Results of the UK NEQAS for Molecular Genetics reference sample analysis

Abstract

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.

Consistency and reproducibility of next‐generation sequencing in cytopathology: A second worldwide ring trial study on improved cytological molecular reference specimens

“…In fact, the median value of AF was 3.30% on slide A, 2.00% on slide B, and 0.30% on slide C. In this setting, however, the second‐look analysis, which entailed a visual inspection of all the slides, revealed a significant difference in the mutation detection between the first and second looks at all the slides (slide A, P = .014; slide B, P = .006; and slide C, P < .001). The challenge of detecting simultaneous mutations occurring in the BRAF gene has recently been highlighted by a study performed on a manufactured human cell line reference sample . Indeed, in this study, only 2 of 53 participating laboratories correctly identified all 8 variants engineered in the BRAF gene .…”

From Somatic Variants Toward Precision Oncology: An Investigation of Reporting Practice for Next-Generation Sequencing-Based Circulating Tumor DNA Analysis

“…Furthermore, many reports have also lost the information of methodological details, particularly assay performance characteristics (such as LOD [52/66, 78.8%] and minimal depth of sequencing coverage [58/66, 87.9%]), and critical quality control (QC) metrics (52/66, 78.8%). It is significant to state that the assay performance characteristics and QC metrics were critical to reliably interpret the results as negative or positive and thus have a direct impact on treatment decisions . However, several reports overinterpreted the absence of a relevant mutation, without providing such information.…”

“…Because circulating tumor DNA (ctDNA) can provide a comprehensive profiling of tumor aberrations, ctDNA analysis is emerging as a valuable tool for the guidance of targeted therapy , the observation of tumor dynamics , and the assessment of resistance to treatment . So far, many different approaches, including the amplification refractory mutation system (ARMS), digital polymerase chain reaction (dPCR), and next‐generation sequencing (NGS), have been applied for the detection of tumor aberrations in ctDNA . Because of the increased affordability, accessibility, high throughput, and unrestricted detection range, NGS‐based ctDNA analysis is now replacing ARMS, dPCR, and other ctDNA analysis approaches stepwise and is being extensively used in the clinical setting .…”

“…So far, many different approaches, including the amplification refractory mutation system (ARMS), digital polymerase chain reaction (dPCR), and next‐generation sequencing (NGS), have been applied for the detection of tumor aberrations in ctDNA . Because of the increased affordability, accessibility, high throughput, and unrestricted detection range, NGS‐based ctDNA analysis is now replacing ARMS, dPCR, and other ctDNA analysis approaches stepwise and is being extensively used in the clinical setting .…”

“…Currently, an increasing number of clinical and commercial laboratories have been performing NGS‐based ctDNA analysis and issuing corresponding clinical reports to provide information to oncologists for the care of their patients . However, because of the use of different NGS panels (commercial NGS panels or laboratory‐developed test panels), target enrichment strategies (targeted capture or multiplex PCR), sequencing platforms, improvement countermeasures, bioinformatics analysis pipelines, and databases (public databases or self‐built databases) by different laboratories, the detected variants and relevant information regarding medication in each report also differ .…”