Bladder cancer (BC) is the fifth most common human malignancy, with an incidence of 81.2 per 100,000 individuals[1]. At initial presentation, approximately 70% of patients present with non-muscle invasive bladder cancer (NMIBC), in which the tumor is superficial (stage carcinoma in situ [CIS]), or limited to the mucosa (Ta) and submucosa (T1), and up to 90% of NMIBC cases are urothelial neoplasms[2, 3]. Urothelial neoplasms are polymorphic in nature and can appear as solitary or multiple, flat, or exophytic, at any site on the surface of the bladder urothelium[4]. Bladder cancer usually has a good prognosis, particularly with early detection at stage Ta or T1. Although white light endoscopy, which is recommended by cancer prevention guidelines, is an easily accessible and non-invasive method to detect BC, up to 30% of tumors can be missed, particularly small satellite tumors and flat CISs[5]. These missed residuals lead to a high recurrence rate of 70%, and relapsed tumors may progress to infiltrative cancer[6], which has a significant impact on patient morbidity and quality of life. In addition, BC can incur high treatment expenses and is a burden on medical care systems. Therefore, improving the detection of BC, particularly those tumors that are often overlooked by current methods, would improve the prognosis of patients with NMIBC.
There are several barriers to overcome in the accurate diagnosis of NMIBC, including patient factors, physician factors, and the limitations of currently available diagnostic tests. Cancer prevention guidelines recommend imaging with white light (WL) endoscopy, which is the most commonly used diagnostic method for NMIBC detection, but this method can still lead to misdiagnosis and missed diagnosis[7–9]. The main cause of missed diagnosis is that the lesions cannot be seen under WL, particularly in the case of multifocal flat cancers[10, 11]. A missed diagnosis can also affect the accurate detection and complete resection of tumors, resulting in recurrent or advanced carcinoma[10, 12]. The presence of inflammatory diseases can lead to misdiagnosis[13–15]. Under WL cystoscopy, it is difficult to distinguish between inflammatory lesions and cancer, as well as benign and malignant lesions, which often leads to misdiagnosis[16]. Although clinicians can make a reliable diagnosis by biopsy, the need for invasive biopsy is a barrier to quick detection of bladder cancer. Additionally, it is impractical to perform a biopsy assessment of every lesion. Therefore, it is necessary to develop new detection methods to improve the diagnosis of NMIBC.
Recently, new imaging technologies have been developed to enhance tumor identification. Endoscopic methods, such as narrow-band imaging[17] and blue light cystoscopy[18], were developed to enhance cancer identification and guide endoscopic resection[19]. However,, these methods are limited by high false-positive rates due to their low specificity[20]. Microscopic imaging techniques, including confocal laser endomicroscopy[21] and optical coherence tomography[22], can provide information on tissue microarchitecture and cellular morphology but are not practical for surveying the entire bladder due to their narrow field of view. Near-infrared (NIR) fluorescence imaging with targeted probes has emerged as a promising technique for precise imaging[23]. NIR fluorescent probes consist of NIR fluorescein and a targeting agent[24]. NIR fluorescein emits light beyond the visible light spectrum and is not interfered with by endogenous substances, thus improving the accuracy of NIR imaging. We previously developed a NIR-based cancer-specific agent[25] that had promising efficacy in preliminary tests. The targeting agent was developed based on molecular changes that are specific to cancer, thus providing high specificity and sensitivity. In the present study, we demonstrate the potential of a NIR-based imaging strategy for improving NMIBC detection using targeted contrast agents.
CD44 is a multifunctional transmembrane glycoprotein that is highly expressed in BC and is involved in cell adhesion, cell migration, tumor progression, and metastasis[26]. CD44v6, a CD44 isoform containing the CD44v6 exon, is also highly expressed in BC[27]. CD44v6 is a co-receptor of HGF in collaboration with the tyrosine kinase c-Met, and together they promote the migration and invasion of BC cells[28]. CD44v6 is a marker of poor prognosis in patients with BC[29]. In addition, CD44v6 is closely related to the degree of malignancy and the stage of BC. Therefore, targeting CD44v6 has tremendous potential for early BC diagnosis[30]. In a previous study, we identified a CD44v6-specific peptide, PLSWT (CSDRIMRGC), and labeled it with a NIR fluorescein, IRDye800CW, to synthesize the BC-specific NIR imaging probe PLSWT7-DMI[25]. As a tool for non-invasive in vivo identification of BC, the specificity of the probe has been confirmed in BC cells, tumor-bearing mice, and ex vivo human bladders. The ex vivo results revealed that CD44v6-targeted NIR imaging can pinpoint multifocal lesions. PLSWT7-DMI has a high sensitivity (up to 91.2%) and specificity (up to 90%). Specific peptides and NIR fluorescence provide a biological basis for enhanced contrast and can achieve desirable tumor-to-background ratios (TBR) for in vivo detection.
Preclinical evaluation of PLSWT7-DMI has strongly supported its feasibility for clinical translation. In the present study, we report our initial findings with NIR fluorescence-guided endoscopy using the imaging probe PLSWT7-DMI (Chinese Clinical Trials Database registration number: ChiCTR-DDD-17012616). The purity, efficacy, safety, and nontoxicity of the agent were confirmed before clinical application. We performed a first-in-human clinical study using this probe, and demonstrated that it can bind specifically to BC tissue for visualization by NIR fluorescence endoscopy, thus improving the detection of easily overlooked BC lesions.