A variety of methods have been employed as potential alternatives to the historical standard for EGFR mutation testing, direct sequencing. In practice, the choice of testing method should be based primarily on the nature of the sample to be tested including tumour content (particularly for cytology material), the testing laboratory's expertise and available equipment, and whether detection of known activating EGFR mutations only is considered sufficient ().
Factors determining the choice of EGFR mutation testing method.
Reflecting its position as the historical standard, direct sequencing was used as the comparator method in many of the studies identified by our search. The variability observed between studies when comparing sequencing to other mutation testing methods may be due to technical differences, such as primers or reagents used by individual laboratories. Perhaps the primary limitation of direct sequencing is its low sensitivity; indeed, a mutation should generally be present in approximately 20% of all DNA in a sample to be reliably detected by this method.78
In our experience, the approximate tumour content of lung tissue samples can range from 5 to 100%. Therefore, a limit of detection of 20% means that direct sequencing may offer insufficient sensitivity.80
Preparation of samples by macro-dissection or laser capture micro-dissection prior to DNA extraction, however, can enrich tumour cell content and thereby increase the utility of sequencing as a routine pre-treatment test.81
While relatively cost-effective from a reagent perspective compared with targeted methods,82
these preparatory enrichment methods are labour intensive and time consuming and provide a relatively marginal improvement. Alternative screening methods to direct sequencing include dHPLC and HRMA. Although dHPLC appears to have higher analytical sensitivity than direct sequencing,20
dHPLC requires extra processing steps after PCR amplification and the use of expensive instrumentation.22
HRMA has been proposed as an alternative, and is able to detect mutant genes at levels of 2.5–10%.22
HRMA is relatively inexpensive; however, samples testing positive by HRMA must then be analysed by direct sequencing to ensure that mutations are properly identified.24
This can be problematic when levels of mutant DNA do not permit analysis by direct sequencing.
Targeted methods, which detect specific mutations only, tend to be more sensitive in terms of limit of detection than screening techniques. ARMS, for example, is a simple PCR-based testing method shown to be more sensitive and robust than direct sequencing for the assessment of common EGFR
mutations in FFPE tumour tissue.31
One other targeted method to be validated is fragment length analysis. While fragment length analysis is used widely in practice, it can only detect insertions or deletions and does not allow detection of point mutations in EGFR
. Compared with some other methods, mutant-specific IHC is fast, cost-effective, and can be performed in most pathology laboratories.44
IHC, however, has not been widely adopted for EGFR
mutation analysis amid concerns that it is not as sensitive or as specific as DNA-based molecular techniques. A study comparing EGFR
mutation testing between 15 centres in France (the Evaluation of the EGFR Mutation Status for the Administration of EGFR-TKIs in Non-Small Cell Lung Carcinoma (ERMETIC) project) suggested that detection accuracy is dependent upon sample quality rather than the sequencing method used, highlighting the importance of good sample collection and processing techniques.83
For poor quality samples, care must be taken to ensure the selection of tumour cells, in addition to the DNA concentration, as test results are frequently dependent on both these parameters.
The availability of targeted methods with high sensitivity offers the potential for accurate, rapid, and high-throughput analysis of clinical samples. The main theoretical drawback of these techniques is their inability to detect all activating EGFR
mutations. The majority of clinical evidence to date robustly supports the use of EGFR TKIs in patients with the two most common activating mutations in EGFR
(exon 19 deletions and the L858R point mutation in exon 21), and most targeted methods are specifically designed to detect these mutations. However, clinical data on less common mutations are emerging and further research is required to fully inform predictable outcomes on EGFR TKIs, aided by the use of sample micro dissection followed by screening methods, to ensure identification of all known mutations.18
While targeted methods can fail to detect some of the rare mutations which are detected by screening, it is anticipated that rare mutations demonstrated to have therapeutic implications will subsequently be included in targeted screening approaches, thus ensuring all patients will benefit from the appropriate therapy. Both screening and targeted methods have been used to identify EGFR
mutations in clinical trials of EGFR TKIs in patients with advanced NSCLC.6–10
These trials were not identified by our search due to our focus on method comparison studies. In practice, laboratories can opt to use commercially available kits or to develop their own tests. Testing kits such as those utilising the ARMS method have the advantages of being validated, ready for use and quality controlled. Laboratory-developed tests, many of which were identified by our search, may be less expensive, but take time to develop and validate and may have limited quality control. If procedures for EGFR
mutation testing are not established at a local level, use of one of a number of global testing laboratories may be considered. Such laboratories use a variety of methods for EGFR
mutation testing including commercially available kits and laboratory-developed tests.
Our literature search confirmed that cytology samples are suitable testing material for EGFR
mutation testing and that detection rates appear to be as high as those obtained with traditional tissue samples. The suitability of cytology samples for routine clinical practice has been recognised in published recommendations for EGFR
Of note, in the recent study of Goto and colleagues, published after we performed our literature search, five different EGFR
testing methods (PCR-Invader, PNA-LNA-PCR clamp, PCR-direct sequencing, cycleave PCR, and ARMS) showed comparable performance in the assessment of tissue and cytology samples. Furthermore, the concordance between matched tumour and cytology samples was extremely high.86
There is a growing trend toward the extensive molecular characterisation of tumours so that the most appropriate therapy can be selected. This is exemplified in the Biomarker-integrated Approaches of Targeted Therapy for Lung cancer Elimination trial, in which patients are adaptively randomised to various treatments based on relevant molecular biomarkers.87
This approach has been made possible by the availability of methods such as Sequenom MassArray,88
and arrays of mutation-specific PCR assays (eg, qBiomarker Somatic Mutation PCR Array), and through the use of next-generation sequencing. These methods can rapidly and sensitively detect many known mutations in a relatively small amount of DNA. Using such gene panel approaches will no doubt increase our knowledge of pharmacogenetic predictive biomarkers and therefore improve patient outcomes by ensuring that each patient is given a treatment with the most likely chance of success. To date, no point-of-care devices are available for EGFR
mutation testing; the future development of such devices would be welcome and would help ensure that treatment is not delayed while test results are awaited.