Prognostic significance of erythrocyte protein band 4.1-like5 expression in upper urinary tract urothelial carcinoma

Urologic Oncology: Seminars and Original Investigations ] (2017) ∎∎∎–∎∎∎

Original article

Prognostic significance of erythrocyte protein band 4.1-like5 expression in upper urinary tract urothelial carcinoma Tatsuaki Daimon, M.D.a,1, Takeo Kosaka, M.D., Ph.D.a,1,*, Eiji Kikuchi, M.D., Ph.D.a, Shuji Mikami, M.D., Ph.D.b, Yasumasa Miyazaki, M.D.a, Ari Hashimoto, Ph.D.c, Shigeru Hashimoto, Ph.D.c, Ryuichi Mizuno, M.D., Ph.D.a, Akira Miyajima, M.D., Ph.D.a, Yasunori Okada, M.D., Ph.D.d, Hisataka Sabe, Ph.D.c, Mototsugu Oya, M.D., Ph.D.a,1,* a

Department of Urology, Keio University School of Medicine, Tokyo, Japan Division of Diagnostic Pathology, Keio University Hospital, Tokyo, Japan c Department of Molecular Biology, Hokkaido University Graduate School of Medicine, Sapporo, Japan d Department of Pathology, Keio University School of Medicine, Tokyo, Japan b

Received 14 December 2016; received in revised form 5 April 2017; accepted 9 April 2017

Abstract Objectives: The erythrocyte protein band 4.1-like5 (EPB4.1L5) regulates E-cadherin in cancer invasion and metastasis inducing epithelial-to-mesenchymal transition. This study aimed to investigate the biological significance of EPB4.1L5 in upper urinary tract urothelial carcinoma (UTUC). Methods: Retrospective analysis of the clinical records of 165 patients with UTUC (Ta-4N0M0) subjected to radical nephroureterectomy and immunohistochemical examination of EPB4.1L5 expression in those tissues. Results: The median follow-up period was 62.2 months (interquartile range ¼ 77.0). The score of EPB4.1L5 significantly correlated with tumor grade, pathological T stage, and lymphovascular invasion (all P o 0.001). The 5-year Kaplan-Meier recurrence-free survival and cancer-specific survival rates were 54.1% and 59.5% in patients with high EPB4.1L5 expression, compared with 81.6% and 87.2%, (all P o 0.001) in their counterparts. Multivariate analyses revealed that high expression of EPB4.1L5 was one of the independent prognostic factors for tumor recurrence (P ¼ 0.022, HR ¼ 2.40) and cancer-specific survival (P ¼ 0.015, HR ¼ 2.94). Conclusion: High EPB4.1L5 expression was related to worse clinical outcome in patients with UTUC. These results indicated that EPB4.1L5 could provide prognostic information in patients with UTUC regarding epithelial-to-mesenchymal transition. r 2017 Elsevier Inc. All rights reserved.

Keywords: Erythrocyte protein band 4.1-like5; Epithelial-mesenchymal transition; Upper urinary tract urothelial carcinoma; Recurrence; Survival

1. Introduction Upper urinary tract urothelial carcinoma (UTUC) is relatively rare, accounting for only 5% to 10% of all This work was supported in part by Grant-in-Aid for Young Scientists (JSPS KAKENHI Grant Number JP26861299 to T. K. and JP15K20111 to T. D.) from Japan Society for the Promotion of Science (JSPS), Japan. 1

These authors contributed equally. Corresponding authors. Tel.: þ81-33-353-1211; fax: þ81-33-2251985. E-mail addresses: [email protected] (T. Kosaka), moto-oya@ z3.keio.jp (M. Oya). *

http://dx.doi.org/10.1016/j.urolonc.2017.04.008 1078-1439/r 2017 Elsevier Inc. All rights reserved.

urothelial cancers [1,2]. Radical nephroureterectomy with excision of the bladder cuff is the standard procedure for clinically localized UTUC. The prognosis of patients who undergo radical nephroureterectomy still remains poor because of the risk of local or distant recurrence even after curative surgery [3]. Many investigators have previously reported the possible predictors of tumor progression, including local or distant recurrences of UTUC [4–7]. The clinicopathologic parameters of UTUC, such as tumor stage, histologic grade, and lymphovascular invasion (LVI), have been reported to be independent prognostic factors of clinical outcome following radical surgery [2,4–8].

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T. Daimon et al. / Urologic Oncology: Seminars and Original Investigations ] (2017) ∎∎∎–∎∎∎

Epithelial-to-mesenchymal transition (EMT) is a key process in cancer development, progression, and metastasis [9,10]. Tumor cells transform from a noninvasive to an invasive phenotype through a series of metastatic steps, whereby epithelial cells lose polarity and invade the lymphovasculature; especially necessary is disruption of E-cadherin-based cell-cell adhesion to allow cell detachment from their neighbors [11]. We previously demonstrated that erythrocyte protein band 4.1-like5 (EPB4.1L5) plays important roles in EMT and cell migration during mouse gastrulation [12]. EPB4.1L5 coordinates the downregulation of E-cadherin-mediated cell-cell adhesion in a posttranscriptional manner by binding to p120ctn through its N-terminal FERM domain, inhibiting p120ctnE-cadherin binding, and relocalizing E-cadherin into Rab5-positive vesicles (endocytosis) [12]. We recently reported that in clear cell renal cell carcinoma, EPB4.1L5 promotes EMT by binding AMAP1 through the Arf6 mesenchymal pathway [13]. Recently, researchers have reported that several transcription factors, such as Twist, Snail, Slug, SIP1, and E2A (E47/E12), play a significant role in EMT through regulation of E-cadherin [14,15]. On the contrary, though it has been said that posttranscriptional events must also play vital roles in EMT, the mechanism of such events is not still largely investigated in detail [12,13,15,16]. In this study, we examined the expression of EPB4.1L5 in UTUC specimens acquired by primary surgery and retrospectively investigated them to assess the prognostic significance of EPB4.1L5 in patients with UTUC.

2. Materials and methods 2.1. Patient selection The medical records of patients operated on between 1984 and 2007 and archived at Keio University Hospital (Shinjuku-ku, Tokyo, Japan) were, retrospectively, reviewed after obtaining Institutional Review Board approval. During this period, nephroureterectomy was performed for more than 200 patients with UTUC at Keio University Hospital. In our study population, we identified a total of 165 patients with UTUC (pTa-4N0M0) whose medical records could be used. The median follow-up of the whole cohort was 5.2 ⫾ 4.8 years. A total of 131 patients (79.4%) had undergone open nephroureterectomy and 34 patients (20.6%) laparoscopic nephroureterectomy. Patients were assessed by urine cytology and cystoscopy every 3 months for 2 years following nephroureterectomy, every 6 months for the next 3 years, and then every 6 to 12 months thereafter. CT scan was conducted every 6 months for 5 years and annually thereafter. Disease recurrence was defined as any recurrence documented by radiograph or pathology-proven failure in nonbladder lesions such as contralateral kidney, operative site, regional lymph nodes,

or distant metastases. The cause of death was determined by the treating physicians. The independent variables included in the present study were age, gender, tumor location, and pathologic factors (tumor grade, pathologic T stage, and LVI). Tumor location was divided into 2 areas: the renal pelvis and the ureter depending on the location of the dominant lesion.

2.2. Tissue samples All the specimens were fixed in 10% formalin and embedded in paraffin, and all tissue preparations were reviewed again by genitourinary pathologists. Tumors were staged according to the 2002 tumor-node-metastasis staging system [17]. Histologic grades were assigned according to the 3-tier World Health Organization classification, namely low (G1 and G2) and high (G3) grades [18]. Lymphovascular invasion was defined as the presence of tumor cells within an endothelium-lined space without an underlying muscular wall.

2.3. Immunohistochemistry All the tissue samples were fixed in 10% formalin, embedded in paraffin, and cut into 4 mm thick sections and placed on silane-coated glass slides. Samples were first deparaffinized in xylene, and hydrated through graded alcohols and finally distilled water. After washing in TBS buffer, antigen retrieval was carried out in 10 mmol/l Tris buffer (Dako target retrieval solution pH 6.0), heated in autoclave at 1211C for 10 minutes. Endogenous peroxidase activity was blocked by 0.3% hydrogen peroxidase/methanol for 20 minutes at room temperature. The tissue sections were then incubated for 15 minutes at room temperature in a blocking solution of 6% dry milk in PBS. After washing in TBS buffer, they were stained for 60 minutes at room temperature with the primary antibody. After that they were stained for 30 minutes at room temperature with the secondary antibody. As the primary antibody, we use rabbit polyclonal antibody against human EPB41L5. It was generated using a GST-fused peptide corresponding to amino acids 541–733. The resulting sera were first adsorbed with GST protein and then affinity-purified using the antigen peptides. It was prepared by our coauthors (A.H., S.H., and H.S.), and it was diluted at 1:1,000 in 1% BSA/PBS. After washing with PBS, the tissue sections were incubated with the secondary antibody against mouse IgG conjugated to a peroxidase-labeled polymer (Histofine Simple Stain MAX PO (R), undiluted solution, for 30 minutes. Color was developed with 3, 30 -diaminobenzamine in 50 mmol/l Tris-HCl (pH 7.5) containing 0.005% hydrogen peroxide. The sections were counterstained with hematoxylin.

T. Daimon et al. / Urologic Oncology: Seminars and Original Investigations ] (2017) ∎∎∎–∎∎∎

2.4. Evaluation of immunohistostaining Immunohistochemically stained slides were viewed at a magnification of 200. Olympus BH2 (Olympus, Tokyo, Japan) was used for the analysis. When we evaluated EPB4.1L5 staining, cancer cells showing positive staining in the cytoplasm were scored, and the mean percentage of stained cancer cells and staining intensity stratified from 0 to 3 (0, no staining; 1, slight staining; 2, medium staining; and 3, strong staining) were estimated. Expression of EPB4.1L5 was assessed using a semiquantitative scoring system. Histoscore (EPB4.1L5 score) was calculated by applying the following formula: staining intensity  percentage (range: 0–300). Two authors (T.D. and S.M.) independently evaluated immunoreactivity. We scored in 5 representative fields and took an average. We were blinded to the clinical information of the patients, and the average of the numbers counted by the 2 investigators was used for subsequent analysis. 2.5. Statistical analysis The association between each clinicopathologic parameter and EPB4.1L5 expression of the tumor was analyzed. Chisquare test or Mann-Whitney U test were used when validating these association. Recurrence-free survival (RFS) and cancer-specific survival (CSS) rates were estimated using the Kaplan-Meier method and were compared using the log-rank test. Survival time was calculated from the date of surgery. Multivariate analysis was conducted using the Cox proportional hazard model. P o 0.05 was considered statistically significant. These analyses were conducted with the SPSS version 22.0 statistical software package (SPSS Corporation). 3. Results 3.1. Patient characteristics Table 1(A) shows the association of clinicopathological parameters with EPB4.1L5 expression in our study population. A total of 122 patients (73.9%) were men. The mean age of the patients was 66.4 years (range: 36–89 years). Pathologic analysis revealed 100 patients (60.6%) with tumors in the renal pelvis and 65 patients (39.4%) with tumors in the ureter. Tumor grade was low in 49 cases (29.7%) and high in 116 (70.3%). In 86 patients (52.1%), the disease was in pT2 stage or lower; 63 patients (38.2%) were positive for LVI. Tumor recurrence occurred in 37 patients (22.4%), and among these, 31 (18.8%) died of the disease during follow-up. 3.2. EPB4.1L5 expression in UTUC To analyze the association between clinicopathological background and EPB4.1L5 in UTUC, we evaluated the

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expression of EPB4.1L5 in UTUC (Fig. 1) immunohistochemically. EPB4.1L5 staining of normal urothelial cells was negative or very low in many cases of superficial and low-grade UTUC. We observed high EPB4.1L5 expression in the most invasive part of the tumor in many cases. In this report, we termed the edge of the tumor as the “most invasive part” or “invasive front” in this report. For pT2–T4 UTUCs, these were where the tumor lesions invaded, and for pT1 UTUCs these were where the tumor lesions invaded subepithelial connective tissue. For pTa UTUCs, they were the edge between tumor and normal tissue like roots of pedunculated tumors. The mean EPB4.1L5 score was 161.2 ⫾ 61.1 in the 165 patients. We defined patients with 160 or more EPB4.1L5 expression score as the high expression group and others with 150 or lower score as the low expression group. In Table 1B, we showed the clinicopathological characteristics of the 2 subgroups (71 high vs. 94 low expressed) and used a chi-square test to compare. Patients with high-grade, high tumor stage, lymphvascular invasion, tumor recurrence, and cancer death had significantly higher EPB4.1L5 score. 3.3. Prognostic significance of EPB4.1L5 expression in all patients with UTUC in our study We conducted univariate and multivariate analyses to determine the predictive factors for tumor recurrence after surgery (Table 2). In univariate analysis, we revealed that high tumor grade, pT3 or higher, presence of LVI, and high EPB4.1L5 score were significant predictive factors of tumor recurrence. In multivariate analysis, we revealed that high tumor grade (P ¼ 0.026, HR ¼ 5.29, 95% CI: 1.21–23.0), high tumor stage (P ¼ 0.015, HR ¼ 2.66, 95% CI: 1.21– 5.84), and high EPB4.1L5 score (P ¼ 0.022, HR ¼ 2.40, 95% CI: 1.14–5.08) were independent predictive factors of tumor recurrence. We next conducted univariate and multivariate analyses to determine the predictive factors of CSS. Univariate analysis showed that high tumor grade, presence of LVI, pT3 or higher, and high EPB4.1L5 score were also significant predictors of CSS. Multivariate analysis confirmed that high tumor stage (P ¼ 0.036, HR ¼ 2.79, 95% CI: 1.07–7.30), and high EPB4.1L5 score (P ¼ 0.015, HR ¼ 2.94, 95% CI: 1.23–7.06) were also independent predictors of CSS. The 5-year Kaplan-Meier RFS and CSS rates were 54.1% and 59.5% in patients with high EPB4.1L5 expression, compared with 81.6% (log rank, P o 0.001) and 87.2% (log rank, P o 0.001) in their counterparts (Fig. 2). 3.4. Risk stratification for UTUC according to independent prognostic factors We examined risk stratification for UTUC according to independent prognostic factors for both PFS and CSS, EPB4.1L5 expression score, and pathologic tumor stage. We divided patients into 3 risk groups: high risk (EPB4.1L5

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Table 1 Correlation of clinicopathological parameters and EPB4.1L5 expression in the 165 study patients (A) and the clinicopathological characteristics of the 2 subgroups (high EPB4.1L5 expression vs. low expression) (B) (A) Characteristics Age o70 Z70 Gender Male Female Tumor location Renal pelvis Ureter Side Left Right Pathologic tumor stage Lower than pT2 pT3 or higher Grade Low High Lymphvascular invasion Positive Negative (B) Characteristics Age o70 Z70 Gender Male Female Tumor location Renal pelvis Ureter Side Left Right Pathologic tumor stage Lower than pT2 pT3 or higher Grade Low High Lymphvascular invasion Positive Negative Recurrence Yes No Cancer death Yes No

(%)

EPB4.1L5 score (mean ⫾ SD)

94 71

57.0 43.0

155.9 ⫾ 61.8 168.3 ⫾ 60.0

0.118

122 43

73.9 26.1

157.9 ⫾ 59.2 170.7 ⫾ 66.0

0.258

100 65

60.6 39.4

156.2 ⫾ 61.0 168.9 ⫾ 61.0

0.189

95 70

57.6 42.4

162.1 ⫾ 63.2 160.0 ⫾ 58.6

0.774

86 79

52.1 47.9

137.0 ⫾ 55.1 187.6 ⫾ 56.5

o0.001

49 116

29.7 70.3

125.1 ⫾ 44.9 176.5 ⫾ 60.8

o0.001

49 116

29.7 70.3

189.4 ⫾ 59.6 148.1 ⫾ 57.3

o0.001

No. of patients

EPB4.1L5 Z 160

EPB4.1L5 o 160

P value

94 71

71 36 35

94 58 36

122 43

47 24

75 19

0.073

100 65

39 32

61 33

0.203

95 70

41 30

54 40

1.000

86 79

21 50

65 29

o0.001

49 116

6 65

43 51

o0.001

51 114

33 38

18 76

o0.001

37 128

26 45

11 83

o0.001

31 134

23 48

8 86

o0.001

No. of patients

P value

0.204

SD ¼ standard deviation.

score Z 160, pT3 or higher), low risk (EPB4.1L5 score o 160, lower than pT2), and intermediate risk (others). The 5-year Kaplan-Meier rates of RFS for each risk group were 91.0% (low risk), 73.9% (intermediate risk), and 47.2% (high risk) and for CSS were 93.3% (low risk),

74.1% (intermediate risk), and 57.1% (high risk). Among the 3 groups, significant differences were observed in RFS (P ¼ 0.013 for the low-risk vs. intermediate-risk group, P ¼ 0.009 for the intermediate-risk vs. high-risk group, and P o 0.001 for the low-risk vs. high-risk group) and

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Fig. 1. (A) EPB4.1L5 expression. Immunostaining for EPB4.1L5 in the case of UTUC. (Intensity: (a) 0 no staining, (b) 1 slight staining, (c) 2 medium staining, (d) 3 strong staining). Bar, 50 mm. (B) Typical staining pictures with pathological information. Weak (e and g) and strong (f and h) staining patterns. Low (e and f) and high (g and h) pathologic tumor stage. Bar, 50 mm. IHC ¼ immunohistochemistry. (Color version of the figure available online.)

CSS (P ¼ 0.025 for the low-risk vs. intermediate-risk group, P ¼ 0.002 for the intermediate-risk vs. high-risk group, and P o 0.001 for the low-risk vs. high-risk group) (Fig. 3).

4. Discussion In this retrospective study, we evaluated the effect of EPB4.1L5 expression by immunohistochemistry in a series of patients with UTUC treated in a single center, and our result suggested that EPB4.1L5 expression, which was closely related to tumor grade, stage, and LVI, was one of the significant prognostic factors. High EPB4.1L5

expression was related to patients' shorter survival. As far as we know, this is the first study evaluating the prognostic value of EPB4.1L5 expression in patients with UTUC. EMT is a vital and essential step in tumor invasion and metastasis, and disruption of E-cadherin-mediated cell-cell adhesion is closely related to EMT in some cancers. In UTUC, some reports have demonstrated that E-cadherin was a significant prognostic marker and correlated with tumor stage and grade [19–21]. E-cadherin is regulated by several transcription factors such as Twist, Snail, and Slug. In UTUC, nuclear Snail was found to be significantly associated with LVI, grade, tumor stage, and a lower CSS, and it was an independent prognostic factor [6]. On the contrary, we have recently reported that EPB4.

Fig. 2. Kaplan-Meier recurrence-free survival (A) and cancer-specific survival (B) of the patients after surgery for UTUC according to EPB4.1L5 score (Ta-4N0M0).

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Table 2 Univariate (A) and multivariate (B) analysis for recurrence-free survival and cancer-specific survival (A) Characteristics

Age o70 Z70 Gender Male Female Tumor location Renal pelvis Ureter Side Left Right Pathologic tumor stage Lower than pT2 pT3 or higher Grade Low High Lymphovascular invasion Positive Negative EPB4.1L5 score Low High (B) Characteristics Age o70 Z70 Gender Male Female Tumor location Renal pelvis Ureter Side Left Right Pathologic tumor stage Lower than pT2 pT3 or higher Grade Low High Lymphovascular invasion Positive Negative EPB4.1L5 score Low High

No. of patients

(%)

Recurrence-free survival

Cancer-specific survival

P value

P value

94 71

57.0 43.0

0.346

0.423

122 43

73.9 26.1

0.108

0.147

100 65

60.6 39.4

0.881

0.480

95 70

57.6 42.4

0.706

0.479

86 79

52.1 47.9

o0.001

o0.001

49 116

29.7 70.3

o0.001

o0.001

63 102

38.2 61.8

o0.001

o0.001

94 71

57.0 43.0

o0.001

o0.001

No. of patients

(%)

Recurrence-free survival HR (95% CI)

Cancer-specific survival P value

HR (95% CI)

P value

2.79 (1.07–7.30)

0.036

94 71

57.0 43.0

122 43

73.9 26.1

100 65

60.6 39.4

95 70

57.6 42.4

86 79

52.1 47.9

2.66 (1.21–5.84)

0.015

49 116

29.7 70.3

5.29 (1.21–23.0)

0.026

63 102

38.2 61.8

1.69 (0.85–3.37)

0.135

1.81 (0.85–3.85)

0.124

94 71

57.0 43.0

2.40 (1.14–5.08)

0.022

2.94 (1.23–7.06)

0.015

HR ¼ hazard ratio.

1L5 regulates E-cadherin posttranscriptionally; it disrupts E-cadherin-based cell-cell adhesion in a posttranscriptional manner by binding to p120ctn, and it leads to endocytosis.

We have also reported the possibility that EPB4.1L5 is associated with tumor invasion and metastases through EMT [12].

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Fig. 3. Risk stratification for UTUC according to independent prognostic factors, EPB4.1L5 score, and pathological tumor stage. Patients were divided into 3 risk groups: low risk (EPB4.1L5 score o 160, lower than pT2), high risk (EPB4.1L5 scored Z 160, pT3 or higher), and intermediate risk (others). Kaplan-Meier recurrence-free survival (A) and cancer-specific survival (B) of the patients after surgery for UTUC according to the risk stratification.

In this study, we found significant correlations between EPB4.1L5 expression and LVI, grade and pathologic tumor stage in UTUC. Additionally, we demonstrated that higher EPB4.1L5 expression was significantly associated with a lower RFS and CSS, and our result indicated that it was an independent prognostic factor. When we especially examined about the recurrence case, the mean EPB4.1L5 score of 37 patients who experienced tumor recurrence was 189.19 ⫾ 64.3, and it was significantly higher compared with the score of others (P ¼ 0.002). The mean EPB4.1L5 score of 31 patients with cancer death was 186.7 ⫾ 61.2, and the score of others was 201.7 ⫾ 84.2 (P ¼ 0.335). In this cohort, there was no significant difference between the patients who experience cancer death and others in clinicopathological parameters including EPB4.1L5 expression score. However, according to our risk stratification, especially, high-risk group (EPB4.1L5 scored Z 160, pT3 or higher) was related to worse clinical outcome in patients with UTUC. These results indicate that EPB4.1L5 in UTUC might play important roles for its progression and the establishment of metastasis. For high-risk groups, aggressive therapy and frequent surveillance might be needed. This study has several limitations. This study was a retrospective study, and the number of patients is limited. Thus, a prospective study in a large population is warranted to clarify the prognostic role of EPB4.1L5 expression in UTUC. It is also necessary to elucidate how EPB4.1L5 is regulated in cancer cells in vitro. In conclusion, our study demonstrated that high EPB4.1L5 expression is related to worse clinical outcome in patients with UTUC regarding EMT.

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