EGFR‑TK mutation testing in adults with locally advanced or metastatic non-small-cell lung cancer

Therascreen EGFR RGQ PCR Kit

4.1 The therascreen EGFR RGQ PCR Kit (Qiagen) is a CE‑marked real-time polymerase chain reaction (PCR) assay for the targeted detection of 29 mutations in exons 18 to 21 of the epidermal growth factor receptor tyrosine kinase (EGFR‑TK) gene:

4.2 To ensure it complies with the CE marking, the DNA is first isolated from a specimen of formalin-fixed paraffin-embedded tissue using the QIAamp DNA FFPE Tissue Kit. The total amount of DNA in the sample is assessed by a control assay. The therascreen EGFR RGQ PCR Kit then uses 2 technologies for detecting mutations: ARMS (amplification-refractory mutation system) for mutation-specific DNA amplification; and Scorpions for detecting amplified regions. Scorpions are bi-functional molecules containing a PCR primer covalently linked to a fluorescently labelled probe. A real-time PCR instrument (Rotor‑Gene Q 5‑Plex HRM Platform for consistency with CE marking) is used to perform the amplification and to measure fluorescence.

4.3 The limits of detection (the per cent mutant DNA present in a background of wild-type DNA, at which 95% or more replicates were determined positive) reported by the manufacturer for the different mutations ranged from 0.5% to 7.0%.

4.4 An older version of the test exists (the therascreen EGFR PCR Kit), which was inherited by Qiagen when they acquired DxS Ltd. This older version uses the same methods as the newer therascreen EGFR RGQ PCR Kit, and detects 28 of the same mutations, but is not designed to detect the resistance mutation T790M. The limit of detection claimed by the manufacturer for the therascreen EGFR PCR Kit is 1% mutant DNA in a background of wild-type DNA. This version is no longer being actively marketed by Qiagen, was not used in any of the studies included in this review and has been superseded by the therascreen EGFR RGQ PCR Kit. Further, an earlier version of the therascreen EGFR PCR Kit, which did include an assay for T790M, was used to analyse all samples in the IPASS trial. This version is no longer available, but is considered equivalent to the therascreen EGFR RGQ PCR Kit for the purpose of this assessment.

Cobas EGFR Mutation Test

4.5 The cobas EGFR Mutation Test (Roche Molecular Systems) is a CE‑marked real-time PCR test for the targeted detection of 41 mutations in exons 18 to 21 of the EGFR‑TK gene:

4.6 The tumour tissue is first processed using the cobas DNA Sample Preparation Kit. The second step is PCR amplification and detection of EGFR‑TK mutations using complementary primer pairs and fluorescently labelled probes. The PCR is run using the cobas z 480 Analyzer, which automates amplification and detection. Cobas 4800 software provides automated test result reporting.

4.7 The limits of detection (lowest amount of DNA [nanogram] per reaction well to achieve a 95% or higher 'mutation detected' rate), as reported by the manufacturer for the different mutations, ranged from 0.78 nanograms to 3.13 nanograms of DNA per well.

Sanger sequencing of samples with more than 30% tumour cells and therascreen EGFR RGQ PCR Kit for samples with lower tumour cell contents

4.8 In this test strategy, Sanger sequencing of exons 18 to 21 (described in section 4.19) is used to detect EGFR‑TK mutations in test samples with more than 30% tumour cells, and the therascreen EGFR RGQ PCR Kit (described in sections 4.1 to 4.4) is used to detect EGFR‑TK mutations in samples with less than 30% tumour cells.

Sanger sequencing of samples with more than 30% tumour cells and cobas EGFR Mutation Test for samples with lower tumour cell content

4.9 In this test strategy, Sanger sequencing of exons 18 to 21 (described in section 4.19) is used to detect EGFR‑TK mutations in test samples with more than 30% tumour cells, and the cobas EGFR Mutation Test (described in sections 4.5 to 4.7) is used to detect EGFR‑TK mutations in samples with less than 30% tumour cells.

Sanger sequencing followed by fragment length analysis and PCR of negative samples

4.10 Sanger sequencing of exons 18 to 21 is used as an initial test to screen for mutations. Fragment length analysis to detect exon 19 deletions and real-time PCR to detect the exon 21 mutation L858R are then used on samples that produce a negative result using Sanger sequencing.

Pyrosequencing and fragment length analysis

4.11 This test strategy combines in-house methods of pyrosequencing (to detect point mutations) with in-house methods of fragment length analysis (to detect deletions and insertions) for EGFR‑TK mutation detection.

4.12 Pyrosequencing involves extracting DNA from the sample and amplifying it using PCR. Nucleotides are added sequentially to the amplified PCR product. A series of enzymes incorporates nucleotides into the complementary DNA strand, generates light proportional to the number of nucleotides added and degrades unincorporated nucleotides. The DNA sequence is determined from the resulting pyrogram trace.

4.13 In fragment length analysis, DNA is extracted from the sample, and then amplified and labelled with fluorescent dye using PCR. Amplified DNA is mixed with size standards and analysed using capillary electrophoresis. The fluorescence intensity is monitored as a function of time, and analysis software can determine the size of the fragments. The presence or absence of deletions and insertions can then be reported.

Therascreen EGFR Pyro Kit

4.14 The therascreen EGFR Pyro Kit (Qiagen) is a CE‑marked pyrosequencing kit. It is a targeted method of mutation detection designed to detect and distinguish between:

4.15 The kit provides all primers, controls, buffers and reagents necessary to perform the assay. Samples are analysed on the PyroMark Q24 System and a plug-in report tool that simplifies analysis of the pyrogram trace is available.

Single-strand conformation polymorphism analysis

4.16 Single-strand conformation polymorphism analysis is a screening method of mutation detection. The DNA is first extracted from the sample and amplified using PCR. The PCR product is then prepared for analysis by heat denature and analysed using capillary electrophoresis under non-denaturing conditions. Sequence variations (single-point mutations and other small changes) are detected through electrophoretic mobility differences.

High-resolution melt analysis

4.17 High-resolution melt analysis is a screening method of mutation detection. The DNA is first extracted from the sample and amplified using PCR. The PCR product is then precisely warmed so that the 2 strands of DNA 'melt' apart. Fluorescent dye, which only binds to double-stranded DNA, is used to monitor the process. A region of DNA with a mutation will 'melt' at a different temperature from the same region of DNA without a mutation. These changes are documented as melt curves and the presence or absence of a mutation can be reported.

Next-generation sequencing

4.18 Next-generation sequencing is a screening method of mutation detection. The concept is similar to Sanger sequencing (described in section 4.19), but the sample DNA is first fragmented into a library of small segments that can be sequenced in parallel reactions.

Sanger sequencing

4.19 Sanger sequencing (also called direct sequencing) is a screening method of mutation detection. Sanger sequencing is a commonly used method, but there is a lot of variation in how it is carried out. In general, after DNA is extracted from the sample, it is amplified using PCR. The PCR product is then cleaned up and sequenced in both forward and reverse directions. The sequencing reaction uses dideoxynucleotides labelled with coloured dyes, which randomly terminate DNA synthesis, creating DNA fragments of various lengths. The sequencing reaction product is then cleaned up and analysed using capillary electrophoresis. The raw data are analysed using software to generate the DNA sequence. All steps are performed at least in duplicate to increase confidence that an identified mutation is real. It should be noted that sequencing only works well when viable tumour cells constitute 25% or more of the sample.