1. Introduction

Urothelial bladder cancer (UC) represents the ninth most common cancer across the globe, with over 500,000 new cases and 200,000 deaths annually¹. In confirmed cases, bladder tumors that are confined to the mucosal layer of the bladder (carcinoma in situ (CIS)/Ta) or the lamina propria (T1) and do not invade the muscularis propria are classified as NMIBC. Approximately 75% of patients were diagnosed with NMIBC at their initial visit, with Ta, T1, and CIS accounting for 70%, 20%, and 10%, respectively. According to the American Urological Association (AUA) risk classification, NMIBC is categorized into four levels: low, intermediate, high, and very high risk². Among them, the 10-year cancer-specific survival (CSS) rate of high-risk patients reaches 70% to 85%³. The 5-year recurrence rate is up to 31% to 78%, and 17% to 45% of cases progress to MIBC or even distant metastasis⁴. These patients are difficult to treat and have a poor prognosis, posing a significant economic burden on patients and society. Conservative treatment with TURBT and postoperative bladder instillation of BCG are recommended for high/very high-risk NMIBC patients, who initially respond well to the treatment, but approximately half of the patients receiving this regimen suffer from recurrence within five years, with 21% of them progressing to MIBC, and achieving a CSS rate of only 35%^5,6. Local side effects occur in about 70% of patients after BCG infusion, and the discontinuation rate due to AEs of BCG infusion ranges from 13% to 20%^7,8. Therefore, RC is recommended for this population⁹. The European Association of Urology (EAU) even recommends that patients with NMIBC at greater risk of recurrence and progression, including those with T1 HG BC, be treated with RC, since RC at the time of progression to MIBC is associated with a worse prognosis¹⁰. Although RC can improve the survival rate of NMIBC patients and prevent local recurrence and distant metastasis, it is associated with a number of complications that significantly affect patients' quality of life, and the perioperative mortality rates at 30 and 90 days are as high as 3.2% and 5.2%, respectively¹¹. This is why most patients refuse to receive this surgery¹². Moreover, a significant number of patients cannot tolerate RC because of factors such as advanced age or comorbidities¹³. Therefore, to address the specific conditions of the high/very high-risk NMIBC population, alternative bladder preservation strategies to improve the prognosis and quality of life of high/very high-risk NMIBC patients are urgently needed.

While several studies have confirmed that intravesical BCG infusion after TURBT significantly reduces the risk of disease recurrence and progression in NMIBC, patients’  outcomes are still unsatisfactory. One intensive study revealed that HER-2 overexpression (2+/3+) was an independent predictor of failure of BCG infusion in NMIBC patients, with a 5-year RFS rate of merely 19.0% and a 5-year PFS of only 58.2% against HER2-negative patients¹⁴. In addition, residual tumor after initial TURBT is considered a risk factor for tumor recurrence and progression, and the majority of NMIBC patients are confirmed to have residual tumor at the time of re-TURBT¹⁵.

The EAU guidelines recommend the use of re-TURBT in patients with high risk NMIBC to lower 5-year postoperative recurrence and progression rates⁵. Recently, immune checkpoint inhibitors (ICIs) and antibody‒drug conjugates (ADCs) have shown significant efficacy for the treatment of a wide array of solid tumors, providing a new therapeutic option for NIMBC patients who refuse or do not respond to BCG perfusion and who are not indicated for RC. The KEYNOTE-057 study reported that Pembrolizumab achieved a 46% 12-month complete remission (CR) rate and a 43.5% disease-free survival (DFS) rate, with BCG-naïve high-risk NMIBC patients attaining a median remission duration of 16.2 months. The treatment demonstrated a good antitumor activity and was recommended by the National Comprehensive Cancer Network (NCCN) for the management of BCG-naïve high-risk NMIBC patients¹⁶. The BladderGATE study reported that 36 patients with high-risk NMIBC who were refractory to BCG therapy and received Atezolizumab in addition to continued BCG infusion accomplished a 2-year DFS rate of 72.8% while maintaining a favorable safety profile¹⁷. The above study has shown that ICIs in combination with Atezolizumab have satisfactory efficacy and safety in high-risk NMIBC patients.

HER2 plays an important role in the development and progression of many malignancies, including mammary, gastric, and urothelial cancers¹⁸, and mounting studies have confirmed that HER2 expression is an important independent prognostic factor in UC and that treatment can be individualized according to HER2 expression¹⁴. Anti-HER2 targeted therapy is recommended for patients with HER2 overexpression. Distitamab Vedotin is a novel humanized anti-HER2 antibody coupled to monomethylornithine protease inhibitor E (MMAE) via a cleavable linker¹⁹. RC48-C005 and RC48-C009 studies have demonstrated that Distitamab Vedotin can therapeutically benefit UC patients over-expressing HER2²⁰. Previous studies have shown that a significant proportion of NMIBC patients have HER2 overexpression (IHC 2+/3+). Therefore, Distitamab Vedotin holds promise to be used for the treatment of NMIBC.

Disitamab Vedotin in combination with ICIs in local or locally advanced UC was demonstrated to have a HER-2-positive (1+/2+/3+) pCR rate of 62.5%. The ORR was 62.5%, the ORR was 100%, the median radiographic progression-free survival (rPFS) was 12.0 months, and the incidence of grade 3/4 AEs was only 22.2%²¹. This regimen exhibited a 68.75% cCR and a 90.91% disease control rate (DCR) as a neoadjuvant regimen in patients with HER2-expressing (1+/2+/3+) MIBC²², whereas 16 patients (66.7%) achieved CR and 8 patients (33.3%) attained disease stabilization (SD) in patients with high-risk NMIBC who were contraindicated for surgery. Only 16.7% of patients experienced Grade 3-4 treatment-related adverse events (TRAEs)²³. As a result, RC48-ADC plus PD-1 inhibitor treatment can produce synergistic antitumor effects, achieved good clinical efficacy and demonstrated good safety profiles, further verifying the advantages of ADCs combined with PD-1 inhibitor in the treatment of UC, especially in HER-2 positive and high/very high-risk patients.

To date, evidence is limited regarding use of Disitamab Vedotin in combination with Tislelizumab and re-TURBT for the treatment of high-risk or very high-risk HER-2 2+-3+ NMIBC. Therefore, we elect to conduct this Phase II, multicenter, prospective study to examine the efficacy and safety of Disitamab Vedotin in combination with tislelizumab and re-TURBT in high/very high-risk NMIBC with HER-2 2+-3+ status. We hypothesize that Disitamab Vedotin in combination with tislelizumab and re-TURBT will not affect patients' quality of life or increase the economic burden, while safely and effectively improving the prognosis of patients with high/very high-risk NMIBC.

2. Methods and analysis

2.1 Study design

This is a Phase II, multicenter, single-arm, open-label clinical trial, which uses competitive enrollment. We enroll 40 patients who satisfy the eligibility criteria between October 2024 and October 2025. The subjects are high/very high-risk NMIBC patients who are pathologically confirmed and have HER-2 overexpression (2+/3+). They are admitted to Fujian Medical University Union Hospital, Fujian Provincial Hospital, Zhangzhou Hospital, Zhejiang Provincial Tumor Hospital, Ningbo Yinzhou No. 2 Hospital, Fujian No. 2 People's Hospital, Longyan No. 1 Hospital, Sanming City No. 1 Hospital, Xiapu County Hospital and Xiamen Hospital of Traditional Chinese Medicine. Patients who meet the eligibility criteria are recruited upon signing an informed consent form, and are then put on a follow-up treatment program. The study design is illustrated in Figure 1.

Figure 1. Study design

2.2 Inclusion and exclusion criteria

The inclusion and exclusion criteria are detailed in Table 1.

2.3 Outcomes

The primary endpoint of the study was 1-year EFS, and the secondary endpoints were 3-year EFS, 2-year BIDFS, time to RC, RFS, OS, QLQ-C30 score and incidence of AEs. Any serious AE was defined according to the rules of good clinical practice and was immediately reported to the lead center (Fujian Medical University Union Hospital) and subsequently communicated to the other participating centers. The 1-year EFS was defined as the probability of patients not having experienced any events for at least 12 months after receiving treatment, including death, disease progression (tumor growth or the appearance of new lesions), switching to chemotherapy (the treatment plan is changed to chemotherapy before the end of the planned treatment.), the addition of other treatments (Other treatments are added before the planned treatment ends.), fatal or intolerable side effects (A patient withdraw from the trial due to fatal or intolerable side effects), etc. Two-year BIDFS is defined as the probability of patients remaining free from muscle-invasive recurrence, regional lymph node recurrence, distant metastasis, the need for radical cystectomy, or death at least 24 months after the initiation of treatment. RFS is defined as the time from the first TURBT to the first occurrence of bladder tumor recurrence, with the main time points being 1 year, 2 years, and 3 years. OS was defined as the time from the start of treatment to death from any cause. The time to radical cystectomy was defined as the time from the start of treatment to radical cystectomy. Radiographic progression will be assessed against the Revised Criteria for the Evaluation of Response in Solid Tumors (RECIST) version 1.1²⁴. AEs are assessed according to the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. Quality of life is assessed based on the European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire (QLQ-C30)²⁵.

2.4 Sample size calculation

A review of domestic multicenter randomized controlled studies, revealed that the 1-year recurrence rate of BCG perfusion after high-risk NMIBC surgery was 21.91%, with a historical 1-year EFS of 78%. The target value for this study is 92%. Using StatBox, a specialized software from ClinicalTrialStatistics.com, with two-sided α=0.05, 1–β= 0.8, registration period of 3 years and a follow-up period of 3 years, at least 32 patients are required for sampling. Accounting for a 20% drop-out rate, the total sample size is 40 patients.

2.5 Interventions

Patients first undergo an initial maximum TURBT and receive a single infusion of 50 mg pirarubicin during 24 hours immediately after the procedure, and tumor features  such as size, location, morphology, and the presence or absence of multiple tumors are recorded. Maximum TURBT refers to a total or segmental resection of a bladder tumor, covering the exophytic part of the tumor, the underlying bladder wall, and the edges of the resection area. Two experienced urologists intraoperatively evaluate the eligibility of the patient against the indications for maximum TURBT. NMIBC patients with a pathological diagnosis of HER-2 overexpression after the day of surgery, as assessed by the clinician on the basis of AUA, are to be put on the protocol of Tislelizumab (200 mg) + Disitamab Vedotin (120 mg [≤60 kg] or 2.0 mg/kg [>60 kg]), every 3 weeks (Q3W) for 3‒4 cycles. The HER2 status of urothelial carcinoma is interpreted on the basis of the Chinese Expert Consensus on HER2 Testing in Urothelial Carcinoma (2021). Safety evaluation, including routine blood tests, biochemical panels, thyroid function tests, and cortisol tests at 8:00 am, is performed during every cycle, and routine blood and biochemical panels were performed on day 10 after the first cycle of dosing. Efficacy evaluation was conducted every two cycles, including lung CT plain cans and MR plain scans and enhancement scans of the whole bladder. Patients with no disease progression or improvement on imaging assessment are subjected to re-TURBT within 6 weeks after the last medication. Patients are removed from the group if imaging reveal disease progression (bladder tumor recurrence or new lymph node metastasis, distant metastasis) and are switched to a systemic treatment regimen or underwent RC.

After re-TURBT, pathological evaluation revealed that the patient was free of residual tumor and treatment continued with 2–4 cycles of Disitamab Vedotin in combination with Tislelizumab. If the pathology of the re-TURBT suggested residual tumor, the lesion is re-staged and evaluated. If pathology suggested MIBC, systemic therapy was given or total bladder excision is performed. If pathology suggests NMIBC, the treatment is switched to another systemic treatment regimen or BCG instillation, follow-up is terminated, and RC is performed if subsequent disease progression occurs.

2.6 Follow-up

At each study setting, a follow-up specialist is designated to follow up all patients included in this trial. All participants received standard care, such as smoking cessation counseling, to reduce the risk of tumor recurrence. Patients are followed for 3 years after re-TURBT, with efficacy being followed up by cystoscopy (every 24 weeks); urine cytology/biopsy/imaging (every 12 weeks on Years 1-2; every 24 weeks on Year 3); and survival followed up every 12 weeks. Each of the above tests are performed at the study center where the patient underwent surgery, and tests are so arranged that they did not preclude the possibility of examination at other hospitals. If it is an external hospital review, results of each examination is followed up and recorded by a follow-up specialist. The overall examination results are used to record and evaluate the postoperative survival status and smoking status in all patients. If patients refuse to accept the aforementioned follow-up plan, they are listed as lost cases and analyzed together with cases that met the study criteria at the end of the study. The patients are followed up until death or the end of follow-up (3 years after treatment). The endpoint of the study is defined as the last follow-up date of the last participant. The estimated end time of the study is October 2028. All the data are independently cross-checked by two researchers with hard copy data stored in a locked cabinet and soft copy data on encrypted hard drives. All the collected data are used solely for research purposes and were accessible only to researchers. All the data are to be destroyed 5 years after the end of the study.

2.7 Statistical analysis

The primary endpoint involves the relapse rate in patients receiving Disitamab Vedotin combined with Tislelizumab and re-TURBT, and the rate is later analyzed based on the intention-to-treat (ITT) population. Descriptive statistics are used for clinical pathological characterization and safety assessment. Consecutive endpoints are presented in the mean, standard deviation (SD), and confidence intervals, and if necessary, the minimum and maximum values P25, median and P75 frequency and percentage distributions for discrete data. The Kaplan‒Meier method is employed to estimate EFS BIDFS, PFS, AE, and OS. As this study is a multicenter clinical trial, a multicenter consistency test is needed for quantitative indicators, and a mixed effects model is used. For qualitative indicators, the CMH method is utilized. For level variables, a level logistic regression model is used for evaluation and calibration. For survival time variables, a Cox regression model is used for evaluation and calibration. Univariate and multivariate survival analyses use Cox proportional hazards models to calculate HRs and 95% CIs. Statistical analysis is performed by using STATA and R software V.3.6.2 (http://R-project. org). The significance level is set to a p ≤ 0.05, and all the statistical tests are two-sided. To avoid data missing, we endeavor to perfect the design of the trial and to collect and manage raw data with great care. Multiple interpolation is used to estimate missing outcome data for patients who drop out or are lost to follow-up.

2.8 Adverse events and management plan

In this research, the intervention drugs have undergone safety testing and are already in clinical use. As a result, participants are not exposed to any additional risks. Nonetheless, potential side effects such as rash, alopecia, and peripheral neuritis and vomiting might occur. Patient safety is be closely monitored throughout the study. In the event of any serious adverse events, these events will be promptly reported to the sponsor, and affected patients would receive appropriate compensation.

Symptomatic and topical treatments are used for mild adverse events (Grade 1 or 2). Persistent low-grade events or serious events (grade ≥ 3) should be treated with systemic corticosteroids such as prednisone or intravenous equivalents. In the case of multiple concurrent low-grade adverse events that do not require discontinuation of medication, the decision to discontinue medication is made by the investigator.

The investigators evaluate the severity of adverse events via the CTCAE version 5.0 grading criteria and adjust it according to the medication. The general adverse events and management recommendations for Disitamab Vedotin and Tislelizumab are outlined in Table 2, Table 3 and Table 4.

2.9 Management and quality control

The lead center regularly organizes training on cystoscopy, magnetic resonance imaging (MRI) plain and enhancement scan, pathological examination, staging, and response assessment. All pathological sections are reviewed and confirmed by two experienced physicians at or above the level of deputy director in each center, and ultimately followed up by the lead center. AUA assessment and corresponding clinical work are conducted by two physicians at the deputy director level or above who are familiar with BC treatment. Additionally, a pharmacologist, a statistician, an ethicist, and a radiologist perform their corresponding work. The central review of observations and evaluations is conducted independently. The clinical research assistants independently evaluate protocol compliance, research safety, and data collection accuracy. This monitoring is conducted once a year.

3. Discussion

TURBT combined with full dose BCG infusion is still recommended by many treatment guidelines for high-risk or very high-risk NMIBC^26,27. High rates of failure, side effects and discontinuation of BCG infusion therapy in HER-2-overexpressing NMIBC have been confirmed in previous studies. In addition, the high cost and increasing shortage of BCG in recent years have led to low BCG utilization in NMIBC patients^14,28,29. However, studies using low-dose BCG suggest that low-dose BCG infusion leads to a shorter DFS than full-dose regimens³⁰. The efficacy of immunomonotherapy is limited in BCG-resistant high-risk NMIBC patients³¹, while a regimen of immunotherapy combined with chemotherapy results in reduced tolerance because of the inevitable superposition of chemotherapy-related adverse reactions and immunotherapy-related adverse reactions. Therefore, novel treatment strategies are urgently needed. In a previous study, patients with high HER-2 expression who received adjuvant treatment with Disitamab Vedotin after TURBT achieved a 12-month RFS rate of 100%, suggesting that Disitamab Vedotin has shown promise as an adjuvant for high-risk NMIBC patients³². ADC combined with immunotherapy has a good ORR, and the associated side effects are milder than those of chemotherapy combined with immunotherapy, and are better tolerated by patients, rendering this regimen a neoadjuvant treatment option for MIBC²⁸. Therefore, we hypothesized that ADC combined with immunotherap, as a treatment regimen for high-risk or very high-risk NMIBC patients with HER overexpression, could achieve high response rates, low toxicity, and low recurrence and progression rates. Re-TURBT can effectively reduce the risk of recurrence and progression of NMIBC³³, but more unfavorable pathological results may be found after the first pathological resection. Moreover, the adverse pathological results of re-TURBT may increase the risk of recurrence and progression of high-risk NMIBC³⁴. Therefore, our triple therapy can improve patient survival and quality of life. This combination can give full play to the synergistic effect of the ADC combined with ICIs, and locally clear suspected residual lesions after the drug has achieved therapeutic effect, thereby greatly lowering the risk of recurrence.

In summary, this single-arm, open-label phase II study is designed to evaluate the safety and efficacy of Disitamab Vedotin in combination of Tislelizumab and re-TURBT in high-risk or very high-risk NMIBC patients with HER-2 2+-3+ status. Such protocol promises to be treatment strategy. If this study achieves satisfactory results, we will go further to conduct a follow-up study. If the protocol fails or leads to intolerable toxic reactions, or if the patients’ preferences change, we will compare notes with the patient whether to proceed immediately with other back-line treatments, such as RC.

Potential side effects will be closely monitored for early detection and timely intervention, to achieve better therapeutic outcomes without compromising quality of life or increasing toxicity of the protocol. We will also record and track patients' smoking to understand changes in their smoking behaviors and take appropriate measures to promote smoking cessation and reduce patients' risk of relapse.

In summary, we conduct a prospective, multicenter, phase II, single-arm exploratory trial to investigate the efficacy and safety of the combination of Disitamab Vedotin with Tislelizumab and re-TURBT for the treatment of HER-2 2-3 high/very high-risk NMIBC. To our knowledge, this is the first study to explore the efficacy of this regimen in patients with high/very high-risk NMIBC with HER-2 2-3, which may provide a new treatment strategy for patients with high/very high-risk NMIBC with HER-2 2-3.