AimsIn addition to providing external quality assessment (EQA) schemes, United Kingdom National External Quality Assessment service (UK NEQAS) for Molecular Genetics also supports the education of laboratories. As an enhancement to the Molecular Pathology EQA scheme, a human cell-line reference sample, manufactured by Thermo Fisher Scientific (AcroMetrix), was provided for analysis. This contained many variants, present at frequencies between 1% and 17.9%.MethodsOne hundred and one laboratories submitted results, with a total of 2889 test results on 53 genes being reported. Known polymorphisms, 46/2889 (1.59%) results, were excluded. Variants detected in the seven most commonly reported (and clinically relevant) genes, KRAS, NRAS, BRAF, EGFR, PIK3CA, KIT and PDGFRA, are reported here, as these genes fall within the scope of UK NEQAS EQA schemes.ResultsNext generation sequencing (NGS) was the most commonly performed testing platform. There were between 5 and 27 validated variants in the seven genes reported here. Eight laboratories correctly reported all five NRAS variants, and two correctly reported all eight BRAF variants. The validated mean variant frequency was lower than that determined by participating laboratories, with single-gene testing methodologies showing less variation in estimated frequencies than NGS platforms. Laboratories were more likely to correctly identify clinically relevant variants.ConclusionsOver 100 laboratories took the opportunity to test the ‘educational reference sample’, showing a willingness to further validate their testing platforms. While it was encouraging to see that the most widely reported variants were those which should be included in routine testing panels, reporting of variants was potentially open to interpretation, thus clarity is still required on whether laboratories selectively reported variants, by either clinical relevance or variant frequency.

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Introduction: Application of targeted NGS assays to comprehensively screen cancer genes has highlighted the need for a well-characterized control to ensure consistency in variant calls. CLIA and CAP guidelines mandate that labs utilize positive run controls which are currently limited to known patient samples or cell lines containing 10's of mutations. These materials do not cover the breadth of potential mutations that could be observed in current highly mutliplexed NGS tests. Materials and Methods: AcroMetrix® Oncology Hotspot Control is a DNA control containing more than 500 COSMIC (Catalogue of Somatic Mutations in Cancer) variants in 53 genes. Covering SNP and indels, the control is designed to function on many platforms and technologies. Formulated as a mixture of synthetic and genomic DNA, the control mimics the complexity of patient samples. Variants were confirmed by Sanger sequencing and quantified by digital PCR. To benchmark product performance, two lots of the controЀ were tested in 19 labs worldwide. Testing was performed using the Ion AmpliSeq™ Cancer Hotspot Panel v2 (CHPv2) on the Ion Torrent Personal Genome Machine® as well as with the Illumina® TruSeq® Amplicon Cancer Panel (TruSeq) and TruSight® Tumor Panel (TruSight) on the MiSeq® instrument. Each lab utilized an established assay and their in house bioinformatics pipeline. Results: In internal tests, good concordance was observed between expected and detected SNP's with greater than 90% being detected on the CHPv2 (382/385), TruSeq® Amplicon Cancer Panel (TSCAP) (319/351) and TruSight® Tumor Panel (TSTP) (209/230). For deletions, detection varied from 66.7%(12/18) for CHPv2 and TruSight® to 76%(16/21) for TruSeq® tests. For insertions, detection was 70%(7/10) for TruSight® and 54% each for CHPv2(6/11) and TruSeq® (7/13). Labs varied in their ability to detect variants; while some labs, running the TSTP, detected over 87% (241/277) of total variants, others detected less than half of expected variants (122/277). Similar variations in detections rates were observed in TSCAP (38%-86%) and CHPv2 (40%-90%). Refining the bioinformatics in variant calling allowed one lab to improve their variant detection by 130% (157 variants detected prior and 410 variants after improvement). Conclusions: AcroMetrix® Oncology Hotspot Control provides a wide variety of variants, including many that are difficult to source. This control will enable laboratories to monitor variation between assay lots and operators, and will facilitate result comparison across laboratories. The control also ensures that bioinformatics pipelines are performing acceptably over time and through updates to ensure reliable variant calling. The availability of controls that target clinically-relevant mutations will significantly improve NGS adoption rates and confidence in NGS results. AcroMetrix® Oncology Hotspot Control is For In Vitro Diagnostic Use. Citation Format: Nakul Nataraj, Mona D. Shahbazian, Aron Lau, Mahjabeen Yucekul, Patty Chiang, Suzy Van Le, Kara Norman. A highly multiplexed control material to satisfy the complex needs of targeted next generation sequencing (NGS) oncology assays. [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 4900. doi:10.1158/1538-7445.AM2015-4900