Genomic complexity of urothelial bladder cancer revealed in urinary cfDNA

Article

European Journal of Human Genetics advance online publication 13 January 2016; doi: 10.1038/ejhg.2015.281
Genomic complexity of urothelial bladder cancer revealed in urinary cfDNA
EJHGOpen

Fiona S Togneri1, Douglas G Ward2, Joseph M Foster3, Adam J Devall2, Paula Wojtowicz1, Sofia Alyas1, Fabiana Ramos Vasques1, Assa Oumie3, Nicholas D James4, K K Cheng5, Maurice P Zeegers6, Nayneeta Deshmukh2, Brendan O'Sullivan7, Philippe Taniere7, Karen G Spink3, Dominic J McMullan1, Mike Griffiths1 and Richard T Bryan2

1West Midland Regional Genetics Laboratory, Birmingham Women’s NHS Foundation Trust, Birmingham, UK
2Institute of Cancer & Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
3Affymetrix UK Ltd, High Wycombe, UK
4Cancer Research Unit, University of Warwick, Coventry, UK
5School of Health and Population Sciences, University of Birmingham, Birmingham, UK
6Department of Complex Genetics, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Centre, The Netherlands
7Department of Histopathology, University Hospitals Birmingham NHS Foundation Trust, Birmingham, UK

Abstract

Urothelial bladder cancers (UBCs) have heterogeneous clinical characteristics that are mirrored in their diverse genomic profiles. Genomic profiling of UBCs has the potential to benefit routine clinical practice by providing prognostic utility above and beyond conventional clinicopathological factors, and allowing for prediction and surveillance of treatment responses. Urinary DNAs representative of the tumour genome provide a promising resource as a liquid biopsy for non-invasive genomic profiling of UBCs. We compared the genomic profiles of urinary cellular DNA and cell-free DNA (cfDNA) from the urine with matched diagnostic formalin-fixed paraffin-embedded tumour DNAs for 23 well-characterised UBC patients. Our data show urinary DNAs to be highly representative of patient tumours, allowing for detection of recurrent clinically actionable genomic aberrations. Furthermore, a greater aberrant load (indicative of tumour genome) was observed in cfDNA over cellular DNA (P<0.001), resulting in a higher analytical sensitivity for detection of clinically actionable genomic aberrations (P<0.04) when using cfDNA. Thus, cfDNA extracted from the urine of UBC patients has a higher tumour genome burden and allows greater detection of key genomic biomarkers (90%) than cellular DNA from urine (61%) and provides a promising resource for robust whole-genome tumour profiling of UBC with potential to influence clinical decisions without invasive patient interventions. Introduction

Urothelial bladder cancer (UBC) is the seventh most common cancer in Western societies with a rising global incidence.

1 Disease management poses numerous challenges because of the following:
(i) the propensity for non-muscle-invasive bladder cancer (NMIBC) to recur, necessitating long-term surveillance;
(ii) a variable risk of NMIBC progression, associated with poor 5-year survival;2, 3
(iii) a lack of proven biomarker prognosticators to identify those subsets of patients who will suffer tumour recurrence, progression and death; and (iv) the radical therapies required to treat muscle-invasive disease (MIBC).4 UBCs are thus highly heterogeneous in their clinical characteristics and this is mirrored in their genomics, characteristics of which traverse conventional grade and stage groupings.5

Typically, genomic aberrations in tumours have been characterised using formalin-fixed paraffin-embedded (FFPE) or fresh-frozen tumour tissue, with such analyses elucidating promising biomarkers and suggesting genomic signatures with potential to influence future therapeutic interventions.6, 7, 8 Identifying such genomic complexity in a non-invasive manner could be highly advantageous for facilitating the diagnosis, treatment and surveillance of patients with NMIBC or MIBC.9, 10

Genetic changes in UBCs have previously been investigated non-invasively using genetic material present in the urine. Both genetic material from exfoliated cells (which pellet upon centrifugation) and cell-free DNA (cfDNA; which remains in the supernatant following centrifugation) have been studied. Most studies to date have focused on exfoliated cells, with data giving a specific read out, for example, the presence or absence of UBC.11 Urine tests looking at genomic copy number (CN) include the FISH-based UroVysion test (Abbott, Des Plaines, IL, USA; FDA-approved UBC diagnosis),12 which uses individual exfoliated tumour cells isolated from urine, and the CGH-based BCA-1 test, which uses DNA extracted from these exfoliated cells. BCA-1 has been used to examine more detailed CN data in bladder cancer patients than that provided by UroVysion, and shows some promise.13, 14 Unfortunately, obtaining sufficient cellular material for analysis is not always possible, hindering the clinical applicability of such tests. A small number of studies have therefore also investigated urinary cfDNA for UBC analysis with mixed results, and it has previously been suggested that due to its origin, cfDNA may be enriched for tumour-specific biomarkers with reduced contamination from germline DNA of non-cancerous cells.15

cfDNA in blood plasma, arising through cancer cell death (necrotic or apoptotic cells) and actively released DNA,16, 17 has been well studied as a liquid biopsy for various solid tumours. cfDNA in urine of bladder cancer patients has also been studied in this setting.15 This nucleic acid resource has been proposed to be predominantly necrotic in origin and quantitative changes in necrotic-specific cfDNA levels have been studied to discriminate between cancer and non-cancer patients.18

In this study, we report the utilisation of Affymetrix’s OncoScan FFPE Assay Kit (Affymetrix, Santa Clara, CA, USA) for detailed genomic profiling of UBC using matched FFPE tumour-derived DNA, cellular DNA from urine cell pellets and cfDNA from urine supernatant. We demonstrate that the complex genomics and important clinically actionable aberrations that are evident in FFPE tumour material (currently the predominant diagnostic biospecimen for solid tumours) are echoed in urinary DNAs, and that the tumour genome is enriched in cfDNA compared with cellular DNA. These data illustrate that urinary cfDNA may represent a reliable resource for non-invasive genomic profiling of bladder cancer.