Comparison of urine cytology and bladder tumor antigen test with histopathology in the diagnosis of suspected cases of urinary bladder carcinoma

INTRODUCTION

Urinary bladder (UB) carcinoma has risen globally to the 9^th most diagnosed cancer, with increasing incidence and mortality. It is the sixth most common cancer in men and eighth in women, accounting for 3.1% of all new cases. In India, UB carcinoma constitutes 1.6% of all cancers and is the 17^th most prevalent malignancy.^[1]

Urinary tract tumors range from benign to aggressive lesions influenced by genetic and environmental factors. Most cases occur in individuals over 50, with men affected more frequently than women. Hematuria, either gross or microscopic, is the primary symptom, often accompanied by dysuria in high-grade tumors due to bladder wall involvement.^[2] UB carcinoma is notorious for recurrence and progression, incurring high costs over a patient’s lifetime. While 70% of cases are initially non-invasive, about half recur within 3 years, with some progressing to muscle-invasive disease.^[3]

Etiological factors include smoking, arylamine exposure, Schistosoma haematobium infection, and prolonged analgesic use. Smokers face a 3–4 times higher risk, with smoking accounting for 31% of male and 16% of female bladder cancer deaths. Occupational exposures in industries such as rubber and dye production further contribute to the risk.^[4,5]

Diagnostic approaches include imaging (computed tomography, magnetic resonance imaging, and ultrasonography [USG]), cystoscopy, and transurethral resection of bladder tumors (TURBTs), with histopathology essential for staging and grading. Cystoscopy remains the gold standard for detecting and monitoring bladder cancer.^[5,6]

Urine cytology is a cost-effective, non-invasive tool for UB carcinoma detection, particularly in high-grade cases.^[7] Sensitivity ranges from 25% to 97%, depending on lesion grade and reporting criteria.^[8] Ancillary techniques such as the bladder tumor antigen (BTA) stat test and cytokeratin-20 immunohistochemistry (CK20 IHC) enhance sensitivity, particularly in atypical urothelial cells (AUCs).^[3,9] The Paris System (TPS) for urine cytology standardizes reporting, emphasizing high-grade urothelial carcinoma (HGUC). The 2022 revision, TPS 2.0, refined the criteria for detecting HGUC and introduced the risk of high-grade malignancy.^[10,11]

This study evaluates the diagnostic efficacy of urine cytology and the BTA stat test, comparing their results to histopathological findings to improve the detection of high-grade UB carcinoma.

MATERIALS AND METHODS

This was an observational cross-sectional study, which was conducted in the Departments of Pathology and Urology at a tertiary healthcare center in North India, from April 01, 2023, to June 30, 2024, after receiving approval from the institutional ethical committee. Patients were included if they provided voided urine samples and were suspected to have UB carcinoma based on clinical or imaging findings, or if they were TURBT-treated UB carcinoma patients under surveillance for recurrence. Cases involving upper urinary tract or urethral malignancies, as well as bladder wash or ileal conduit samples, were excluded.

Suspected UB carcinoma cases were identified, and their clinical data were recorded after obtaining informed consent. Fresh midstream urine samples were collected on 3 consecutive days in sterile containers and thereafter processed within 30 min to 1 h. The BTA test was performed using the BTA stat® kit (Polymed Therapeutics Inc., Houston, Texas, USA) by placing untreated urine in a test device, with results read and interpreted as per the manufacturer’s protocol. The kit was procured through local distributors, and as of 2024, the approximate cost per test cassette was INR 300–350 (USD 3.5–4.2), which may vary slightly depending on the supply region and the quantity purchased.

Cytological processing was done by cytocentrifuge (CytoTek, Sakura) to create cell smears, which were fixed using air-drying or ethanol and subsequently stained with Giemsa and Papanicolaou stains, respectively. For cases with AUC, cell blocks were prepared using 50% ethanol and formalin.^[12] A vial half-filled with 50% ethanol was flushed with 12–15 mL of voided urine. The sample was centrifuged at 2000 rpm for 3 min, the supernatant discarded, and the sediment fixed in a mixture of formalin and ethanol (1:9) for 45 min. This was followed by another centrifugation at 2000 rpm for 3 min. The resultant cell button was then submitted for routine histopathological processing.^[12] Immunohistochemistry staining for cytokeratin-20 (CK20) (mAb clone Ks20.8, product no. PM062, Biocare, IntelliPATH) was done on cell block sections. Strong positivity of CK20 in >10% tumor cells in the cell block was considered as diffuse positive, and less intense, focal positivity in <10% tumor cells was considered as focal positive.^[13] The TPS system was used to categorize the cases as non-diagnostic, negative for HGUC (NHGUC), AUC, suspicious for HGUC (SHGUC), HGUC, low grade urothelial carcinoma (LGUC), and others.^[10] TURBT specimens, where available, were analyzed histopathologically according to the World Health Organization 2022 classification of urothelial tumors.^[14]

Data were statistically analyzed using the Statistical Package for the Social Sciences version 28. Sensitivity, specificity, positive predictive value, and negative predictive value of urine cytology and BTA tests were calculated. These parameters were also assessed separately for HGUC. Results with a P < 0.05 were considered statistically significant.

RESULTS

A total of 230 patients suspected of UB carcinoma based on imaging or cystoscopy findings, as well as follow-up cases of TURBT-treated UB carcinoma patients, were included in the study. Their mean age was 56.67 ± 13.58 years, with most cases in the 51–60-year age group (32.6%). Male patients predominated (89.1%). Smoking was the leading risk factor (67.0%), followed by tobacco chewing (35.2%) and alcohol abuse (19.6%), while 24.3% had no identifiable risk factors. Painless gross hematuria was the most common symptom (77.4%), with painful hematuria (23.9%), dysuria (33.9%), and burning micturition (40.4%) also reported. Radiological imaging revealed papillary masses in 182 cases (79.1%), with 86.82% exceeding 5 cm in size. Cystoscopy in 153 patients identified exophytic growths in 80.4% and endophytic patterns in 19.6%, with multifocal lesions predominating (84.42%).

The BTA stat test was positive in 31.74% of cases. Urine cytology was categorized (TPS 2.0) 55.2% as NHGUC, 20% as AUC, 7.3% as SHGUC, and 17.4% as HGUC [Figures 1 and 2]. A strong correlation (P < 0.01) was observed between cytology findings and BTA positivity in high-grade cases [Table 1].

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Figure 1:

(a) In a case of LGUC; few atypical urothelial cells (AUC) (Papanicolaou, 400x). (b) The BTA stat test result was negative. (c) IHC on urine cell block section shows focal positive staining for CK20 (100x). (d) In a case of HGUC, urine cytology smear shows few AUCs with high N:C ratio (>0.5) and hyperchromasia (Papanicolaou, 400x). e: The BTA stat test result was positive. f: IHC shows diffuse positive stain for CK 20 (200x).

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Figure 2:

(a) In a case of HGUC, urine cytology smear shows loose clusters and singly dispersed atypical urothelial cells with high N:C ratio (>0.7), hyperchromatic nuclei and irregular nuclear membrane with scant amount of cytoplasm; high-grade urothelial carcinoma (HGUC) (Papanicolaou, 400x). (b) The BTA stat test result was positive. c: In a case of HGUC, urine cytology smear shows few benign urothelial cells and inflammatory cells; negative for high grade urothelial carcinoma (NGHUC) (Giemsa, 200x). d: The BTA stat test result was positive.

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Among 153 cases with histopathology results, 80.4% were malignant, with 36.6% diagnosed as HGPUC and 43.8% diagnosed as LGPUC. For statistical analysis, cases categorized as AUC, SHGUC, or HGUC on cytology were considered malignant. BTA stat test and urine cytology results were compared with histopathology [Table 1]. Significant correlations were noted between histopathology and both the BTA stat test (P < 0.001) and urine cytology (P < 0.001).

CK20 IHC on 46 AUC cases revealed diffuse expression and focal expression. Cases lacking CK20 expression were tumor-free. However, histopathological correlation was available in only 43 cases. Whereas follow-up was not available for three cases. Out of 43 cases, diffuse expression was observed in 18 cases (41.86%) and focal in 16 cases (37.21%). Negative CK20 expression was found in nine cases (20.93%). It was observed that CK20 IHC in malignant cells with diffuse positive staining was seen in HGUC [Figure 1], and focal staining was noted in LGUC [Figure 1]. CK20 IHC on the cell block for the diagnosis of malignancy was found to be a statistically significant correlation (P < 0.001).

The BTA test showed a sensitivity of 58.53% and specificity of 96.67%, with higher sensitivity (98.21%) for HGUC. Urine cytology had a sensitivity of 68.29% and specificity of 66.67%, with improved sensitivity (89.29%) in HGUC.

DISCUSSION

UB carcinoma is the second most common urological malignancy worldwide, following prostate cancer in males. According to Globocan 2022, the global burden includes approximately 614,298 new cases and 220,596 deaths annually.^[1] The disease predominantly affects men, with a male-to-female ratio of 4:1 globally and 8:1 in this study.^[4]

The mean patient age in our cohort was 56.67 ± 13.58 years, consistent with prior findings.^[4,6] Smoking emerged as the most significant risk factor, observed in 67% of cases, followed by tobacco chewing. Notably, 24.35% of patients had no identifiable risk factors, highlighting the multifactorial etiology of the disease.^[4,15]

Clinically, painless gross hematuria was the most common presenting symptom (77.4%), followed by dysuria and burning micturition. Radiological evaluation identified papillary growths in 80% of cases, most frequently on the bladder’s posterior wall and base. These findings are comparable to the findings of previous studies.^[4,6,15-17]

The BTA stat test demonstrated a sensitivity of 58.53% and specificity of 96.67% for UB carcinoma. In cases of HGUC, sensitivity increased to 98.21%, with an overall diagnostic accuracy of 87.58%. These results are consistent with existing literature, which reports sensitivity ranging from 45% to 85% and specificity from 65% to 85%.^[8,9,17,18] However, the BTA test’s utility is limited by its cost compared to urine cytology.

Urine cytology, standardized through TPS 2.0, remains a valuable diagnostic method for UB carcinoma, particularly for HGUC. In the present study, urine cytology demonstrated a sensitivity of 68.29% and specificity of 66.67%, with sensitivity rising to 89.29% for HGUC, which is similar to existing literature.^[19,20] TPS implementation has improved reporting consistency, reduced the proportion of cases categorized as AUC, and increased HGUC detection rates.^[10,11] Nevertheless, urine cytology is less sensitive for low-grade urothelial carcinoma (LGUC), as observed in several cases misclassified as negative or AUC, or NHGUC.^[11,19]

Histopathological analysis confirmed malignancy in 80.4% of cases, predominantly HGUC and low-grade papillary urothelial carcinoma (LGUC). Interestingly, five cases were negative on urine cytology but positive on BTA stat and confirmed as HGUC histologically. CK20 IHC proved invaluable in AUC cases, especially as an ancillary test to establish malignancy on cytology, which was statistically significant. In addition, a staining pattern may provide further clues for the differentiation of HGUC (diffuse staining) from LGUC (focal staining). Few previous studies have shown on histopathological sections that CK-20 IHC has intense cytoplasmic positivity in high-grade UB carcinomas and less intense positivity in low-grade ones.^[20,21] However, few studies have done a similar evaluation on voided urine specimens, and thus, a multi-observer evaluation and larger sample size are required for validation of the staining pattern.

To the best of our knowledge, the present study is the first in the existing literature to compare urine cytology (reporting as per TPS 2.0) and BTA stat test with histopathology for HGUC. The meager number of existing studies, for the aforesaid comparison, have utilized urine cytology, without the application of TPS or utilizing the older system to compare with the BTA stat test.^[19,22,23] Thus, the lack of uniformity of reporting of urine cytology across studies makes an objective comparison difficult.

Comparison with existing studies revealed variability in diagnostic metrics, influenced by sample size, demographics, and disease spectrum [Table 2]. Dhakhwa et al. reported higher sensitivity (90.48%) for urine cytology in HGUC, comparable to our findings.^[19] This variability underscores the importance of multi-modal diagnostics in high-grade disease.^[20,23] The higher number of high-grade cases in the present study may reflect the tertiary care setting, where referred cases often have advanced disease.

The implementation of TPS 2.0 has enhanced diagnostic precision, particularly for HGUC, by providing clear criteria for cytological interpretation. However, challenges remain in diagnosing LGUC due to overlapping cytological features and inconsistent BTA stat results.^[11] In such cases, CK20 IHC emerged as a pivotal adjunct, especially for ambiguous cytology cases, particularly when focal staining patterns may suggest LGUC, while diffuse patterns may indicate HGUC.^[20]

CONCLUSIONS

The present study underscores the diagnostic value of combining urine cytology (TPS 2.0), CK 20 IHC, and BTA stat tests for diagnosing HGUC in voided urine samples. While the BTA test demonstrated higher sensitivity for HGUC, urine cytology remains a cost-effective method for diagnosis and surveillance of recurrence. The utilization of urine cytology or BTA stat test, or their combination in larger studies and different hospital infrastructure may validate their comparative efficacy as a mass screening tool.