MMR assays identify patients with a low risk of recurrence. KRAS mutational analysis provides useful additional risk stratification to guide use of chemotherapy.

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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.

Simple digital morphometry applied to a single representative H&E section identifies CRC patients with a >50% higher risk of disease recurrence. This technique can reliably partition patients into subpopulations with different risks of tumour recurrence in a simple and cost-effective manner. Further prospective validation is warranted.

Background:The influence of EGFR pathway mutations on cetuximab-containing rectal cancer preoperative chemoradiation (CRT) is uncertain.Methods:In a prospective phase II trial (EXCITE), patients with magnetic resonance imaging (MRI)-defined non-metastatic rectal adenocarinoma threatening/involving the surgical resection plane received pelvic radiotherapy with concurrent capecitabine, irinotecan and cetuximab. Resection was recommended 8 weeks later. The primary endpoint was histopathologically clear (R0) resection margin. Pre-planned retrospective DNA pyrosequencing (PS) and next generation sequencing (NGS) of KRAS, NRAS, PIK3CA and BRAF was performed on the pre-treatment biopsy and resected specimen.Results:Eighty-two patients were recruited and 76 underwent surgery, with R0 resection in 67 (82%, 90%CI: 73–88%) (four patients with clinical complete response declined surgery). Twenty–four patients (30%) had an excellent clinical or pathological response (ECPR). Using NGS 24 (46%) of 52 matched biopsies/resections were discrepant: ten patients (19%) gained 13 new resection mutations compared to biopsy (12 KRAS, one PIK3CA) and 18 (35%) lost 22 mutations (15 KRAS, 7 PIK3CA). Tumours only ever testing RAS wild-type had significantly greater ECPR than tumours with either biopsy or resection RAS mutations (14/29 [48%] vs 10/51 [20%], P=0.008), with a trend towards increased overall survival (HR 0.23, 95% CI 0.05–1.03, P=0.055).Conclusions:This regimen was feasible and the primary study endpoint was met. For the first time using pre-operative rectal CRT, emergence of clinically important new resection mutations is described, likely reflecting intratumoural heterogeneity manifesting either as treatment-driven selective clonal expansion or a geographical biopsy sampling miss.

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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