Urothelial carcinoma is the extremely prevalent cancer of the urinary system, accounting for the majority of bladder cancers globally¹. It is a heterogeneous disease characterized by a spectrum ranging from low-grade, non-invasive lesions with relatively favorable outcomes to high-grade, muscle-invasive tumors associated with poor prognosis and high mortality². Accurate assessment of tumor grade and depth of invasion is essential for prognostication and treatment planning³. While histological grading remains the gold standard, its limitations in predicting tumor behavior have led to growing interest in molecular and immunohistochemically biomarkers to aid in early diagnosis and risk stratification⁴.

One such marker is E-cadherin, a calcium-dependent transmembrane glycoprotein that plays a critical role in cell-to-cell adhesion and maintenance of epithelial integrity⁵. It is mostly expressed at the basolateral surface of epithelial cells and is encoded by the CDH1 gene.⁶. In normal tissues, E-cadherin ensures cellular polarity and suppresses tumor invasion by anchoring cells together and inhibiting epithelial-to-mesenchymal transition (EMT)⁷. Loss or reduction of E-cadherin disrupts these adhesive interactions, facilitating tumor cell detachment, migration, and invasion into surrounding tissues⁸. Therefore, decreased E-cadherin expression is considered an early marker of invasiveness and poor differentiation in many carcinomas, including those of the urinary bladder⁹.

In the context of urothelial carcinoma, studies have shown that loss of E-cadherin expression is associated with high histologic grade and greater invasiveness, both of which predict worse clinical outcomes¹⁰. The immunohistochemical evaluation of E-cadherin has thus gained traction as a potentially valuable prognostic tool, particularly in distinguishing aggressive tumors from indolent ones¹¹. Despite these findings, there remains limited local data from Pakistani populations on E-cadherin expression in urothelial carcinoma and its correlation with established histopathological parameters such as tumor grade and depth of invasion¹².

This study addresses the lack of regional data on E-cadherin expression patterns in urothelial carcinoma and aims to evaluate its association with tumor grade and depth of invasion in a Pakistani population.

Study Design and Setting: This cross-sectional study was conducted at the Department of Pathology, Pakistan Institute of Medical Sciences (PIMS), Islamabad. The study duration was 24 months, spanning from August 2018 to August 2020.

Sample Size Calculation: Using OpenEpi software, the sample size was determined with a 95% confidence level, a 10% margin of error, and a 50% expected frequency of Ecadherin expression in urothelial cancer. These criteria determined that 96 instances, which were added one after the other throughout the study period, was the minimal sample size needed.

Sampling Technique: A non-probability consecutive sampling method was employed. All formalin-fixed paraffin-embedded (FFPE) tissue specimens diagnosed as urothelial carcinoma on histopathological examination, obtained through transurethral resection of bladder tumor (TURBT) or cystectomy, were included.

Inclusion and Exclusion Criteria: All cases with adequate tissue and definitive diagnosis of urothelial carcinoma were included in the study. Poorly preserved specimens, biopsies with inadequate tumor tissue, or ambiguous diagnoses were excluded to maintain diagnostic and staining accuracy.

Histological Grading and Invasion Assessment: Tumors were graded as low-grade or high-grade according to the 2016 WHO/ISUP classification system. Depth of invasion was assessed histologically and recorded as invasion into lamina propria, muscularis propria, or no invasion based on hematoxylin and eosin (H&E) stained sections.

Immunohistochemical Staining for E-Cadherin: Immunohistochemical analysis for E-cadherin was performed on 3–4 μm thick tissue sections using a monoclonal E-cadherin antibody. Sections were deparaffinized, rehydrated, and subjected to antigen retrieval. After incubation with the primary antibody and subsequent steps with secondary antibody and chromogen, the slides were counterstained and mounted. Positive and negative controls were included with each staining batch to validate the staining quality.

Interpretation of E-Cadherin Expression: E-cadherin expression was evaluated semi-quantitatively based on membranous staining of tumor cells. Staining was considered preserved if ≥90% of tumor cells showed strong membranous positivity, and reduced if <90% of cells displayed weak or absent staining. Expression was further correlated with tumor grade and depth of invasion.

Data Collection and Statistical Analysis: SPSS version 21 was used for the statistical analysis. Patient age, gender, tumor grade, invasion status, and E-cadherin expression results were all recorded using a structured proforma. Chi-square analysis was utilized to evaluate
relationships between invasion depth or tumor grade and E-cadherin expression. Statistical significance was defined as a p-value of less than 0.05.

A total of 96 cases of urothelial carcinoma were included in this study, with a mean patient age of 64.1 ± 11.1 years and an age range of 36 to 86 years. The majority were male (75 cases; 78.1%) compared to 21 females (21.9%), yielding a male-to-female ratio of 3.6:1. Based on histological grading, 52 cases (54.2%) were classified as high-grade and 44 cases (45.8%) as low-grade. In terms of invasion, 78 cases (81.2%) exhibited lamina propria invasion, while 33 cases (34.4%) demonstrated muscularis propria invasion. As illustrated in Figure 2, Immunohistochemical analysis of E-cadherin expression revealed that 53 cases (55.2%) showed preserved expression, whereas 43 cases (44.8%) displayed reduced expression, defined as weak, patchy, or absent membranous staining in more than 10% of tumor cells. In contrast, preserved expression was considered when strong membranous positivity was observed in 90% or more of the tumor cells. This distribution indicates that nearly half of urothelial carcinoma cases exhibit reduced E-cadherin expression, reflecting altered cell adhesion, which is a recognized hallmark of tumor progression and invasiveness. These baseline findings provided the foundation for subsequent analysis of E-cadherin expression in relation to tumor grade and depth of invasion.

A highly significant association was observed between E-cadherin expression and tumor grade as shown in Table 1, among low-grade tumors, 36 cases (81.8%) maintained preserved expression, while only 8 cases (18.2%) exhibited reduced expression. In contrast, high-grade tumors showed preserved expression in only 17 cases (32.7%), while 35 cases (67.3%) had reduced expression. The difference was statistically significant with a p-value < 0.001, confirming that loss of E-cadherin is strongly linked to higher histological grade. This suggests that E-cadherin can serve as a useful adjunct marker to histologic grading in predicting tumor aggressiveness.

Table 1: Association between E-Cadherin expression and Tumor Grade

There was a statistically significant association between lamina propria invasion and E-cadherin expression (p = 0.041). As shown in Table 2, among the 78 cases with lamina propria invasion, 40 (51.3%) had preserved and 38 (48.7%) had reduced E-cadherin expression. In comparison, 13 out of 18 cases (72.2%) without invasion showed preserved expression, and only 5 (27.8%) had reduced expression. These findings imply that reduced E-cadherin expression is more frequently associated with early tumor invasion, even before w layer involvement. This supports the theory that cell adhesion loss contributes to early infiltration of bladder wall structures.

Table 2: E-Cadherin expression and Lamina Propria invasion

A strong and significant correlation was also found between muscularis propria invasion and E-cadherin expression (p < 0.001). As Illustrated in Figure 2, among 33 cases with muscle invasion, only 9 (27.3%) had preserved E-cadherin expression, while a majority of 24 (72.7%) showed reduced expression. Conversely, in non-invasive tumors, 44 out of 63 cases (69.8%) maintained preserved expression, with only 19 (30.2%) showing reduced expression. These results indicate that loss of E-cadherin is highly predictive of deeper invasion, consistent with its role in promoting tumor cell migration and tissue infiltration. This strengthens its value as a prognostic indicator for invasive behavior.

When tumor grade and invasion depth were considered together, the pattern of E-cadherin expression became even more distinct.  As shown in Table 3, among low-grade, non-invasive tumors, 30 of 35 cases (85.7%) preserved E-cadherin expression. In contrast, high-grade, muscle-invasive tumors showed preserved expression in only 7 out of 33 cases (21.2%), while 26 cases (78.8%) demonstrated reduced expression. The p-value was <0.001, indicating a statistically significant trend. This integrated view highlights the compounding effect of grade and invasion on cell adhesion loss. It confirms that E-cadherin downregulation is a hallmark of aggressive tumor biology in urothelial carcinoma.

Table 3: Combined Effect of Tumor Grade and Invasion on E-Cadherin Expression

In urothelial carcinoma, this study showed a statistically significant correlation between E-cadherin expression and both tumor grade and depth of invasion. Reduced membranous expression of E-cadherin was observed in a majority of high-grade tumors (67.3%) and in tumors with muscularis propria invasion (72.7%). In contrast, most low-grade tumors (81.8%) and non-invasive tumors exhibited preserved E-cadherin expression. The p-values for these associations were all highly significant (<0.001), indicating that loss of E-cadherin is strongly linked with tumor aggressiveness and invasiveness. Additionally, tumors showing both high grade and muscularis propria invasion had the highest frequency of E-cadherin reduction (78.8%), reinforcing its role in advanced tumor behavior.

When compared to global research, the results are consistent with previous findings demonstrating that loss or reduction of E-cadherin expression is commonly associated with poor tumor differentiation, high grade, and increased invasiveness¹³. Similar studies have shown that tumors with reduced E-cadherin tend to lose cellular cohesion, undergo epithelial-mesenchymal transition (EMT), and display a greater ability to invade surrounding tissues¹⁴. Research has also established that E-cadherin is not only a structural protein but also plays an active role in suppressing tumor progression through intracellular signaling pathways¹⁵. Reduced E-cadherin expression has been linked to enhanced tumor migration, lymphovascular invasion, and metastasis, making it a critical molecular event in bladder cancer pathogenesis¹⁶. Furthermore, immunohistochemical evaluation of E-cadherin has been increasingly accepted as a reliable surrogate marker for tumor aggressiveness, especially in cases where morphological grading alone may be insufficient¹⁷.

This study’s findings further support the concept that E-cadherin immunostaining can be used as an adjunct to histological evaluation in urothelial carcinoma. Particularly under-resource settings, where molecular diagnostics may be difficult to obtain, E-cadherin serves as a cost-effective prognostic tool¹⁸. The consistent pattern of E-cadherin loss in high-grade and deeply invasive tumors reinforces its potential role in risk stratification and treatment planning, including decisions regarding early cystectomy or intensified surveillance protocols¹⁹.

Limitations and Future Suggestions: The study's one limitation is that it was only carried out at one location with a small sample size (n=96), which would limit how broadly the findings can be applied. Additionally, no molecular or genetic assays were performed to correlate immunohistochemical findings with CDH1 gene status. Future studies should include multi-center cohorts with larger sample sizes and molecular confirmation of E-cadherin gene mutations or promoter methylation. It is also recommended to explore co-expression profiles of other markers, such as β-catenin, N-cadherin, and vimentin, to better understand the EMT spectrum and its impact on tumor biology in urothelial carcinoma.

According to the study's findings, high-grade urothelial carcinoma and deeper invasion, encompassing both the lamina propria and the muscularis propria, are substantially correlated with decreased E-cadherin expression. The results validate the function of E-cadherin as a useful prognostic immunomarker, helping to predict tumor aggressiveness and invasiveness. Loss of E-cadherin expression reflects disruption in cell-to-cell adhesion and may contribute to epithelial-mesenchymal transition, facilitating tumor progression. Therefore, E-cadherin immunohistochemistry can be a valuable adjunct to conventional histopathological evaluation in guiding clinical management. Such findings imply that the expression of E-cadherin should be a part of the standard diagnostic and prognostic evaluation of urothelial carcinoma.

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