Molecular Classification of Bladder Urothelial Carcinoma Using NanoString-Based Gene Expression Analysis.

Antonio Lopez-Beltran,Liang Cheng,Ana Blanca,Rajan Gogna,Rodolfo Montironi,Alessia Cimadamore

doi:10.3390/cancers13215500

Abstract

Simple SummaryOur study aimed to apply a quantitative method based on mRNA counting as nCounter (NanoString Technologies, Inc). This method can obtain precise and accurate measures of RNA expression compared to RT-PCR, and which might represent an alternative to the NGS-genomic/transcriptomic profiling frequently used to generate molecular data in bladder cancer and provide clinically meaningful datasets for the molecular classification of bladder cancer. The current study generated a four-gene classifier, incorporating GATA3 and KRT20 (typically related to luminal molecular subtype) and KRT5 and KRT14 (typically related to basal molecular subtype). This methodology allowed us to explore differences in clinicopathologic parameters and potential sensitivities to ICI immunotherapy in a cohort series of 91 urothelial carcinomas of the bladder.Molecular classification of bladder carcinoma is a relevant topic in modern bladder cancer oncology due to its potential to improve oncological outcomes. The available molecular classifications are generally based on transcriptomic profiles, generating highly diverse categories with limited correlation. Implementation of molecular classification in practice is typically limited due to the high complexity of the required technology, the elevated costs, and the limited availability of this technology worldwide. We have conducted a gene expression analysis using a four-gene panel related to luminal and basal subtypes in a series of 91 bladder cancer cases. NanoString-based gene expression analysis using typically luminal (GATA3+/KRT20+) and basal markers (KRT14+/KRT5+/GATA3low/-/KRT20low/-) classified urothelial bladder carcinoma samples as luminal, basal, and a third category (KRT14-/KRT5-/GATA3-/KRT20-), null/double negative (non-luminal/non-basal). These three categories were meaningful in terms of overall cancer-specific survival (p < 0.0001) or when classified as conventional urothelial carcinoma and variant histology urothelial carcinoma (p < 0.0001), NMIBC vs. MIBC (p < 0.001), or by AJCC stage category Ta (p = 0.0012) and T1 (p < 0.0001) but did not reach significance in T2-T4 (p = 0.563). PD-L1 expression (low vs. high) was also different according to molecular subtype, with high PD-L1 expression mostly seen in basal and null subtypes and carcinomas with variant histology (p = 0.002). Additionally, the luminal subtype was enriched in NMIBC with favorable cancer-specific survival (p < 0.0001). In contrast, basal and null subtypes resulted in aggressive MIBC tumors with shorter cancer-specific survival (p < 0.0001), some of which presented variant histology. In conclusion, a comprehensive evaluation of a gene classifier related to molecular taxonomy using NanoString technology is feasible. Therefore, it might represent an accessible and affordable tool in this rapidly expanding area of precision genomics.

Highlights

Bladder carcinoma has been traditionally classified as non–muscle-invasive bladder cancer (NMIBC), including Ta, T1, and urothelial carcinoma in situ, and muscle-invasive bladder cancer (MIBC), including T2-T4 disease [1,2,3]
Patient follow-up and survival status ranged from 2–125 months and 8–125 months, respectively, and cancer specific survival (CSS) ranged from 2–71 months (Figure 2)
Following the major subtypes observed in breast carcinoma, these two categories were recognized in urothelial carcinomas: luminal and basal molecular subtypes [13,28]

Summary

Introduction

Bladder carcinoma has been traditionally classified as non–muscle-invasive bladder cancer (NMIBC), including Ta, T1, and urothelial carcinoma in situ, and muscle-invasive bladder cancer (MIBC), including T2-T4 disease [1,2,3]. In an attempt to better define urothelial carcinoma, molecular classification of these tumors might provide meaningful information to stratify prognostically relevant categories or to define the proper treatment in a given patient [13,20]. In this clinical scenario, the development of the molecular taxonomy of bladder cancer probably represents the most fascinating and important novelty in decades. These two wide categories present differences in prognosis and sensitivity to the current therapies, with the basal subtype being more aggressive than luminal [22,25,27,29,30]

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Discussion

Conclusion