A Simple Immunohistochemical Panel Could Predict and Correlate to Clinicopathologic and Molecular Subgroups of Urinary Bladder Urothelial Carcinoma

Abstract

Background

Although gene expression profiling provided a comprehensive molecular characterization of different subtypes of bladder urothelial carcinoma (UC), which are distinct in their biological features and prognosis, such a system is not yet applicable for routine clinical practice. This study aimed to examine the expression of these molecular classes of UC using simple panel of immunohistochemical markers.

Materials and Methods

Tissue sections from 192 specimens of UC were stained with FGFR3, CK5, CCNB1, HER-2, and P53. The molecular classes identified were correlated with clinicopathologic characteristics and patient survival.

Results

The most frequent class in our cohort was urobasal B (UroB) (44.1%), followed by squamous cell carcinoma-like (SCCL) (22%), genomically unstable (GU) (20.3%), and urobasal A (UroA) (13.6%). Patients with SCCL were significantly younger (P < .0001). Both the SCCL and GU types were of significantly higher histopathologic grade (P < .0001). UroA tumors were mainly of the T1 stage (75%), whereas 61.5% of the SCCL and 58.3% of the GU types were of stage T2 (P < .001). Prognosis was significantly different among groups. The SCCL class showed the lowest overall survival (38.5%; P = .030) and metastasis-free survival (69.2%; P = .017). The best prognosis was for UroA, with an overall survival of 75% and no metastatic events.

Conclusion

The distribution of UC subtypes in our study was uniquely different from other studies. This simple immunohistochemical panel could be suggested as a clinically applicable tool that has the potential to be used routinely in guiding individualized treatment of UC.

Introduction

Bladder cancer is the ninth most common malignancy worldwide,1, 2 with a high rate of recurrence and progression, resulting in a significant burden on health care systems.³

On the therapeutic front, the management of non–muscle-invasive (NMIBC) and muscle-invasive tumors (MIBC) remained unchanged for decades. However, the lack of significant improvement in patient survival indicates the need for the identification of new features that can predict the prognosis and biological behavior of the tumor.⁴

Global analysis of mRNA expression has emerged as an efficient tool for classification of tumors, according to the gene expression profile, into classes distinct in their biological characteristics and prognosis.⁵ The application of such a modality on breast cancer translated into more accurate prognostic and therapeutic stratification of patients.⁶ Similar research on bladder cancer has been started, with promising results obtained. Initial gene expression profiling experiments on urothelial carcinoma (UC) identified 4 prognostically distinctive classes (Lund classification); urobasal A (UroA), genomically unstable (GU), urobasal B (UroB), and squamous cell carcinoma-like (SCCL).⁷ Distinct gene changes were found in each class irrespective of the pathologic stage or grade.

This initial classification that was based on gene expression was later on translated into immunohistochemical classification using markers that can identify such classes based on protein expression.⁸ UroA tumors show positive expression of keratin 5 (CK5), P-cadherin (P-Cad), and epidermal growth factor receptor (EGFR) expression confined to basal cells, and cell cycle activity (CCNB1) restricted to the tumor-stroma interface. In contrast, the SCCL subtype uniformly expresses CK5, P-Cad, EGFR, CK14, and cell cycle genes throughout the tumor parenchyma. Unlike the first 2 classes, the GU shows absence of CK5, P-Cad, and EGFR expression but shows high expression of ERBB2 (HER-2) and E-Cad with high tumor cell proliferation. UroB tumors showed features between the UroA and SCCL subtypes.⁸

Transcriptomic analyses have yielded gene expression profiles that were translated into distinct molecular subtypes by different research groups. A common theme in different classifications is the emergence of luminal and basal subtypes. Luminal UC was found to be matching the UroA class described earlier. Basal class (matching the SCCL class in the Sjodhal study⁸), was characterized by expression of basal cytokeratines and squamous differentiation. The discovery of other classifiers and clusters includes: claudin-low cluster in the updated University of North Carolina (UNC) taxonomy; luminal papillary, luminal infiltrated, and neuronal subtypes in the new The Cancer Genome Atlas (TCGA) classification; the P53-like and double-negative subtypes in the MD Anderson (MDA) classification; and the extended new taxonomy of Lund University.⁹

Aine et al compared the different types of classes among classification systems and concluded that there is extensive overlap among classes in different systems and that the Lund classification has the highest resolution.¹⁰ Recently, the Lund taxonomy was extended to include 6 categories instead of 4. It included 2 minor new subtypes: SCCL/mesenchymal infiltrated (SCCL/Mes-inf) and small cell/neuroendocrine-like (Sc/NE). By immunohistochemistry (IHC), the SCCL/Mes-inf type is negative for basal cell-related and SCC-related markers but is positive for ZEB2 and VIM, even though they belong to the same global mRNA tumors of SCCL. Moreover, only one-half of the cases designed as the Sc/NE type showed expression of neuroendocrine markers, and all cases shared features with the GU subtype, so they suggested that both the Sc/NE and GU tumors should be in one consensus cluster.¹¹

It is known that genomic studies to classify patients are complex, costly, and unsuitable for clinical application.¹¹

The aims of the present study were to: (1) Evaluate the distribution pattern of the identified 4 main molecular classes of UC according to the Lund classification (UroA, UroB, SCCL, and GU) in patients with UC using a simple validated panel of immunohistochemical markers; and (2) Assess the association between these classes and the clinicopathologic characteristics of tumors and patient survival.