Here we review one of these companion tests, the Roche cobas® EGFR mutation test v2, from a methodological point of view, also exploring its. “The cobas® EGFR Mutation Test v2 is a companion diagnostic test that supports IRESSA® as an additional therapeutic option for patients and. The U.S. Food and Drug Administration (FDA) recently approved the cobas EGFR Mutation Test v2 as a companion diagnostic test with gefitinib.

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Correspondence should be addressed to Kyung-A Lee ; ca. This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Liquid biopsies to genotype the epidermal growth factor receptor EGFR for targeted therapy have been implemented in clinical decision-making in the field of lung cancer, but harmonization of detection methods is still scarce among clinical laboratories.

We performed a pilot external quality assurance EQA scheme to harmonize circulating tumor DNA testing among laboratories. From November to Juneseven clinical diagnostic laboratories participated in the EQA program. Quantitative results from the cobas assay were positively correlated with allele frequencies derived from digital droplet PCR measurements and showed good reproducibility among laboratories.

Careful interpretation is particularly important for p. TM detection in the setting of relapse. Individual laboratories should optimize NGS performance to maximize clinical utility. Circulating tumor DNA ctDNA carries the same molecular alterations as the tumor itself and can be used to select treatment, assess the emergence of drug resistance, and monitor lung cancer patients in routine clinical practice [ 1 ].

The fraction of tumor-derived cell-free DNA cfDNA in blood plasma varies according to tumor stage, tumor burden, vascularization of the tumor, biological features of the tumor such as apoptotic rate, and the metastatic potential of the cancer cells [ 2 ]. Tumor-derived ctDNA often represents a small percentage of the total cfDNA and can be present at allele fractions as low as 0. Therefore, highly sensitive methodologies have been developed to detect low abundance epidermal growth factor receptor EGFR mutations, including p.

A sensitive method is needed to detect the p. TM mutation in relapsed tumors because of tumor heterogeneity [ 6 ]. To ensure optimal quality molecular testing, clinical laboratories should evaluate the technical performance of ctDNA testing according to the standards from formal accreditation bodies, such as Clinical Laboratory Improvement Amendments CLIA and ISO [ 89 ].

External quality assessment EQA is a way to standardize interlaboratory results and to monitor and improve testing processes across laboratories [ 10 ]. The workflow of the study process is shown in Supplementary Figure S1. Details are provided in Supplementary Table S1. TM HDp. LR HDand p. Details are provided in Supplementary Table S2. Torrent Suite software covas molecular coverage depth fgfr well as read coverage depth at target bases to increase detection sensitivity for low-frequency variants [ 1112 ].

Amplification and detection were performed using the rgfr z analyzer Roche Molecular Systems, Inc. Data were interpreted by the cobas z software if positive and negative controls showed valid results.

When a mutation was detected, semiquantitative index SQI values for each mutation are reported automatically by the software using the observed threshold cycle for the target mutation. These test samples had expected mutant allele frequencies of 3. Each laboratory director requested the amount of Cobass material needed according to the number of methods planned for plasma EGFR testing.


The number of reactions per test method among participating laboratories was Submitted qualitative results were evaluated as acceptable muration for expected mutations or negative for unexpected mutations or unacceptable negative for expected mutations or positive for unexpected mutationsaccording to the mutqtion and validated target mutations in this study Table 1 and Supplementary Table S2.

LOD level 4 material, which had an expected mutant allele frequency of 0. For the cobas assay, the mean, standard deviation, coefficient of variation CVmedian value, minimum value, and maximum value of data from the peer group and the standard deviation index of the data from the laboratory were provided in the evaluation reports.

Correlations between SQI from cobas assay and mutant allele frequency were analyzed using Spearman rank-correlation test. All values were two-sided, egdr values less than 0. In a deep sequencing run, all four quality control samples were sequenced with high median coverage depth of more than 86,X.

There was sufficient coverage at all target mutations to detect variants with allele frequencies of 0. All targeted mutations were cbas in levels 1—4 materials at similar allele frequencies to what were expected.

Seven of nine cobas assays All eight measurements of Test 4 material detected the exon 19 deletion. For all mutations, SQI values from the cobas assay exhibited a strong positive correlation with the expected mutant allele frequencies derived from digital droplet PCR measurements Spearman rank-correlation coefficient, 0.

In AprilEQA materials were made and distributed to each laboratory. A month after distributing the EQA materials, all results were emailed from each laboratory to an organizing director.

In Juneevaluation reports were distributed to participating muration. All results obtained using the cobas assay were concordant except for detection of EGFR exon 19 deletion rest p. LR in LOD level 4 material. Among seven laboratories, only six laboratories had a positive result for exon 19 deletion detection rate LR detection rate TM and exon 20 insertion mutations were not detected in LOD level 4 material by any of the laboratories. The precision of SQI is summarized in Table 4.

The cobas assay generally showed good reproducibility with a CV between 1. Mutant allele frequencies were calculated from the submitted depth of coverage data from NGS Table 5.

Results from two laboratories were consistent with the expected mutant allele frequencies calculated from absolute allele frequencies measured using digital droplet PCR. Exon 19 deletion and exon 20 insertion mutations were detected at 0. LR mutations were not detected, despite the fact that total egrr coverage depth was not lower for these loci than other loci 65,X for p.

TM and 70,X for p. TM and exon 20 insertion mutations were not detected in level 3 material unacceptable result. None of the four target mutations were detected in level 4 material. In the present study, we confirmed the LODs of the cobas assay for each target mutation. The analytical sensitivities of the cobas assay were not identical mutatoin the different target mutations, similar to previous reports [ 1415 ]. In our pilot EQA, the cobas assay showed a higher detection rate xobas lower imprecision for exon 19 deletion and p.

LR than for p.

Analytical performance of the cobas EGFR mutation assay for Japanese non-small-cell lung cancer.

TM and exon 20 insertion. LR were not detected, despite adequate depth of coverage of the target site compared to other loci. This difference in assay performance according to target mutation might be due to the assay design and characteristics of the target regions [ 1415 ]. This finding is an important issue for detection of p.

Previous studies reported that it is challenging to detect the p. Therefore, caution is warranted in the setting of tumor relapse, and additional efforts should be made to optimize the experimental conditions to increase the sensitivity of p.


mutattion The cobas assay showed reliable and robust test performance in all laboratories. SQI showed a positive correlation with mutant allele frequency derived from digital droplet PCR measurements. This finding is consistent with that of a previous study that evaluated clinical samples with NGS and the cobas assay [ 13 ].

Therefore, SQI could be useful for patient monitoring. Thus, this assay can be used for rapid and reliable plasma ctDNA analysis in clinical diagnostic laboratories. A limitation of this study is the small number of laboratories that participated, especially laboratories performing NGS. It was unclear whether unacceptable responses were due to the performance of specific NGS methods or the laboratory.

However, coverage depth results from two laboratories indicate that more read coverage depth is required to detect low-frequency variants in samples.

In our validation experiment using the Oncomine Lung cfDNA Assay, all mutations were detected in level 4 material when the coverage depth was more than ,X.

Our data and previous reports indicate that high coverage depth is essential to improve the detection of low-level targets [ 1819 ]. NGS generally requires more time than IVD, although it differs depending on batch yest and muttion platform used. Using NGS, rare e. Tes resistance mutation [ 20 ] or novel mutations in Mtuationas well as other genes, can be identified [ 21 ].

Moreover, advanced NGS technology enables detection of not only cobsa mutation but also gene fusions and amplifications [ 2223 ]. In the era of companion diagnostics, more mutations will be used as predictive markers to determine patient eligibility for molecular-targeted therapies. As a result, rigorous quality controls to avoid inappropriate patient treatment will become increasingly important in clinical diagnostic laboratories. EQA is critical for quality assurance and continuous improvement of individual laboratory performance [ 9 ].

Analytical performance of the cobas EGFR mutation assay for Japanese non-small-cell lung cancer.

Recently, Haselmann et al. The authors suggested that method sensitivity correlates with diagnostic accuracy. TM testing included pyrosequencing, digital PCR, and several allele-specific PCR platforms, using four levels of spiked materials [ 25 ]. Although we used limited number of methods of two NGS and one IVD platform, we suggested more delicate means of EQA workflow tailored to ctDNA testing, using strictly designed low-level materials to assess assay sensitivity and precision in individual laboratories.

Larger trial including more cobbas platforms including digital PCR with our sample preparation protocol is worthy of further investigation. The authors declare that there are no conflicts of interest regarding the publication of this paper. Indexed in Science Citation Index Expanded. Subscribe to Table of Contents Alerts. Table of Contents Alerts. Abstract Liquid mutxtion to genotype the epidermal growth factor receptor EGFR for targeted therapy have been implemented in clinical decision-making in the field of lung cancer, but harmonization rest detection methods is still scarce among clinical laboratories.

Introduction Circulating tumor DNA ctDNA carries the same molecular alterations as the tumor itself and can be used to select treatment, assess the emergence of drug resistance, and monitor lung cancer patients in routine clinical practice [ 1 ].

Materials and Methods 2. Preparation of EQA Materials 2. Copy numbers and frequencies of mutant muattion are provided in Supplementary Table S3.