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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.

BackgroundAdoption of new technology in both basic research and clinical settings requires rigorous validation of analytical performance. The OncoScan® FFPE Assay is a multiplexing tool that offers genome-wide copy number and loss of heterozygosity detection, as well as identification of frequently tested somatic mutations.MethodsIn this study, 162 formalin fixed paraffin embedded samples, representing six different tumour types, were profiled in triplicate across three independent laboratories. OncoScan® formalin fixed paraffin embedded assay data was then analysed for reproducibility of genome-wide copy number, loss of heterozygosity and somatic mutations. Where available, somatic mutation data was compared to data from orthogonal technologies (pyro/sanger sequencing).ResultsCross site comparisons of genome-wide copy number and loss of heterozygosity profiles showed greater than 95% average agreement between sites. Somatic mutations pre-validated by orthogonal technologies showed greater than 90% agreement with OncoScan® somatic mutation calls and somatic mutation concordance between sites averaged 97%.ConclusionsReproducibility of whole-genome copy number, loss of heterozygosity and somatic mutation data using the OncoScan® assay has been demonstrated with comparatively low DNA inputs from a range of highly degraded formalin fixed paraffin embedded samples. In addition, our data shows examples of clinically-relevant aberrations that demonstrate the potential utility of the OncoScan® assay as a robust clinical tool for guiding tumour therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-015-0079-z) contains supplementary material, which is available to authorized users.

BackgroundThe importance of accurate and affordable mutation calling in fixed pathology samples is becoming increasingly important as we move into the era of personalised medicine. The Affymetrix OncoScan® Array platform is designed to produce actionable mutation calls in archival material.MethodsWe compared calls made using the OncoScan platform with calls made using a custom designed PCR panel followed by next-generation sequencing (NGS), in order to benchmark the sensitivity and specificity of the OncoScan calls in a large cohort of fixed tumour samples. 392 fixed, clinical samples were sequenced, encompassing 641 PCR regions, 403 putative positive calls and 1528 putative negative calls.ResultsA small number of mutations could not be validated, either due to large indels or pseudogenes impairing parts of the NGS pipeline. For the remainder, if calls were filtered according to simple quality metrics, both sensitivity and specificity for the OncoScan platform were over 98%. This applied even to samples with poorer sample quality and lower variant allele frequency (5–10%) than product claims indicated.ConclusionsThis benchmarking study will be useful to users and potential users of this platform, who wish to compare technologies or interpret their own results.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-017-0254-5) contains supplementary material, which is available to authorized users.

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Objectives Copy number (CN) and somatic mutation (SM) analysis in tumors is rapidly gaining importance in cancer management as a tool for differential diagnosis, determination of prognosis, and selection of therapeutic. Genome-wide copy number and LOH detection as well as a panel of frequently tested somatic mutations can be detected with OncoScan® FFPE Assay Kit. We report on a validation study of OncoScan FFPE Assay Kit CN and SM data by orthogonal technologies (FISH and NGS, respectively) to estimate the sensitivity and specificity parameters of the platform. In addition, we assessed the reproducibility of the platform across three different sites in the UK: Leeds Institute of Cancer and Pathology (LICP), West Midlands Regional Genetics Laboratory (WMRGL), and Almac Diagnostics (Almac). Methods Validation of CN data was performed on a cohort of cancer samples identified as Her2 positive/ambiguous by FISH. The panel of SMs available was validated by a custom targeted amplicon NGS panel on a diverse collection of samples derived from multiple cohorts across several tumor types sourced from both LICP and WMRGL. For the reproducibility study, 162 samples encompassing six different tumor types (breast, colorectal, lung, melanoma, prostate, and ovarian) were collected from LICP and WMRGL. DNA was extracted and plated in triplicate and distributed to the three testing sites: LICP, WMRGL, and Almac. Data from all sites was analyzed for reproducibility of genome-wide CN/LOH calls and SM calls. Results Cross-site comparisons of genome-wide CN and LOH profiles on 162 FFPE solid tumor samples showed greater than 95% average agreement between three sites (LICP, WMGRL, and Almac), while SM classification concordance between the sites averaged 97%. Initial orthogonal validation of Her2 amplification by FISH showed greater than 90% concordance, as did initial test samples used for validating OncoScan SM calls by a targeted amplicon NGS panel. Conclusion In this study we validated both CN and SM calls using OncoScan FFPE Assay Kit and demonstrated a high degree of agreement with orthogonal methods in all aspects. Reproducibility of whole-genome CN, LOH, and SM data using OncoScan FFPE Assay Kit has also been demonstrated for a range of FFPE samples, including highly degraded samples. This study is a step forward in evaluating the potential clinical utility of a platform combining genome-wide copy number and somatic mutation calls within the national health service of the UK. Citation Format: Joseph M. Foster, Assa Oumie, Fiona S. Togneri, Morag Taylor, Sofia Alyas, Paula Wojtowicz, Henry Wood, Emma Tinkler-Hundal, Katie Southward, Dominic McMullan, Phil Quirke, Katherine E. Keating, Mike Griffiths, Karen G. Spink, Fiona Brew, Eric Fung, Jeanette Schmidt. Cross-site reproducibility and orthogonal validation of copy number and somatic mutation calls of OncoScan® FFPE Assay Kit in solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 626. doi:10.1158/1538-7445.AM2015-626

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