Evolution of RAS Testing Over Time: Factors Influencing Mutation Rates in Metastatic Colorectal Cancer Patients

Abstract

Aim: Correct identification of RAS gene variants is key for targeted treatment decisions in patients with metastatic colorectal cancer. Published RAS mutation rates differ and could be influenced by several factors including testing methods. This study aimed to describe the performance of laboratories to correctly identify RAS variants over time and to understand how RAS testing has evolved in Europe. Materials & methods: Misclassification and test failure rates were calculated and related to the used test methodology for 239 unique laboratories participating in external quality assessment for metastatic colorectal cancer between 2013 and 2018. In addition, 33 laboratories completed a survey aiming to obtain more details on their routine testing strategies, number of samples analyzed and RAS mutation rates between 2013 and 2017. Results: The mutation status was correctly analyzed in 96.1% (N = 5471) RAS and BRAF tests. A total of 4.6% (N = 2860) RAS tests included false-negative results. In 1.6% (N = 5562) RAS and BRAF tests, an analysis failure occurred. Misclassifications and technical failures both decreased between 2013 and 2018. The number of next-generation sequencing users increased from 6.9% (N = 130) in 2013 to 44.6% (N = 112) in 2018. Over time, more codons were included in the methodologies, yet 23.2% (N = 112) did not offer full RAS testing (exon 2, 3, 4) in 2018. Based on the survey the overall RAS mutation rate was estimated as 45.2% (N = 27,325). Conclusion: This is the largest observational study reporting RAS mutation rates to-date. There was no trend of RAS mutation rates over time despite having a clear shift to more sensitive tests and increased quality of testing.

Implementation of Novel Methods, Markers or Sample types in Molecular Pathology – Quality Assurance Aspects

“…Laboratories reported to frequently participate to EQA schemes to validate their methods, and train their personnel to perform the actions associated with validation or verification. Frequent participation to EQA as well as training in the form of workshops have previously been reported to exert a beneficial effect on test performance [28,29,31,47].…”

“…to the occurrence of resistance mechanisms upon progression to TKIs. Second, recent analysis in mCRC stressed that not all laboratories have well implemented rare RAS variants in exons 3 and 4, resulting in higher error rates [30,31]. Third, the analysis of underlying causes of incorrect tests revealed that laboratories mentioned insufficient method sensitivities or omission of rare variants in their kit to be at basis of the false negative results [32].…”

“…For instance, even though several commercial kits for mutation analysis have the advantage that they include a positive and negative control, they only report the test outcome and users have limited insight into troubleshooting without contacting the manufacturer (Table 4). Over time however, EQA schemes have reported on an increased use of NGS [28,29,31]. In the meantime, NGS-specific EQAs have been organized and guidelines for NGS implementation were established [22-24, 50, 51].…”

“…Participation to external quality assessment (EQA) programs can further aid laboratories in validating their test methods, by monitoring and comparing their performances to international peers and receiving individual feedback [10]. EQA schemes organized by the European Society of Pathology (ESP) have revealed increased error rates upon the implementation of novel markers, both for NSCLC and mCRC [28][29][30][31]. Error rates also varied between the technique type used, and were higher for IHC compared to FISH [28], but lower for next-generation sequencing (NGS) based techniques compared to other mutation detection assays [28,29,31].…”

“…EQA schemes organized by the European Society of Pathology (ESP) have revealed increased error rates upon the implementation of novel markers, both for NSCLC and mCRC [28][29][30][31]. Error rates also varied between the technique type used, and were higher for IHC compared to FISH [28], but lower for next-generation sequencing (NGS) based techniques compared to other mutation detection assays [28,29,31]. More errors were also reported for methods that do not include all required variants for testing by the FDA and EMA, stressing the importance of careful method validation [30].…”