Definition of EGFR
The epidermal growth factor receptor (EGFR), sometimes referred to as HER1 or ERBB1, is a member of the HER family of membrane bound receptor tyrosine kinases. Physiological activation of the receptor is through ligand binding by epidermal growth factor (EGF) or transforming growth factor beta (TGFβ). This leads to homodimersation with other EGFR molecules or heterodimerisation with other HER family members, HER2 forming the favoured heterodimer. Dimerisation leads to activation of the internal domain tyrosine kinase. The active kinase can then activate RAS and thus the RAS-RAF-MAPK pathway, and PI3K and thus the PI3K-AKT-TSC1/2-mTOR pathway. These effects and other downstream signalling involving the STAT pathway promote cell proliferation and protein synthesis, and inhibit apoptosis through p53. Consequently, any abnormal upregulation of the EGFR tyrosine kinase could have oncogenic effects.
EGFR Protein Expression in Non-Small Cell Lung Cancer (NSCLC)
EGFR protein may be detected by immunohistochemistry. Relatively high levels of EGFR protein may be found in squamous cell carcinoma and in adenocarcinomas. Definitions of ‘high expression’ vary but expression is increased in anywhere between 40-75% of cases. Typically the protein is most strongly expressed on tumour cell membranes.
Claims have been made that high EGFR protein expression may be either a good or a poor prognostic factor, the latter being more frequently claimed, but meta-analysis failed to demonstrate a convincing effect.
In the FLEX trial, high levels of EGFR protein demonstrated by immunohistochemistry (IHC), and expressed as an H score over 200, were associated with more frequent responses to cetuximab, a therapeutic monoclonal antibody against EGFR, when combined with chemotherapy. Currently however, this treatment has failed to receive regulatory approval.
EGFR Gene Copy Number
NSCLC may show increased gene copy number, either as a result of polysomy or through gene amplification. Once again, definitions of increased gene copy number vary, depending on whether and to what extent polysomy, as opposed to amplification, is included in the cohort. Prevalence is reported at anywhere between 15–50% and very much depends on the case mix and the proportion of cases which show mutation of the EGFR gene (see below). There is some correlation between increased gene copy number and high protein expression.
Both increased gene copy number and higher EGFR protein expression were considered as potential biomarkers for predicting response to therapy in NSCLC using first generation small molecule EGFR receptor tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib. The predictive power of high IHC protein expression is, however, poor, but for EGFR gene amplification, the association is stronger. Of course, neither are routinely used clinical predictive biomarkers since EGFR gene mutations are the best predictive markers with regulatory approval. In the presence of gene mutation, the mutant allele is frequently preferentially amplified, leading to an association between EGFR amplification and mutation, and therefore, response to EGFR TKIs.
EGFR Gene Mutation in NSCLC
Mutations in exons 18-21 of the EGFR gene coding for the internal receptor tyrosine kinase domain of EGFR have a variable ability to activate the TK in the absence of ligand binding. This constitutive activation provides a powerful oncogenic drive. The aetiology of such mutations is not known but it is not associated with tobacco carcinogenesis. This oncogenic change is almost exclusively seen in the context of peripheral lung epithelial carcinogenesis and is thus almost exclusively associated with the adenocarcinoma phenotype. Such mutations seem to occur exclusive of other oncogenic mutations and are regarded as addictive mutations. EGFR mutations are commonest in East Asian patients, never smokers and females.
In Caucasian populations where the vast majority of squamous cell lung carcinomas (SCC) are tobacco induced, EGFR mutation in SCC is exceedingly rare. EGFR mutations are uncommonly reported in SCC in East Asian populations where tobacco carcinogenesis is less often associated and where the squamous cancers may derive from the peripheral lung epithelium. EGFR mutations are reported in 10-15% of Caucasian adenocarcinomas (all cases regardless of smoking history), in 40–60% of adenocarcinomas in East Asian populations, and in 50% of Caucasian never smokers with adenocarcinoma.
EGFR Mutation as a Predictive Biomarker
A majority of the activating mutations occurring exons 18-21 of the EGFR gene are also sensitizing to blockade of tyrosine kinase activity by small molecule TKIs, especially exon 19 deletion mutations and the L858R substitution mutation in exon 21, which collectively account for 75-90% of EGFR mutations. Several other mutations, mostly in exon 18 may also confer some less sensitivity to EGFR TKI blockade. Rare activating mutations, mostly in exon 20, the most common of which is the T790M mutation, confer resistance to EGFR TKIs. T790M is a rare mutation and usually co-exists with another activating mutation. However, in tumours which recur or relapse on TKI therapy, the T790M is found in 50-60% of cases, probably due to outgrowth of a pre-existing minor clone of resistant tumour cells. EGFR mutations are the approved predictive biomarker for EGFR TKI therapy.
EGFR Mutation Testing in NSCLC
Most guidelines recommend that all patients with an adenocarcinoma, probable or possible adenocarcinoma should have their tumour tested for EGFR mutations in exons 18-21. In never or long time ex-smokers it is reasonable to consider testing squamous cell carcinomas.
Clinical parameters – smoking habit, gender, ethnicity etc. should not be used to select patients for testing. In many Caucasian cohorts, excluding male smokers with adenocarcinoma would risk missing a substantial proportion of treatable mutations.
Although EGFR TKI therapy is generally the preserve of stage IV disease, testing is often carried out regardless of stage, for practical purposes, or in the absence of stage information. Reflex testing, driven by the pathologist on making the appropriate histological diagnosis, is widely practice but, for a variety of reasons, local practice may favour ‘bespoke’ testing driven on request by the oncologist or tumour board.
There are many different methods that may be used to provide an accurate and valid EGFR mutation test result. Some of these methods are allele specific and only find those specific mutations the technology in use is designed to detect. If such methods are used (many commercially available ‘black box’ platforms are of this type), users should be aware of the mutation coverage of the panel used (what it covers, which mutations it misses). Some of these solutions may also fail to provide detailed information of the particular mutation. Other methods will screen the entire four exons and any mutation will be detected, with caveats.
As well as coverage, the other key parameter of a testing methodology is its sensitivity, that is, the % of mutant alleles detected in a background of wild type alleles which are always present. Some commercial allele specific PCR based solutions claim sensitivity as low as 1-3%. Sanger sequencing often requires 20% or more mutant alleles but in expert hands may be good down to 10%.
Samples for Testing
Any pathological samples containing tumours cells may be used: biopsy samples, cytology material, surgically resected tumour. The key factors, assuming a standalone EGFR mutation test, are as follows:
- The material has been processed or prepared in a way that does not preclude testing.
- The material for extraction is prepared ensuring no cross contamination from other patients samples.
- The material submitted for DNA extraction definitely contains tumour and the molecular lab is informed of the % of nuclei in the extraction sample that are of tumour.
- Steps are taken wherever possible to enrich tumour cells in the sample for DNA extraction (macrodissection etc.).
- The actual number of tumour cells in the extraction sample is reasonable (fewer than 20 tumour cells may pose extreme challenges).
Ensuring Timely and Quality EGFR Mutation Testing
There are many steps and many individuals involved in the work chain from the decision to test, through sample handling, submission to the molecular lab, assimilation of the molecular and pathological data and reporting back to the treating physician. Actual analytical time in the molecular lab should not exceed 5 working days but the overall process as outlined can take more than double this time. Efficient communication and working systems are vital for timely diagnosis.
All labs issuing clinical reports should participate in, and perform adequately in external quality assurance programmes. Regular in-house monitoring of performance, and awareness of potential false positive and false negative tests is important.
Reporting EGFR Mutation Test Results
EGFR mutations should be reported in the context of the sample which was submitted to the lab (% tumour cells and cellularity, tumour type) and assimilated into the overall pathology report. Context is vital, to avoid misinterpretation of results.
The report to the treating physician should provide detail of any mutation(s) found and if possible, data on allelic frequency, with respect to the sample tested. Generic comments about the implications of any findings for therapy are acceptable, but specific treatment recommendations should be avoided.
- Prudkin L, Wistuba II. Epidermal growth factor receptor abnormalities in lung cancer. Pathogenetic and clinical implications. Ann Diag Pathol 2006;10,306-315.
- Yatabe Y. EGFR mutations and the terminal respiratory unit. Cancer Metastases Rev 2010;29:23-26.
- Hirsch FR, Dziadziuszko R, Thatcher N, et al. Epidermal growth factor receptor immunohistochemistry. Cancer2008;112,1114-21.
- Hirsch FR. EGFR: A prognostic and / or a predictive marker? J Thorac Oncol 2006;1,395-397.
- Goto K, Satouchi M, Ishii G, et al. An evaluation of EGFR mutation tests utilised for non small cell lung cancer in the diagnostic setting. Ann Oncol 2012;23,2914-2919.
- Kerr KM. Personalized medicine in Lung cancer: Challenges for pathology. Histopathol 2012;60,531-546.
- Lindeman NI, Cagle PT, Beasley MB, et al. Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. J Thorac Oncol 2013;8:823-59.
- Kerr KM, Bubendorf L, Edelman MJ, et al. Second ESMO Consensus Conference on Lung Cancer: pathology and molecular biomarkers for non-small-cell lung cancer. Ann Oncol 2014;25,1681-90.