Bladder cancer (BCa) is the 9th most common cancer worldwide as of 2025 [[1], [2], [3]], and is generally divided into muscle-invasive bladder cancer (MIBC) and nonmuscle-invasive bladder cancer (NMIBC) [4]. BCa has diverse molecular and histologic subtypes, such as micropapillary, plasmacytoid, nested, sarcomatoid, and neuroendocrine bladder cancer [5]. The current gold standard of BCa diagnosis and surveillance is cystoscopy with urine cytology and bladder biopsy. However, the procedure is invasive and costly due to its high recurrence rate and requirement for cystoscopy procedures [6,7].

Urine is a noninvasive liquid specimen that can be used in cytology diagnosis as well as clinical follow up. Urine contains proteins, DNA, RNA, and extracellular vesicles (EVs), which are a heterogeneous group of membrane enclosed vesicles transporting various biomolecules such as RNA, DNA, lipids and proteins [8], making it an ideal specimen for scientific interrogation. This review aims to provide an overview of the current state of urinary cytology diagnosis and urinary biomarkers for bladder cancer management. The focus is on the clinical utility of advanced molecular approaches, including using tumor-related proteins, DNA, RNA, and EV for urine based 'liquid biopsy' in the era of precision medicine and artificial intelligence.

1. Gold standard of bladder cancer diagnosis through urine cytology: The Paris System (TPS 2.0).

There are 2 types of urine cytology specimens: voided urine and cystoscopy wash, both of which are good resources for cytology diagnosis. The Paris System (TPS) Urine cytology is a widely used diagnosis criterion for detecting BCa as an adjunct to cystoscopy. The updated version is TPS 2.0 [9]. Many institutes, but not all, are using TPS 2.0 for urine cytology diagnosis. The guiding principle is to detect HGUC due to its flat lesion feature and high propensity for invasion (Fig. 1). The sensitivity ranges from 18% to 82% [10] and the specificity is as high as 93% [11]. Considering the cost effectiveness and turnaround time, urine cytology remains a reliable screening and diagnostic test for detecting bladder cancer, regardless of the specimen type [9].

There are 5 categories in the TPS 2.0 for urine cytology diagnosis: unsatisfactory, Negative for high grade urothelial carcinoma (NHGUC), Atypical Urothelial Cells (AUC), suspicious for high grade urothelial carcinoma (SHGUC), and positive for high grade carcinoma (HGUC) (Table 1) [12].

The unsatisfactory category is defined as voided urine less than 25 to 30 ml with low cellularity, or instrumental urine with low cellularity and without any significant morphologic findings. The management with this category usually sends a repeat specimen.

The category for negative for high-grade urothelial carcinoma (NHGUC) accounts for 90.5% of voided urine specimens. It is defined as a urinary specimen that is adequate and lacks any cytomorphologic findings for HGUC (Figs. 2 and 3). Because urine cytology is essential in the surveillance of patients for recurrences following therapy, the management of a patient with a diagnosis of NHGUC may be to continue the routine surveillance at intervals that commensurate with the risk of recurrence.

The atypical urothelial cells (AUC) category contains urothelial cells with mild-to-moderate cytologic (not architectural) changes for HGUC, which accounts for 5.6% of voided urine specimens. The diagnosis criteria including one major criteria: Increase in N/C ratio of ≥0.5, but <0.7, due to nuclear enlargement [13], plus one of the minor criteria including nuclear hyperchromasia, irregular nuclear membranes and chromatinic rim or nuclear contour, and irregular, coarse and clumped chromatin (Fig. 4) [14]. The management of AUC should be individualized based on the patient's risk assessment and prompt a thorough evaluation. Reflex molecular tests such as UroVysion FISH and urinary biomarkers are applied in different hospitals.

Suspicious for high-grade urothelial carcinoma (SHGUC) counts for 1.6% of voided urine. The diagnosis is restrictively used for cases of abnormal urothelial cells that quantitatively fall short of the definitive diagnosis of HGUC. The diagnosis criteria are the same as HGUC but with less malignant cells (<5 cells for lower tract and <10 cells for upper tract) (Fig. 5). The management of patients diagnosed with SHGUC should include a thorough evaluation for the presence of HGUC. This management should be decisively guided by the patient's history, clinical context, and cytologic findings.

High-grade urothelial carcinoma (HGUC) requires the presence of more than 5 cells in the lower urinary tract and more than 10 cells in the upper urinary tract that meet specific diagnostic criteria. These criteria include a nucleus-to-cytoplasm ratio greater than or equal to 0.7 due to nuclear enlargement, as well as the presence of at least 2 of the following features: hyperchromasia, irregular nuclear membranes, and coarse chromatin (Fig. 6). The calendar management of HGUC and SHGUC involves investigating and identifying the source of suspicious cells using regular cystourethroscopy and imaging studies such as CT urography, MR urography, and ultrasound with retrograde pyelography [15].