TROY expression is associated with pathological stage and poor prognosis in patients treated with radical cystectomy

2.1 Patient population

From 1990 to 2015, 161 consecutive patients who had undergone radical cystectomy with pelvic and iliac lymphadenectomy at Kitasato University Hospital were included in this retrospective study. In total, 10 patients who had histologic variants including squamous cell carcinoma, adenocarcinoma, and small cell carcinoma and 15 patients who had been previously treated with neoadjuvant chemotherapy were excluded. None of the remaining patients were treated preoperatively with either systemic chemotherapy or radiation therapy. The final study group consisted of 111 (81.6%) male and 25 (18.4%) female subjects, with a median age of 65 years (range, 40–82; mean, 64.1). The median follow-up was 50.7 months (range, 0.7–288.3; mean, 77.3) for patients who were alive at the last follow-up. When a patient died, the cause of death was determined by the patient’s physician, chart review supported by death certificate, or death certificate alone. In total, 33 patients (24.3%) received adjuvant chemotherapy after surgery for adverse pathological characteristics including regional lymph node metastases or extravesical involvement. A total of 34 patients (25.0%) received cisplatin-based treatment for disease recurrence. This study was approved by the ethics committee of Kitasato University School of Medicine and Hospital (B17-010).

2.2 Immunohistochemistry and scoring

Monoclonal antibodies specific for TROY were generated using the previously described hybridoma technology [12]. In brief, 300 μ g of N-terminal, His-TROY protein was injected into the footpad of BALB/c mice using keyhole limpet hemocyanin as an adjuvant. Four weeks later, spleen cells were isolated and fused to the myeloma cell line P3-X63Ag8,653 using polyethylene glycol 1500 (PEG 1500) as previously described [13]. Primary antibodies in the hybridoma culture supernatant were tested by immunoblot analysis with non-tagged recombinant N-terminal protein. Animal experiments were performed ethically according to the Guidelines for Animal Experiments at Yokohama City University.

Formalin-fixed, paraffin-embedded blocks representing the most invasive areas of each tumor were collected for further investigation. Normal urothelia were harvested from cystectomized specimens. The 2002 TNM classification was used for pathological staging, and the 1973 World Health Organization classification was used for pathological grading. Lymphovascular invasion (LVI) determined the presence of cancer cells within the endothelial space. Cancer cells that merely invaded a vascular lumen were considered negative [14].

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Three- μ m thick sections were immunostained by the Bond-Max Automated Immunohistochemistry system and Bond Polymer Refine Detection Kit DC9800 (Leica Biosystems, Newcastle, UK) based on the report from Lu et al. with the following minor modifications [15]. First, tissues were deparaffinized and pre-treated with Bond Epitope Retrieval Solution 2 (Leica Biosystems) at 100 ∘ C for 20 min. After washing, peroxidase blocking was carried out for 10 min. Tissues were again washed and then incubated with 500 × diluted anti-TROY monoclonal antibody with the Bond Primary Antibody Diluent (Leica Biosystems) for 30 min. Tissues were then incubated with Bond Polymer (Leica Biosystems) for 10 min, developed with DAB-Chromogen for 10 min, and then counterstained with hematoxylin for 5 min. Negative controls were reacted with Bond Primary Antibody Diluent replacing first antibody. TROY is located in the cell membrane, and membranous immunostaining of the tumor cell was considered to be positive. All slides were reviewed by two investigators blinded to clinical and pathological data. TROY was scored by the percentage of positive tumor cells and staining intensity. The percentage of tumor cells was scored as 0 (0%), 1 (1–33%), 2 (34–66%), and 3 (67–100%). Staining intensity was also scored as 1 (weak), 2 (moderate), and 3 (strong). The multiply index was obtained by totaling the intensity and percentage scores. In a preliminary study, we assessed the discriminative value for clinical characteristics and prognosis using each value in the multiply index as the cutoff point. Univariate analysis demonstrated that 0 was the only value to yield statistically significant findings (data not shown). Expression scores for TROY were stratified further into negative (score 0) and positive (score 1 or greater) for the purpose of presentation.

We also performed nestin (polyclonal antibody, IBL, Takasaki, Japan) immunohistochemical staining as previously described [16]. Nesting expression scores using the sum index score were stratified into negative (score 0) and positive (score 1 or greater).

For this analysis, age ( ⩽ 65 versus $>$ 65), tumor stage ( ⩽ T1 versus ⩾ T2), grade (grades 1 and 2 versus grade 3), and lymph node status (N0 versus N1 and N2) were evaluated as dichotomized variables. The Fisher’s exact test was used to evaluate the association between TROY, nestin, age, gender, stage, grade, carcinoma in situ (CIS), LVI, and lymph node status. Multivariate survival analyses were performed using the Cox proportional hazards regression model to estimate the association between TROY expression and pathological stage and grade, presence of LVI, and lymph node metastases. All analyses were performed with Stata ver.13 for Windows (Stata, Chicago, IL, USA). Statistical significance in this analysis was fixed at 0.05.

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3.1 Association of TROY expression with clinicopathological characteristics

Representative cases of immunostaining for TROY in normal urothelium and UCB tissues are shown in Fig. 1. Positive UCB tissues demonstrated membranous immunoreactivity (Fig. 1B). Normal urothelium showed negative immunoreactivity for TROY (Fig. 1C).

The clinicopathological characteristics of the 136 patients and the association with TROY expression are shown in Table 1. Positive and negative expressions of TROY were noted in 60 (44.1%) and 76 (55.9%) patients, respectively. Positive expression of TROY was associated with muscle invasive disease and nestin expression ( p = 0.019 and 0.013, respectively). Age, gender, pathological grade, presence of CIS, lymph node metastases, and LVI did not differ significantly between positive and negative expression.

3.2 Association of TROY expressions with prognosis

Disease progression developed in 71 patients (52.2%) at a median time of 13.7 months (range, 0.7–123.3; mean 26.3). Of all patients, 74 (54.4%) had died at the time of analysis at 21.4 months (range, 2.2–224.5; mean 39.7). The cause of death in 65 patients (87.8%) was identified as advanced or metastatic lesions, while 9 patients died of intercurrent causes without evidence of disease progression.

A Kaplan-Meier analysis indicated that positive expression of TROY was associated with increased probability of disease progression and cancer-specific death ( p = 0.042 and 0.008, respectively; Fig. 2).

In the Cox proportional hazards regression analyses, only lymph node metastasis was found to be associated with disease progression and cancer-specific survival (Table 2). Expression of TROY was marginal in terms of cancer-specific survival ( p = 0.058; Hazards ratio = 1.71; 95% confidence interval = 0.983–2.984).