Precision

Precision in mutation testing

Epidermal growth factor receptor (EGFR) activating mutation are found in exons 18 to 21 of the EGFR gene, which is part of the gene coding for the tyrosine kinase domain of the EGFR protein.1,2 In patients diagnosed with advanced non-small cell lung cancer (NSCLC), the most common EGFR mutations are exon 19 deletions and an L858R point mutation in exon 21.3–7

Primary EGFR mutations2,8,9

EGFR Mutation

*EGFR mutations and their frequency is based on COSMIC annotations, some of which can be seen in the same tumour. Because of many data entries in COSMIC reflect studies focused on exons 21 and 19, the frequency of rarer mutations may be underestimated.2

Other uncommon EGFR mutations are known to exist.8

G719X refers to G719A, G719C, G719S

There are several different EGFR mutation tests and the choice of test should be based primarily on the available sample type. Consideration should also be given to the relevant lab expertise and available equipment.

Testing technologies

The choice of EGFR mutation testing method is often influenced by a laboratory’s expertise and available equipment. Labs can use commercially available kits, develop their own tests (lab-developed tests) or send the samples to an external specialist testing laboratory. It is important to consider using highly sensitive testing methods for cytology and plasma samples because of the limited number of tumour cells present in such samples.10-12

To ensure that the EGFR mutation testing process is reliable and consistent, and to ensure any driver mutations are characterised, it is essential to build quality assurance into every step of the procedure. This includes:13

  • Independent quality control of data and reports
  • Validation of new assays
  • Use of good laboratory practice, including control of PCR product contamination
  • Use of positive and negative controls

X


EGFR mutation testing kits

Commercially available tests

There are a number of commercially available testing kits for determining EGFR mutation status; however, not all kits are regulatory approved in-vitro diagnostics (IVDs). Regulatory approval is adopted at a regional level and provides assurance of the integrity of a test’s methodology, and in some cases the clinical utility. In the US, the Food and Drug Administration regulates IVDs (denoted by a US-IVD mark) and in Europe this falls under the European IVD Directive (98/79/EC), denoted by a CE-IVD mark.

The table below contains examples of commercially available, regulatory approved EGFR mutation testing kits. Where applicable, IVD regulatory status for the EU and US is noted. For IVD regulatory status in other regions, please consult local regulatory guidelines.

Kit Manufacturer Mutations detected Sensitivity: limit of detection* Technology Platform(s)

cobas® EGFR Mutation Test V215
CE-IVD
US-IVD

Roche Molecular Systems
www.molecular.roche.com
Exon 18: G719X
Exon 19: 29 deletions and complex mutations
Exon 20: S768I
Exon 20: T790M
Exon 20: 5 insertions
Exon 21: L858R
Exon 21: L861Q
For FFPET specimens, can detect mutations in EGFR exons 18, 19, 20 and 21 with at least 5% mutation level using the standard input of
50 ng per reaction well
PCR amplification and detection of target DNA using complementary primer pairs and oligonucleotide probes labelled with fluorescent dyes Roche cobas z 480 analyser

therascreen® EGFR RGQ PCR Kit16
CE-IVD
US-IVD

Qiagen
www.qiagen.com

Exon 18: G719X
Exon 19: 19 deletions
Exon 20: S768I
Exon 20: T790M
Exon 20: 3 insertions
Exon 21: L858R
Exon 21: L861Q
Exon 18: G719X: 7.4%-10.3%
Exon 19: 19 deletions: 1.6%
Exon 20: S768I: 7.7%
Exon 20: T790M: 9.7%
Exon 20: 3 insertions: 2.4%-11.6%
Exon 21: L858R: 5.9%
Exon 21: L861Q: 2.2%
PCR amplification using ARMS and Scorpions technologies Qiagen
Rotor-Gene
Q MDx 5Plex HRM instrument

EGFR29 Mutation
Detection Kit17
CE-IVD
CFDA-IVD for China)

AmoyDx
www.amoydx.com

Exon 18: G719X
Exon 19: 19 deletions
Exon 20: S768I
Exon 20: T790M
Exon 20: 3 insertions
Exon 21: L858R
Exon 21: L861Q
1% ADx-ARMs technology: two-step PCR amplification procedure and novel fluorescent probe design None specified

PNAClampTM
EGFR Mutation
Detection Kit
18
CE-IVD

Panagene
www.panagene.com

Exon 18: G719X
Exon 19: 26 deletions
Exon 20: S768I
Exon 20: T790M
Exon 20: 6 insertions and H773L
Exon 21: L858R
Exon 21: L861Q

<1% PNA blocking of wild-type DNA amplification; SYBR Green detection Multiple

therascreen® EGFR Pyro Kit19
CE-IVD

Qiagen
www.qiagen.com

Exon 18: G719X
Exon 19: 20 deletions and complex mutations
Exon 20: S768I
Exon 20: T790M
Exon 21: L858R
Exon 21: L861Q
Exon 21: L861R

Refer to manufacturer’s website for details on limits of detection Pyrosequencing Qiagen
PyroMark
Q24 System
Kit cobas® EGFR Mutation Test V215
CE-IVD
US-IVD
Manufacturer Roche Molecular Systems
www.molecular.roche.com
Mutations detected Exon 18: G719X
Exon 19: 29 deletions and complex mutations
Exon 20: S768I
Exon 20: T790M
Exon 20: 5 insertions
Exon 21: L858R
Exon 21: L861Q
Sensitivity: limit of detection* For FFPET specimens, can detect mutations in EGFR exons 18, 19, 20 and 21 with at least 5% mutation level using the standard input of 50 ng per reaction well
Technology PCR amplification and detection of target DNA using complementary primer pairs and oligonucleotide probes labelled with fluorescent dyes
Platform(s) Roche cobas z 480 analyser
Kit therascreen® EGFR RGQ PCR Kit2
CE-IVD
US-IVD
Manufacturer Qiagen
www.qiagen.com
Mutations detected Exon 18: G719X
Exon 19: 19 deletions
Exon 20: S768I
Exon 20: T790M
Exon 20: 3 insertions
Exon 21: L858R
Exon 21: L861Q
Sensitivity: limit of detection* Exon 18: G719X: 7.4%-10.3%
Exon 19: 19 deletions: 1.6%
Exon 20: S768I: 7.7%
Exon 20: T790M: 9.7%
Exon 20: 3 insertions: 2.4%-11.6%
Exon 21: L858R: 5.9%
Exon 21: L861Q: 2.2%
Technology PCR amplification using ARMS and Scorpions technologies
Platform(s) Qiagen
Rotor-Gene
Q MDx 5Plex HRM instrument
Kit EGFR29 Mutation
Detection Kit17
CE-IVD
CFDA-IVD for China)
Manufacturer AmoyDx
www.amoydx.com
Mutations detected Exon 18: G719X
Exon 19: 19 deletions
Exon 20: S768I
Exon 20: T790M
Exon 20: 3 insertions
Exon 21: L858R
Exon 21: L861Q
Sensitivity: limit of detection* 1%
Technology ADx-ARMs technology: two-step PCR amplification procedure and novel fluorescent probe design
Platform(s) None specified
Kit PNAClampTM
EGFR Mutation
Detection Kit
18
CE-IVD
Manufacturer Panagene
www.panagene.com
Mutations detected Exon 18: G719X
Exon 19: 26 deletions
Exon 20: S768I
Exon 20: T790M
Exon 20: 6 insertions and H773L
Exon 21: L858R
Exon 21: L861Q
Sensitivity: limit of detection* <1%
Technology PNA blocking of wild-type DNA amplification; SYBR Green detection
Platform(s) Multiple
Kit therascreen® EGFR Pyro Kit19
CE-IVD
Manufacturer Qiagen
www.qiagen.com
Mutations detected Exon 18: G719X
Exon 19: 20 deletions and complex mutations
Exon 20: S768I
Exon 20: T790M
Exon 21: L858R
Exon 21: L861Q
Exon 21: L861R
Sensitivity: limit of detection* Refer to manufacturer’s website for details on limits of detection
Technology Pyrosequencing
Platform(s) Qiagen
PyroMark
Q24 System

The information in this table is derived from the manufacturer’s product information handbooks and websites.

*Unless otherwise stated, limit of detection (LOD) represents the percent mutation required to achieve 95% mutation detection; the value for the reaction that gave the highest LOD is given; G719X refers to G719A, G719C, G719S; PNA, peptide nucleic acid

A number of other EGFR mutation kits are commercially available, but may not have regulatory approval, often referred to as research use only kits. Examples include EntroGen EGFR Mutation Analysis Kit for Real-Time PCR, Sysmex OncoBEAM EGFR, and Illumina TruSight Tumor 26.20-22

Lab developed tests

Lab-developed epidermal growth factor receptor (EGFR) mutation tests may be less expensive, but they can take time to develop and validate.23 Quality control may be limited compared with commercially prepared kits, which are likely to have been tested for reproducibility prior to commercial distribution.23 Lab-developed tests may only undergo quality assurance in a single lab, meaning that the reproducibility of results is unknown.