Validation of a Novel, Sensitive, and Specific Urine-Based Test for Recurrence Surveillance of Patients With Non-Muscle-Invasive Bladder Cancer in a Comprehensive Multicenter Study.

Rui Batısta ,Ainara Villafruela,Thorsten Ecke,Mario Pual Sanchez Perez,Hakan Öztürk,Valdemar Máximo,Fábio Almeida,Tomé Lopes,Amílcar Sismeiro,Rabehi Abdelmalek,Santiago Moreno Perez De La Cruz,Paulo Conceição,Nuno Fidalgo,Pedro Peralta,Cristina Sampaio,Ricardo Leão,Pascal Stinjs,Pedro Eufrásio,Edwin Romero Gonzalez,João Vinagre,Hugo Prazeres,Carlos Oliveira,Paula Soares,João Pimentel Torres ,M Álvarez Maestro ,Álvaro Gomes ,J.i Monzó ,C González-Enguita ,F Furriel ,B Yu Bidovanets ,P Beardo-Villar ,Alfonso Luis Calle Pascual ,P Azinhais ,P Parra Serván

doi:10.3389/fgene.2019.01237

Abstract

Bladder cancer (BC), the most frequent malignancy of the urinary system, is ranked the sixth most prevalent cancer worldwide. Of all newly diagnosed patients with BC, 70–75% will present disease confined to the mucosa or submucosa, the non-muscle-invasive BC (NMIBC) subtype. Of those, approximately 70% will recur after transurethral resection (TUR). Due to high rate of recurrence, patients are submitted to an intensive follow-up program maintained throughout many years, or even throughout life, resulting in an expensive follow-up, with cystoscopy being the most cost-effective procedure for NMIBC screening. Currently, the gold standard procedure for detection and follow-up of NMIBC is based on the association of cystoscopy and urine cytology. As cystoscopy is a very invasive approach, over the years, many different noninvasive assays (both based in serum and urine samples) have been developed in order to search genetic and protein alterations related to the development, progression, and recurrence of BC. TERT promoter mutations and FGFR3 hotspot mutations are the most frequent somatic alterations in BC and constitute the most reliable biomarkers for BC. Based on these, we developed an ultra-sensitive, urine-based assay called Uromonitor®, capable of detecting trace amounts of TERT promoter (c.1-124C > T and c.1-146C > T) and FGFR3 (p.R248C and p.S249C) hotspot mutations, in tumor cells exfoliated to urine samples. Cells present in urine were concentrated by the filtration of urine through filters where tumor cells are trapped and stored until analysis, presenting long-term stability. Detection of the alterations was achieved through a custom-made, robust, and highly sensitive multiplex competitive allele-specific discrimination PCR allowing clear interpretation of results. In this study, we validate a test for NMIBC recurrence detection, using for technical validation a total of 331 urine samples and 41 formalin-fixed paraffin-embedded tissues of the primary tumor and recurrence lesions from a large cluster of urology centers. In the clinical validation, we used 185 samples to assess sensitivity/specificity in the detection of NMIBC recurrence vs. cystoscopy/cytology and in a smaller cohort its potential as a primary diagnostic tool for NMIBC. Our results show this test to be highly sensitive (73.5%) and specific (93.2%) in detecting recurrence of BC in patients under surveillance of NMIBC.

Highlights

Bladder cancer is the most frequent malignancy involving the urinary system and affects approximately four times more males than females (Miyazaki and Nishiyama, 2017)
We present preliminary results on the highsensitivity screening of KRAS codon 12 and codon 61 alterations achieved through the use of a custom-made mutation detection procedure developed to fibroblast growth factor receptor 3 (FGFR3) hotspot mutation detection procedure, rendering this method suitable for detection of mutations in bladder cancer tumor cells exfoliated to urine
telomerase reverse transcriptase (TERT) promoter mutations were firstly described in sporadic and familial melanoma (Horn et al, 2013; Huang et al, 2013), and since they were reported in several cancers, such as central nervous system (43–51%), hepatocellular carcinoma (59%), thyroid (10%), and notably in bladder cancer (59–80%) (Killela et al, 2013; Liu et al, 2013a; Liu et al, 2013b; Nault et al, 2013; Vinagre et al, 2013; Wu et al, 2014)

Summary

Introduction

Bladder cancer is the most frequent malignancy involving the urinary system and affects approximately four times more males than females (Miyazaki and Nishiyama, 2017). TERT promoter (TERTp) mutations are the most common event across stages and grades in malignant bladder tumors, strongly suggesting its participation in the two major genetic pathways of urothelial tumorigenesis (Allory et al, 2014; Hurst et al, 2014) These features point TERTp mutations as a game changer in bladder cancer and pointed them to be considered as a useful urinary biomarker for disease monitoring and early detection of recurrence, even in low-grade NMIBC, where urinary cytology usually lacks sensitivity (Allory et al, 2014; Hurst et al, 2014; Vinagre et al, 2014; Descotes et al, 2017). KRAS mutations, found in a lower percentage (11.5%) of bladder cancers, are assuming a relevant position since the detection of KRAS mutations in conjunction with the previous alterations could improve the sensitivity of a biomarker panel (Alexander et al, 2012)

Objectives

Methods

Results

Conclusion