are key players in the regulation of energy balance and body weight control and act as a growth factor for specific organs in both normal and disease states. However, LEPR accumulation may be involved in

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carcinogenesis, progression, and metastasis in many cancers. This study evaluated the clinical significance of LEPR expression in upper tract urothelial carcinoma (UTUC). Materials and methods: LEPR expression was examined in 110 tissue samples from

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patients with UTUC, using immunohistochemistry, and an analysis was performed to

clinicopathological variables.

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identify evidence of association between LEPR expression and different

Results: LEPR expression was significantly correlated with patients with increased body mass index (BMI) (P < 0.001) and high serum creatinine levels (P = 0.005). We found, using the log-rank test, that high LEPR expression was associated with poor

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recurrence-free (P = 0.009) and cancer-specific survival (P = 0.001). This finding was supported by our results using Cox regression analysis, which showed that LEPR expression was an independent predictor of poor recurrence-free survival (hazard ratio

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= 2.55, P = 0.011) and cancer-specific survival (hazard ratio = 2.26, P = 0.006).

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Conclusions: Our findings indicate that LEPR expression is a potential biomarker for predicting patient survival in UTUC. Further study is necessary to investigate the role of LEPR in carcinogenesis of upper tract urothelial carcinoma. Key words: Upper tract urothelial carcinoma (UTUC), leptin receptor (LEPR), obesity, BMI, immunohistochemistry (IHC)

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ACCEPTED MANUSCRIPT Introduction Urothelial carcinoma (UC) is the most common malignancy of the urinary tract. The incidence of renal pelvic and ureteral cancer is rare, accounting for only 4 % of all urothelial cancers. In the United States, renal pelvic cancer accounts for

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approximately 8 % of all renal cancers, whereas ureter cancer accounts for approximately 5 % of all urothelial cancers. However, because an increased incidence of upper tract urothelial carcinomas (UTUC) has been reported

1, 2

, it is likely that

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additional genetic and environmental factors are contributing to UTUC in Taiwan. Nephroureterectomy is the standard treatment for UTUC. For advanced stage or

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metastatic cancer, systemic chemotherapy with cisplatin-based regimen is the only effective therapy. But even with the same pathological stage and standard treatment, the patients still have diverged prognosis. The exact molecular mechanisms of UTUC progression remains unclear and no promising prognostic factor is found.

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Obesity prevalence is globally increasing to epidemic levels, and is rapidly becoming a major public health problem. Obesity is a well-known risk factor for a number of cancers including breast, colon, endometrial, and high-grade prostate cancer

3, 4

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Furthermore, epidemiological studies have found associations between obesity and

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incidence and mortality of site-specific cancers 5, 6. In addition, studies have examined the relationship between obesity and cancer mortality in different urologic cancers including kidney, prostate, and bladder cancer

7-9

. In fact, it has been proposed that

obesity may be a potential prognostic biomarker for UTUC 10. Leptin and its receptor have major roles in homeostasis and body weight control 11, 12. Leptin, which is a 16-kDa polypeptide secreted by adipose tissue, binds to the leptin receptor (LEPR) in the cell membrane of hypothalamic neurons. This binding results in a cascade of signal transductions that regulate energy balance and body weight 5

13

.

ACCEPTED MANUSCRIPT Previous studies found that through the activation of signaling pathways such as JAK2/STAT3, MAPK/ERK, and PI3K/AKT, which stimulate cell proliferation and inhibit apoptosis, leptin may have a role in malignancy by stimulating growth, migration, and invasiveness of tumor cells

14-16

. Upon binding to its ligand, LEPR

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activates the canonical transduction pathway and regulates several cancers related genes such as cyclin D1, COX-2, VEGF and potentiates several pro-carcinogenic processes including angiogenesis, anti-apoptosis, cell proliferation, migration and

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mesenchymal transformation. Thus the leptin-LEPR system may participate in tumorigenesis and increase the risk of cancer particularly in obese persons having

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high serum leptin17-19.

Since there is no published study examined the role of LEPR in UTUC, the purpose of this study is to determine whether there is evidence of associations between LEPR expression and clinicopathological factors and survival in our well-characterized

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cohort of UTUC.

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ACCEPTED MANUSCRIPT Materials and Methods Surgical specimens and clinicopathological data The study protocol was reviewed and approved by the Institutional Review Board of Kaohsiung Medical University (KMUH-IRB-20120119). One-hundred and ten

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formalin-fixed, paraffin-embedded tissue samples from patients with UTUC were obtained from the Department of Urology, Kaohsiung Medical University Hospital from 1997-2006. All of the patients in our cohort received nephroureterectomy and

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excision of the bladder cuff. The pathologic grade was classified according to World Health Organization (WHO) histologic criteria [1], and tumor stage was determined

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according to the AJCC Cancer Staging Manual [2]. All data were retrospectively obtained from medical records. The follow-up protocol was determined according to NCCN guidelines. Cystoscopy was performed every 3 months for the first 2 years following surgery and then at increasing intervals thereafter. Bladder recurrence was

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defined with pathological identification of UC. Recurrence-free survival was defined as the time from the date of surgery to the date of bladder recurrence. Immunohistochemical detection of the leptin receptor

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The 4-µm thick tissue sections were cut from the paraffin-embedded tissue blocks and placed on precoated slides. Slides were immersed twice in xylene for 10 min per

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immersion, hydrated in a descending series of ethanol solutions, and washed with distilled water. Antigen retrieval was performed by heating the samples for 15 min at 96 °C in a retrieval solution (Dako North America), followed by incubation at room temperature (RT) for 30 min. Endogenous peroxidase activity was quenched by incubation in 3 % hydrogen peroxide buffer for 10 min. Tissue sections were incubated overnight at 4 °C in a humidification chamber with a 1:500 dilution of the anti-leptin receptor primary antibody, Ob-R (H-300) (sc-8325; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Samples were incubated for 30 min with the 7

ACCEPTED MANUSCRIPT secondary, biotinylated universal link antibody (K0690; Dako North America) followed by a 5-min washing step, and incubation with streptavidin-horseradish peroxidase (K0690; Dako North America) for 30 min at RT. Specific reactivity was visualized by incubation with 3,3ʹ-diaminobenzidine tetrahydrochloride (DAB)

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(K3468; Dako North America) for 3 min. Slides were contrasted using hematoxylin

mounted. Evaluation of immunohistochemical staining

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for 1 min, followed by dehydration using an ethanol series, clearance in xylene, and

Scoring for LEPR-positive staining was determined based on the percentage of

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positively stained cells in four quantitative categories: score 1, ≤25 % positive cells; score 2, 26–50 % positive cells; score 3, 51–75 % positive cells; and score 4, ≥76 % positive cells. Tumor immunostaining was examined by two qualified pathologists who were blinded to the clinical status of the patients. Any discrepancies in scoring

Statistical analysis

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between pathologists were jointly reviewed and a consensus was reached.

All statistical analyses were performed using the SPSS statistical package for PC

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(version 14.0, SPSS, Inc., Chicago, IL, USA). As a representation of indicative LEPR expression, tumors with scores of 1 or 2 were categorized as low expression (i.e., ≤50

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% positively stained cells), whereas tumors with scores of 3 or 4 were categorized as high expression (i.e., >50 %). The chi-square test was used to test for associations between LEPR expression and tumor stage, tumor grade, gender, age at diagnosis, body mass index (BMI), hemodialysis, and serum creatinine level. Survival curves were generated using Kaplan–Meier estimates, and the significance of differences between curves was evaluated using the log-rank test. Furthermore, hazard ratios (HRs) and 95 % confidence intervals (CIs) computed from univariate and multivariable Cox regression models that were used for investigating the relationship 8

ACCEPTED MANUSCRIPT between clinicopathological characteristics and survival. P values less than 0.05 were considered statistically significant. Results Association between leptin receptor expression and clinicopathological features

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Leptin receptor expression in cancer tissues (n = 110 patients) was examined by immunohistochemistry and stratified into four scores (quartiles). Based on scoring, tissues were further categorized into high (scores of 3 and 4; 36.4 %) or low (scores of

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1 and 2; 63.6 %) LEPR expression groups (Fig. 1 and Table 1). To explore the potential role of LEPR in UTUC, we tested LEPR expression for evidence of

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association with different clinicopathological variables including tumor stage, tumor grade, gender, age at diagnosis, BMI, hemodialysis, and serum creatinine level. We found that high LEPR expression in UTUC tissues was significantly associated with increased BMI (P < 0.001) and serum creatinine levels (P = 0.005) (Table 1).

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Survival analysis in UTUC

To evaluate factors related to LEPR expression in patients with UTUC, we performed univariate and multivariable Cox regression analysis. We found statistically

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significant associations between recurrence-free survival and the following three parameters: tumor stage (HR = 3.09, 95 % CI = 1.63–5.83, P = 0.001), tumor grade

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(HR = 3.36, 95 % CI = 1.54–7.35, P = 0.002), and LEPR expression (HR = 2.35, 95 % CI = 1.21–4.57, P = 0.011) (Table 2), using univariate analysis. In our multivariate analyses, we found recurrence-free survival was significantly associated with tumor stage (HR = 2.15, 95 % CI = 1.06–4.35, P = 0.034), tumor grade (HR = 2.67, 95 % CI = 1.12–6.34, P = 0.026), and LEPR expression (HR = 2.55, 95 % CI = 1.24–5.24, P = 0.011) (Table 2). We also tested for correlations between LEPR expression and clinical parameters with cancer-specific survival, using univariate and multivariable Cox regression analysis. We found statistically significant associations between 9

ACCEPTED MANUSCRIPT cancer-specific survival and the following variables: tumor stage (HR = 8.10, 95 % CI = 2.97–22.07, P < 0.001), BMI status (HR = 3.05, 95 % CI = 1.07–8.67, P = 0.036), and LEPR expression (HR = 5.35, 95 % CI = 1.74–16.41, P = 0.003) (Table 3). We found, using multivariate analysis, evidence for association with tumor stage (HR =

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8.42, 95 % CI = 2.58–27.47, P < 0.001) and LEPR expression (HR = 2.26, 95 % CI = 1.67–23.40, P = 0.006) (Table 3).

Next, we investigated LEPR expression patterns in UTUC tissue samples for

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correlations with recurrence-free survival and cancer-specific survival of patients using Kaplan–Meier estimates. Patients were followed up for recurrence-free and

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cancer-specific survival status with a median follow-up of 39 months (range of 1-126 months). Increased recurrence-free and cancer-specific survival rates were observed in the LEPR low expression group as determined using the log-rank test (P = 0.009 and P = 0.001, respectively; Fig. 2). In regards to BMI status, patients with high

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LEPR expression had a significantly worse recurrence-free survival rate in the high (>25) BMI group (P= 0.008; Fig. 3). Similarly, patients with high LEPR expression had a significantly poorer cancer-specific survival rate in the high BMI group (P =

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0.009; Fig. 3). There were no significant differences found in recurrence-free and cancer-specific survival rates in patients and LEPR expression in the low BMI group

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(Fig. 3).

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ACCEPTED MANUSCRIPT Discussion Body mass index (BMI), which is defined as weight (in kilograms) divided by the square of the height (in meters), is the most widely used anthropometric measure of adiposity. We believe that this is the first study to find evidence of an association

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between LEPR expression and UTUC. In this study, LEPR staining by IHC was scored according to the percentage of positive cells. Our results were divided into four quartiles and the cutoff value was defined according to a validated method in our

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previous studies20. Another measure method of LEPR is based on using the staining intensity of positively stained cells in the tissue of UTUC patients. However, our

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results for LEPR staining were almost the same on the staining intensity in the current study. Therefore, we evaluated the percentage of positive cells for LEPR staining in this study. We found that increased LEPR staining in UTUC tissue samples was associated with unfavorable risk factors including high BMI and increased serum

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creatinine levels in patients. We also found statistically significant associations between high LEPR expression and worse recurrence-free and cancer-specific survival rates in patients with UTUC. These findings indicate that LEPR expression

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levels may reflect endogenous instability in cancerous tissues and thus, support a potential role of LEPR in UTUC pathogenesis. In addition, low LEPR expression

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levels may indicate a slowly proliferating, less aggressive tumor phenotype with good prognostic features, whereas high LEPR expression levels may indicate a more aggressive, highly proliferating tumor phenotype and worse prognosis. These findings suggest that leptin receptor signaling may have an important role in UTUC progression. Leptin and its receptor have a major role in energy homeostasis and body weight control 11-13. Long-term exposure to leptin enhances growth of all three major prostate cancer cell lines (LNCaP, DU145, and PC-3) 11

21, 22

. Leptin also induces expression of

ACCEPTED MANUSCRIPT vascular endothelial growth factor (VEGF), transforming growth factor β1, and basic fibroblast growth factor in DU145 and PC-3 cells by stimulating cell survival pathways that lead to proliferation and angiogenesis

23

. As an alternative mechanism

for leptin-induced prostate cell proliferation, leptin affects estrogen metabolism by 24

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increasing estrogen receptor (ER)-α expression and decreasing ER-β expression

.

Leptin was shown to induce cellular migration of human prostate cancer via upregulation of integrin and intracellular signal transduction

25, 26

. The leptin receptor

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is capable of activating IRS1 subsequent to Jak activation by leptin stimulation, further leading to PI3K, AKT, and NF-κB pathway activation

27-29

. Application of

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recombinant leptin to pancreatic cancer cells in vitro resulted in a significant increase in phosphorylation of the AKT signaling pathway suggesting that the leptin receptor was functional in these cells.

Obesity and alterations in diet composition have been reported to affect the growth 29, 30

, and although there is still conflicting evidence

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and onset of many cancers

regarding the association of obesity and cancer risk, it is known that UTUC can be more aggressive in obese patients than in those with a lower BMI. Leptin, a major

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adipokine secreted by adipose tissues, has been linked to progression and metastasis of many cancers through activation of LEPR. Similar to obesity, leptin and the leptin

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receptor correlate with increased cancer risk and tumor development

31, 32

. We found

in this study that patients with high LEPR expression had significantly worse recurrence-free survival and cancer-specific survival rates in the high BMI (>25) group compared to that seen in the lower BMI group. Based on our findings and available evidence, we propose that obesity may not only affect the incidence of UTUC but may have an effect on recurrence of existing cancers. Therefore, a combination of epidemiological and histopathological studies is warranted to determine the role of adipokines and adipokine receptor expression in obesity-related 12

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changes to cancer.

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ACCEPTED MANUSCRIPT Conclusion We found an association between leptin receptor expression and malignant behavior of UTUC in our cohort of 110 patients. We also found that high LEPR expression was independently associated with poor recurrence-free and progression-free survival

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using multivariable Cox regression analysis. These study provided evidence that LEPR was involved in the progression of UTUC. We expected to help prediction of prevalence and prognosis of UTUC, as well as generation of novel targeted therapy,




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Clinical Practice Points

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especially in obese patients with impaired LEPR signaling.

Upper tract urothelial carcinoma and bladder cancer are different diseases with the same pathologic characteristics. The cancer development and prognosis may be different between them.

There are still no reliable molecular prognostic factors for upper tract urothelial carcinoma.

Leptin receptors, which are involved in energy balance and body weight control,

patients.

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could predict prognosis of upper tract urothelial carcinoma, especially in obese

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ACCEPTED MANUSCRIPT Acknowledgments This study was supported by grants from Kaohsiung Medical University “Aim for the Top

Universities”

(KMU-TP104E31, KMU-TP105G00,

KMU-TP105G01,

KMU-TP105G02), the health and welfare surcharge of tobacco products, Ministry of

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Health and Welfare (MOHW105-TDU-B-212-134007), Ministry of Science and Technology (MOST103-2314-B-037-059-MY3), and Kaohsiung Medical University

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Hospital (KMUH101-1R46, KMUH102-2M41, KMUH102-2R40, KMUH103-3T11).

Conflicts of interest

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The authors declare that they have no conflicting interests.

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ACCEPTED MANUSCRIPT Figure Legends Fig 1. Leptin receptor expression in upper tract urothelial carcinomas was determined

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using immunohistochemistry. The degree of expression was divided into four categories: score 1, ≤25 % positive staining of tumor cells; score 2, 26-50 % positive staining of tumor cells; score 3, 51-75 % positive staining of tumor cells; and score 4,

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≥76 % positive staining of tumor cells (×200).

Fig 2. Kaplan-Meier survival curves for recurrence-free and cancer-specific survival

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rates of patients with LEPR expression.

Fig 3. Kaplan-Meier survival curves for recurrence-free and cancer-specific survival

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rates of patients with LEPR expression according to low or high BMI status.

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Mendonsa AM, Chalfant MC, Gorden LD, VanSaun MN. Modulation of the leptin receptor mediates tumor growth and migration of pancreatic cancer cells. PLoS One. 2015;10:e0126686. Cheon EC, Strouch MJ, Barron MR, et al. Alteration of strain background and a

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Cancer Prev. 2015;16:3621-3627. Wang Y, Yang H, Gao H, Wang H. The association between LEPR Q223R polymorphisms and breast cancer risk. Breast Cancer Res Treat. 2015;151:1-6.

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28.

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ACCEPTED MANUSCRIPT Table 1. Correlation of OB-R expression with clinicopathological characteristics in upper tract urothelial carcinoma. Leptin receptor Low

Gender

Age (years)

BMI

Hemodialysis

110 (100)

70

63.6

I/II

67 (60.9)

43

61.4

III/IV

43 (39.1)

27

38.6

Low

39 (35.5)

25

35.7

14

35.0

High

71 (64.5)

45

64.3

26

65.0

Male

59 (53.6)

36

51.4

23

57.5

Female

51 (46.4)

34

48.6

17

42.5

＜65

34 (30.9)

22

31.4

12

30.0

≧65

76 (69.1)

48

68.6

28

70.0

＜25

63 (57.3)

49

70.0

14

35.0

≧25

47 (42.7)

21

30.0

26

65.0

Negative

95 (86.4)

62

88.6

33

82.5

Positive

15 (13.6)

8

11.4

7

17.5

≦1.5

58 (52.7)

44

62.9

14

35.0

52 (47.3)

26

37.1

26

65.0

＞1.5

No

*The P value was calculated by the chi-square test.

%

P value*

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%

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Creatinine (mg/dl)

No.

40

36.4

24

60.0

16

40.0

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Grade

Patient No. (%)

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Stage

Item

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Variables

High

0.883

0.940

0.539

0.876

<0.001

0.372

0.005

ACCEPTED MANUSCRIPT Table 2. Univariate and multivariable analysis of recurrence-free survival for upper tract urothelial carcinoma. Univariate Item

Gender

Age (years)

BMI

Hemodialysis

HR

95% CI

P value

III/IV

3.09

(1.63, 5.83)

0.001

2.15

(1.06, 4.35)

0.034

I/II

1.00

High

3.36

(1.12, 6.34)

0.026

Low

1.00

Female

1.64

(0.93, 3.84)

0.080

Male

1.00

≧65

0.86

(0.45, 1.95)

0.864

< 65

1.00

≧25

1.67

(0.57, 2.23)

0.724

<25

1.00

(0.35, 3.75)

0.825

(0.35, 1.60)

0.447

(1.24, 5.24)

0.011

Positive

0.84

RI PT

P value

1.00

(1.54, 7.35)

0.002

2.67 1.00

(0.88, 3.05)

SC

Grade

95% CI

0.119

M AN U

Stage

HR

(0.45, 1.62)

(0.86, 3.22)

TE D

Variables

Multivariable

(0.33, 2.16)

0.632

0.129

1.89 1.00 0.94 1.00 1.13 1.00

0.723

1.14

Negativ

≦1.5

0.90

＞1.5

1.00

AC C

Creatinine (mg/dl)

1.00

EP

e

Leptin receptor

High Low

2.35 1.00

(0.47, 1.72)

1.00 0.741

0.74 1.00

(1.21, 4.57)

0.011

2.55 1.00

ACCEPTED MANUSCRIPT Table 3. Univariate and multivariable analysis of cancer-specific survival for upper tract urothelial carcinoma. Univariate

Age (years)

BMI

Hemodialysis

95% CI

P value

III/IV

8.10

(2.97, 22.07)

<0.001

8.42

(2.58, 27.47)

<0.001

I/II

1.00

High

2.62

(0.58, 7.43)

0.266

Low

1.00

Female

0.90

(0.32, 2.63)

0.863

Male

1.00

≧65

0.71

(0.44, 3.53)

0.690

< 65

1.00

≧25

3.05

(0.59, 5.72)

0.290

<25

1.00

(0.13, 3.88)

0.681

(0.24, 2.55)

0.683

(1.67, 23.40)

0.006

Positive

1.35

Negative

1.00

≦1.5

1.22

＞1.5

1.00

Leptin receptor

High Low

5.35 1.00

RI PT

HR

AC C

Creatinine (mg/dl)

P value

1.00

(0.96, 7.13)

0.059

2.07 1.00

(0.38, 2.11)

SC

Gender

95% CI

0.806

M AN U

Grade

HR

(0.30, 1.66)

(1.07, 8.67)

TE D

Stage

Item

EP

Variables

Multivariable

(0.40, 4.58)

(0.48, 3.07)

0.430

0.036

0.91 1.00 1.24 1.00 1.84 1.00

0.635

0.70 1.00

0.678

0.78 1.00

(1.74, 16.41)

0.003

2.26 1.00

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT