Noninvasive monitoring of urothelial carcinoma recurrence and progression using somatic mutations in cell-free tumor DNA from urine

At least half of the patients diagnosed with non–muscle-invasive bladder cancer (NMIBC) experience recurrence and approximately 15% will develop progression to muscle invasive or metastatic disease. Major clinical challenges are to identify patients at risk for recurrence, progression, and metastasis, to make timely clinical decision and to monitor treatment response. Therefore, biomarkers for disease surveillance are urgently needed.

The circulating cell-free tumour DNA (ctDNA) contains tumour-specific genomic variants that may be used as unique genetic signatures (biomarkers). Liquid biopsy specimens have already been used to detect specific cancer mutations in ctDNA.  Detection of mutations in ctDNA from the plasma of a standard blood draw may indicate the type of tumor present, thus reducing the need for an invasive tissue biopsy specimen. Longitudinal, quantitative monitoring of cancer mutations in ctDNA provides information about tumor burden for an individual patient during treatment [1]. Cell-free urine contains highly fragmented DNA derived from urothelial cells, inflammatory cells, or DNA fragments cleared from circulation. For bladder cancer (BC) surveillance urine appears to be a more attractive source of genetic material than other biological liquids. Indeed, urine samples are more available and in preliminary studies quantity and quality of ctDNA isolated from urine was better as compared with plasma-derived ctDNA [2].

Recent comprehensive analysis of molecular genetic landscape of urothelial carcinoma pointed to several potential candidates for diagnostic and prognostic biomarkers [3-6]. Based on this knowledge mutational analysis of ctDNA obtained from liquid biopsies (including urine) have been applied by several research groups [2, 7-10].

We chose 2 biomarkers for primary analysis of urine from BC patients. Fibroblast growth factor receptor 3 (FGFR3) that is the highly mutated gene in BC, with an overall frequency of 60%. FGFR3 mutations are associated with stage and grade and are very common in pTa and grade 1/2 tumors. This makes FGFR3 mutation analysis in voided urine a useful tool for diagnosing recurrent disease after a primary non–muscle-invasive bladder cancer (NMIBC) [10]. And Telomerase Reverse Transcriptase (TERT) that is also highly mutated in  in both non–muscle-invasive and muscle-invasive bladder cancers suggests a requirement in all pathways of urothelial tumorigenesis [11]. These biomarkers (mutations FGFR3 and TERT have already been tested) for the ability to predict recurrence or progression of NMIBC [7, 12, 13]. However, suggested herein approach provide comprehensive prospective analysis with highly sensitive, precise and clinically applicable read out assay. This approach may allow noninvasive disease surveillance in patients with NMIBC, using highly sensitive and tumour-specific personalized urine-based assays. Based on these considerations we plan to use digital PCR technique to detect somatic mutations of tumor-derived cell free DNA in urine to discovery the correlation of amount of mutated DNA to disease status.

 In our preliminary studies we confirmed that: I.  dPCR approach allows quantitatively with very high sensitivity to detect genomic DNA with known concentrations. II. This procedure can be used for detection ctDNA in urine.

Based on these results we plan to perform mutational analysis of urine samples from patient diagnosed for BC.

The present study points to several clinical perspectives for the future use of personalized genomic biomarkers. Early detection of tumor recurrence, progression and identification of disease-free patients may improve disease surveillance and patient outcome.


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