Study Protocol for Evaluating the Efficacy of Anti-HER-2 Antibody-Drug Conjugates (ADC) in Locally Advanced or Metastatic Bladder Cancer Based on Organoid Technology.

1. Research Background (Rationale and Significance Based on Domestic and International Research Status)

Bladder cancer is a malignant tumor originating from the bladder urothelium and ranks first in incidence among urogenital tumors in China. Urothelial carcinoma is the most common type, accounting for over 90% of bladder cancer cases [1]. Approximately 5% of bladder cancer patients are diagnosed with metastatic disease, termed metastatic bladder cancer (MBC) [2-4]. Studies have shown that the median progression-free survival (PFS) for MBC patients is 7 months, and the median overall survival (OS) is 15 months [5, 6].

For patients with unresectable locally advanced or distant metastatic bladder cancer, platinum-based combination chemotherapy (CC) remains the standard first-line treatment [7]. In recent years, with continuous advancements in medical technology, the emergence of novel chemotherapeutic agents, targeted drugs, and immune checkpoint inhibitors has significantly increased the therapeutic options available after progression on first-line chemotherapy for MBC, yielding promising efficacy [8]. Anti-HER2 antibody-drug conjugates (ADCs), represented by trastuzumab-based therapies, are among these options. Human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase receptor involved in stimulating cell proliferation, differentiation, and survival [9]. HER2 overexpression plays a significant role in the development and progression of various malignancies, including breast cancer, gastric cancer, and urothelial carcinoma [10], and is often associated with a biologically aggressive tumor phenotype, poor prognosis, increased risk of disease recurrence, and limited benefit from chemotherapy [9, 11-13]. In bladder urothelial carcinoma, the proportion of HER2 protein overexpression (Immunohistochemistry [IHC] 2+ and 3+) ranges from 18.1% to 36% [14-16], and HER2 gene amplification is observed in 10% to 23% of cases [14, 16-17]. Research indicates that anti-HER2 ADC drugs demonstrate good efficacy and safety in treating patients with locally advanced unresectable or metastatic urothelial carcinoma, providing significant clinical benefit for this population [18]. In June 2021, Disitamab Vedotin (RC-48, RemeGen Co., Ltd.) was approved in China for the treatment of HER2-positive locally advanced or metastatic urothelial carcinoma [10]. HER2 protein expression status is typically assessed using IHC [10]. However, there is currently no internationally standardized detection protocol or interpretation criteria for HER2 protein expression status in urothelial carcinoma. This leads to considerable discrepancies in HER2 expression assessment across different clinical centers, directly affecting the selection of patients eligible for anti-HER2 ADC therapy and the standardization of clinical research. Additionally, fluorescence in situ hybridization (FISH) testing for HER2 gene amplification status also holds clinical significance for treatment guidance [10]. Clinical studies suggest that HER2 ADC drugs remain effective in urothelial carcinoma patients with HER2 protein overexpression (IHC 2+) but negative FISH results [18, 19], and clinically, there are patients who benefit despite negative protein detection but positive FISH. These findings indicate that neither IHC nor FISH results alone can fully guide the use of Disitamab Vedotin. When clinical assessment determines a patient's insensitivity to the administered drug, severe toxic side effects have often already occurred, and disease progression may deprive the patient of opportunities for alternative treatments. Therefore, evaluating the response of locally advanced or metastatic bladder cancer patients to anti-HER2 antibody-drug conjugates to guide treatment selection for patients with high drug sensitivity, thereby achieving precise and individualized tumor therapy, has increasingly become a current research focus.

The realization of precise cancer treatment heavily relies on drug sensitivity testing. Through precise and individualized drug screening experiments, the most effective and least toxic treatment regimen can be identified for each patient before therapy begins. Developing patient-specific treatment plans represents a new research direction for achieving precision treatment in bladder cancer and improving drug efficacy. Previously, commonly used preclinical models included traditional tumor cell lines and patient-derived xenograft (PDX) models. Tumor cell line culture is simple but insufficient to simulate the growth state of tumor cells within the patient's body, and drugs screened using these systems have low clinical translation value. While PDX models can simulate in vivo tumor characteristics and preserve the tumor microenvironment, they have significant limitations, such as relatively low stable engraftment rates, long modeling and evaluation cycles (six months to one year), being time-consuming and labor-intensive, making them difficult to generate and utilize for high-throughput drug screening [20-23]. Therefore, to develop more personalized treatment and prevention strategies tailored to an individual's unique genetic, environmental, and lifestyle characteristics, minimizing risks and optimizing medical intervention outcomes, it is imperative to develop drug sensitivity testing models that can simulate the heterogeneity and complexity of bladder cancer.

Organoids, specifically Patient-Derived Organoids (PDOs), are three-dimensional organotypic structures formed through the self-assembly of stem cells in vitro. They can differentiate into multiple organ-specific cell types and exhibit cell-cell interactions, cell-extracellular matrix interactions, and spatial organization, thereby recapitulating key functions and structures of real human organs in vitro while maintaining stable phenotypic and genetic characteristics. Compared to two-dimensional tumor cell lines and PDX models, tumor organoids can be cultured directly from a patient's own tissue. These organoids can effectively replicate key properties of the primary tumor, preserve the pathological morphology and biological mechanisms of the patient's tissue, retain tumor heterogeneity and a more authentic tumor microenvironment, and have the advantage of a short growth cycle [24]. This facilitates their use for sensitivity testing of chemotherapy drugs, molecularly targeted agents, and anti-tumor antibodies in clinical cancer patients, predicting patient response to drugs, and holds potential for assisting clinical treatment decision-making.