Somatic mutations of the epidermal growth factor receptor and non‐small‐cell lung cancer

Abstract

Frequent overexpression of epidermal growth factor receptor (EGFR) in non‐small‐cell lung cancer (NSCLC) makes EGFR a new therapeutic target. Two specific EGFR tyrosine kinase inhibitors, gefitinib (ZD1839, Iressa) and erlotinib (OSI‐774, Tarceva), have been developed and approved by the US Food and Drug Administration for second‐line and third‐line treatment of advanced NSCLC. Clinical trials have shown considerable variability in the response rate between different patients with NSCLC, which led to the discovery of somatic EGFR‐activating mutations. This brief review summarises the discovery and functional consequences of the mutations, their clinicopathological features and significant implications in the treatment and prognosis of NSCLC.

Lung cancer is one of the most common human cancers and the leading cause of death due to cancer worldwide.¹ It claims more lives than colorectal, breast and prostate cancers combined. Fewer than 15% of patients can be cured and have a >5 year survival rate, as the disease is often in an advanced stage at the time of diagnosis and chemotherapy/radiotherapy cannot cure the advanced disease.² Lung cancer is generally classified into two basic types, small‐cell lung cancer and non‐small‐cell lung cancer (NSCLC). NSCLC accounts for approximately 85% of cases and can be further divided into squamous‐cell carcinoma, adenocarcinoma and large‐cell carcinoma.³ In recent years, adenocarcinoma has replaced squamous‐cell carcinoma as the most common histological subtype of NSCLC in the US and many other parts of the world.⁴,⁵

The pathogenesis of lung cancer is yet to be fully understood. Multiple environmental factors are implicated in lung carcinogenesis, such as outdoor and indoor pollution, diet and bacterial and viral infections.⁶ Cigarette smoke has been widely accepted as the major cause of lung cancer and a linear dose‐response relationship has been established between risk of lung cancer and the amount of cigarettes consumed.⁷,⁸,⁹ The occurrence of lung cancer is also closely associated with the accumulation of multiple genetic and/or epigenetic changes.¹⁰,¹¹,¹² Better understanding of molecular mechanisms underlying the pathogenesis of lung cancer would provide pivotal guidance for the treatment and prevention of cancer.

EGFRs and lung cancer

Human tumours often express high levels of epidermal growth factor (EGF)‐related receptors, which include EGFR/HER1, c‐erbB2/HER2, c‐erbB3/HER3 and c‐erbB4/HER4 (table 1).¹⁶,¹⁷,¹⁸,¹⁹,²⁰ All these receptors share a common extracellular ligand‐binding domain, a hydrophobic transmembrane domain and a multifunctional intracellular domain that has an ATP‐binding site and tyrosine kinase activity (HER3, however, does not have tyrosine kinase activity). Receptor activation is generally initiated by the binding of EGF‐related ligands and receptor homodimerisation or heterodimerisation, which activate the intrinsic tyrosine kinase activity of the receptors and lead to autophosphorylation of the tyrosine residues. These residues further activate downstream signalling cascades, such as the Ras‐Raf‐MAP‐kinase, PI3K‐Akt and STAT pathways, which have strong regulatory effects on cell proliferation, differentiation, survival and migration.²¹,²²,²³

Table 1 The family of human epidermal growth factor (EGF)‐related receptors.

	Gene location	Molecular weight	Protein distribution
c‐erb B‐1/EGFR	7p12.3–p12.1	170 kDa	A large variety of cell type or tissues except haemopoietic cells13
c‐erb B‐2/HER‐2	17q21.1	185 kDa	A number of human secretory epithelial tissues14
c‐erb B‐3/HER‐3	12q13	160 kDa	Several human tumour cell lines15
c‐erb B‐4/HER‐4	2q33.3–q34	180 kDa	Lining epithelia of the gastrointestinal, urinary, reproductive, breast cancer cell lines, and normal skeletal muscle, heart, pituitary, brain and cerebellum16

EGFR is generally expressed at a low level in a wide variety of normal tissues. Excessive expression or activation of EGFR is able to induce malignant transformation.²⁴ Overexpression of EGFR has been observed in 40–80% of NSCLCs,²⁰,²⁵ and it is often associated with aggressive clinical behaviours, such as advanced stage, increased metastatic rate, higher tumour proliferation rate and poor prognosis.²⁶,²⁷,²⁸,²⁹ Other than overexpression, a mutant form of EGFR (EGFRvIII) with constitutive tyrosine kinase activity has been identified in NSCLC³⁰,³¹,³² and is implicated in lung tumorigenesis.³³,³⁴

Given that EGFR is often overexpressed in NSCLC and given its potential role in lung carcinogenesis, EGFR has been considered a rational target molecule for the treatment of NSCLC. Specific EGFR tyrosine kinase inhibitors (EGFR‐TKIs), gefitinib and erlotinib, have been approved by the US Food and Drug Administration as monotherapy treatment for advanced or metastatic NSCLC.³⁵,³⁶ Both agents adversely compete with ATP for the critical ATP‐binding site located in the intracellular domain and inhibit receptor phosphorylation.

Gefitinib and EGFR mutations

The effectiveness of gefitinib has been evaluated by two randomised and double‐blinded Phase II clinical trials in patients with advanced NSCLC.³⁷,³⁸ The results suggest that gefitinib is a well‐tolerated oral EGFR‐TKI. It has meaningful antitumour activity and brings about considerable improvement in cancer‐related symptoms in certain subgroups of patients (approximately 10–19%). Females, non‐smokers, Japanese people and patients with lung adenocarcinoma generally have a higher response rate than males, smokers, people of European origin and patients with other histological types of NSCLC.³⁵,³⁷,³⁸ To determine whether somatic mutations in the EGFR gene play a causal role in response to TKI treatment, two research groups have systematically sequenced all 28 exons of EGFR and identified several important activating mutations that show striking correlation with gefitinib response.³⁹,⁴⁰ This discovery has been claimed as the most significant molecular event in lung cancer.⁴¹ It has greatly stimulated research in this area worldwide, and a number of other novel mutations have been identified (table 2).

Table 2 Mutations identified in exons 18–21 of the EGFR gene (RefSeq NM_005228)*.

Sequence change	Amino acid change	Reference
Exon 18
2126A→T	E709V	42, 43
2126A→C	E709A	44, 45
2126A→G	E709G	44, 45
2125G→A	E709K	45
2155G→A	I715S	43
2155 G→T	G719C	40, 42–47
2155 G→A	G719S	39, 43–45
2156 G→C	G719A	42–44, 47, 48
2159 C→T	S720F	43, 48
Exon 19
2225T→C	V742A	49
Del (2236–2244)+2245G→C+2248G→C	E746‐R748 del with E749Q, A750P	47
Del (2235–2249)/del (2236–2250)	E746‐A750 del	39, 40, 42–50
Del (2235–2236)+	E746‐A750 del with I, P ins	48
Del (2242–2248)+2241A→C
Del (2237–2251)+2252C→T	E746‐A750 del with V ins	43
Del (2237–2251)	E746‐T751 del with A ins	44–46, 48, 50
Del (2235–2236)+	E746‐T751 del with I ins	43, 48
Del (2239–2252)/
Del (2235–2252)+
2254T→A+2255C→T
Del (2235–2236)+	E746‐T751del with I, P ins	48
Del (2242–2248)+2241A→C
2237–2238 AA→TC+	E746‐T751 del with V ins	43, 49
Del (2239–2253)
Del (2484–2501)	E746‐S752 del with D ins	44
Del (2237–2254)+2255C→T	E746‐S752 del with V ins	45, 48, 50
Del (2239–2247)	L747‐E749	46
Del (2239–2247)+2248G→C	L747‐E749 del with P ins	39, 42, 43, 45
Del (2240–2248)+2239T→C	L747‐T750 del with P ins	43, 48
Del (2238–2252)/	L747‐T751 del	43, 44, 48, 49
Del (2239–2253)/
Del (2240–2254)
Del (2240–2251)	L747‐T751 del with S ins	40, 46, 48
Del (2239–2256)	L747‐S752 del	44, 48
Del (2239–2256)+2258 C→A	L747‐S752 del with Q ins	43
Del (2238–2255)+2237A→T	L747‐S752 del, E746V	39
Del (2240–2257)	L747‐P753 del with S ins	39, 40, 42–46, 48–50
Del (2240–2257)	L747S, R748‐P753 del	47
Del (2254–2277)	S752‐I759 del	39, 47
2273A→G	E758G	49
Exon 20
2308 ins GCCATA	M766‐A767 with AI ins	47
2308 ins CCAGCGTGG+	A767‐S768 with SVA ins	47
2310C→T silent
2303G→T	S768I	43–45, 47
Dup (2549–2557)	S768‐D770 dup	44
2308–2316 ins GCCAGCGTG	ASV770‐772 ins	43
2320–2322 ins CAC	H774 ins	43
2317–2222 ins AACCCC+	NP773‐774 ins, H775Y	43
2223 C>T
2320–2325 ins CCCCAC	PH774‐775 ins	43
2320–2328 ins AACCCCCAC	NPH774‐776 ins	43
2326C→T	R776C	51
2308 ins (CCAGCGTGG)+	ins779 ASV+P782R	48
2310C→T+ 2315C→G
2311 ins (GCGTGGACA)+	ins780 SVD+P782R	48
2315C→G
2369 C→T	T790M	52–55
Exon 21
2743T→G	L833V	44
2750A→T	H835L	44
2758C→G	L838V	44
2513T→C	L838P	49
2520C→T	A840A	49
2527G→A	V843I	49
2551G→A	V851I	49
2572C→A	L858M	46
2573T→G	L858R	39, 40, 42–50
2582T→A	L861Q	40, 42–45
2593G→A	E856L	49
2612C→T	A871V	49

*Only mutations reported with both nucleotide and amino acid changes are summarised. The beginning of the coding sequence is defined as +1.

Genotyping methods

Two important factors affect the detection of somatic EGFR mutations in clinical cancer samples. The first is the availability of the tumour genome. There is no doubt that frozen surgical tumour samples⁴⁵ and tumour paraffin blocks⁴⁷ are the best samples for mutation analysis, as they are directly resected from corresponding tumours and can provide sufficient tumour nucleic acids for genotyping. However, a large proportion of patients with NSCLC are not eligible for surgery on diagnosis. Therefore, non‐surgical specimens, such as diagnostic biopsy and effusion drainage, are probably as important as surgical specimens in these patients with advanced cancer. Pleural effusion⁵⁶ and needle biopsy/aspiration⁴⁹ have been successfully managed for mutation screening. Asano et al⁵⁷ even showed the feasibility of detecting EGFR mutations with the use of soluble DNA extracted from pleural fluid.

The second factor affecting mutation detection is the purity of the tumour genome. Usually clinical cancer samples contain a large proportion of normal cells, which make up a strong background of wild‐type alleles and seriously dilute the signal from biologically important somatic mutations. Therefore, the sensitivity of genotyping methods is of great importance for the detection of mutations.

Among a number of reported methods, PCR‐based direct sequencing is the most commonly used.³⁹,⁴⁰,⁴³,⁴⁴,⁴⁷ With the help of cloning technology, even samples presenting difficulty in direct sequencing can be sequenced using primers located on vectors. Moreover, tumour RNA can be used for genotype determination, as all the reported EGFR‐activating mutations are exonic.⁴²,⁵⁵ However, RNA is usually more difficult to handle than genomic DNA, because of its rapid degradation and limited quantity.

Single‐strand conformation polymorphism (SSCP) assay is another important method used for EGFR mutation screening. SSCP has been considered to be more sensitive than direct sequencing in mutation analysis.⁵⁸,⁵⁹ The two large studies performed by Marchetti et al⁴⁶ and Sonobe et al⁴⁸ have reported that SSCP assays not only confirmed all the EGFR mutations detected by direct sequencing but also identified additional mutations that were missed in sequencing analysis. Therefore, SSCP assay could be a reliable method for large‐scale diagnostic screening for EGFR mutations in clinical samples.

For detection of known EGFR mutations, a number of methods have been developed, including restriction fragment length polymorphism and length analysis of fluorophore‐labelled PCR products,⁶⁰ peptide nucleic acid–locked nucleic acid PCR clamp,⁶¹ mutant‐allele‐specific amplification⁶² and mutant‐enriched PCR.⁵⁷ The restriction fragment length polymorphism–capillary electrophoresis method can not only detect the mutations but also estimate gene amplification based on the relative height of the mutation peak to the germline peak. The peptide nucleic acid–locked nucleic acid PCR clamp, mutant‐allele‐specific amplification and mutant‐enriched PCR have high sensitivity. They are able to distinguish even one EGFR mutant tumour cell in the presence of up to 1000–2000 normal cells.⁵⁷,⁶¹,⁶²

The pattern and functional consequence of EGFR mutations

Three common types of EGFR mutation—in‐frame deletion, insertion and missense mutation—have been identified. Most of the mutations are located in the tyrosine kinase‐coding domain (exons 18–21). Amino acids 746–753 encoded by exon 19 and amino acid 858 encoded by exon 21 are the two mutation hotspots, comprising >80% of the mutations. All the identified mutations are of somatic origin, and not present in the germline genome.

EGFR mutations have been proposed as an early event in lung carcinogenesis. They are not correlated with the classification of tumour stage.⁴⁶ Well or moderately differentiated tumours have more EGFR mutations than poorly differentiated tumours.⁴⁸ Some of the mutations can even be detected in respiratory epithelia with normal histology.⁶³ The oncogenic characteristics of EGFR mutants have recently been proved by anchorage‐independent growth and focus formation in transfected cells and tumour formation in immunocompromised mice.⁶⁴

EGFR mutants (deletion in exon 19 and L858R) are capable of enhancing EGF‐dependent receptor activation (Tyr¹⁰⁶⁸).⁴⁰ The downstream signalling pathways Akt and STAT are also selectively activated,⁶⁵,⁶⁶ and these have an important anti‐apoptotic function. When mutant EGFR expression is suppressed by specific small interfering RNA or when Akt and STAT pathways are blocked by specific inhibitors, rapid and massive apoptosis occurs. A similar event also happens when EGFR‐TKIs are applied to mutant NSCLC cell lines.⁵¹,⁶⁵,⁶⁶ All these suggest that excessive EGFR signalling plays a critical role in tumorigenesis in patients harbouring an EGFR mutation, and mutant EGFRs drive the growth of cancer cells and maintain their malignant phenotype by the selective activation of Akt and STAT signalling pathways.

EGFR gene amplification is present in approximately 30–40% of patients with NSCLC,⁶⁷,⁶⁸ and it is associated with a susceptibility to gefitinib.⁶⁸,⁶⁹,⁷⁰ The association between EGFR mutations (deletion in exon 19 and L858R in exon 21) and an increased copy number of the EGFR gene has been shown in both preclinical⁶⁶,⁷¹ and clinical investigations,⁵⁰,⁷⁰,⁷¹ which could partially throw light on gefitinib sensitivity in patients with EGFR gene amplification. Genetic instability has been proposed to be the cause of gene amplification and the facilitator of the incidence of EGFR mutations.⁷¹

Factors predisposing to EGFR mutations in NSCLC

The distribution of EGFR mutations in NSCLC has been intensively investigated (table 3). In general, EGFR mutations are more common in patients of oriental origin, in females and in patients with a history of never smoking or with adenocarcinoma.⁷³

Table 3 Overview of 10 large studies on EGFR mutations in non‐small‐cell lung cancer (NSCLC).

	Ethnicity	Subjects (n)	Mutation rate (%)	Deletions in exon 19 (%)	L858R (%)	Mutation rate (%)
						Non‐smokers vs smokers	Female vs male	Adenocarcinoma vs other NSCLC
Marchetti46	White	860	5	46	46	20 vs 2	19 vs 2	10 vs 0
Eberhard47	White	228	13	62	24	25 vs 12	13 vs 13	15 vs 11
Cappuzzuo67	White	89	17	53	47	46 vs 12	26 vs 12	20 vs 10
Shigematsu43	White	158	8	46	39	29 vs 3	20 vs 1	16 vs 1
	East Asian	361	30			56 vs 14	55 vs 19	48 vs 3
Kosaka55	Japanese	277	40	47	44	66 vs 22	59 vs 26	49 vs 2
Sonobe48	Japanese	154	39	57	37	79 vs 16	70 vs 19	55 vs 0
Tokumo72	Japanese	120	32	50	47	69 vs 15	57 vs 20	45 vs 3
Huang44	Taiwanese	101	39	33	51	47 vs 18	44 vs 32	55 vs 3
Sugio62	Japanese	469	29	45	55	54 vs 19	46 vs 21	42 vs 1
Yokoyama45	Japanese	349	29	40	39	54 vs 14	54 vs 15	42 vs 1

Ethnicity

EGFR mutations have significant ethnic variation. The mutation rate is approximately 5–13% among Caucasians,⁴³,⁴⁶,⁴⁷ but 30–40% among East Asians (table 3).⁴³,⁴⁴,⁵⁵ Although the factors that determine more EGFR mutations in Asians are still an enigma, different genetic backgrounds and living environments could provide informative clues. The international HapMap project (http://www.hapmap.org) has genotyped >300 single‐nucleotide polymorphisms across the EGFR gene in Caucasian, Japanese, Chinese Han and Yoruba populations. Considerable interethnic variations in the prevalence of single‐nucleotide polymorphisms have been observed between Japanese and Caucasian populations (fig 1). It remains unknown if the germline variations are correlated with the occurrence of EGFR somatic mutations. However, the association between germline and somatic variations in upper gastrointestinal familial adenomatous polyposis has been reported.⁷⁴

Figure 1 Genotype distribution of germline EGFR polymorphisms in Caucasian and Japanese populations. The genotype datasets are downloaded from the HapMap project and displayed in Visual Genotype (VG) format (http://pga.gs.washington.edu). The columns are the polymorphic sites. The rows are the arrays of samples. Blue represents a homozygote for the common allele, red represents a heterozygote (both common and rare allele) and yellow represents a homozygote for the rare allele.

Smoking status and sex

Cigarette smoke is the most significant risk factor for lung cancer. N‐nitrosamines and polycyclic aromatic hydrocarbons, the two major classes of tobacco‐related carcinogens, can produce as many as 100 mutations per cell genome by means of formation of DNA adducts.⁷⁵ Cigarette smoke, however, is not the mutagen of EGFR, as EGFR mutations are usually more frequent in patients who never smoke or have a low exposure to cigarette smoke (table 3). Risk factors associated with non‐smoking lung cancer, such as pre‐existing lung diseases, a family history of cancer, passive smoking, indoor cooking fumes and occupational exposures⁷⁶ could affect the occurrence of EGFR mutations. More studies are required to identify the causal mutagens and elucidate the potential mutagenic capability.

EGFR mutations are more frequent in female than in male patients, especially among Asians (table 3). This difference could be correlated with their distinct life styles and smoking habits. Generally, women tend to be non‐smokers or light smokers, and they play a heavier role in housework, such as cooking and cleaning, whereas those men who smoke tend to be heavy smokers and are more often involved in social activities. If the occurrence of EGFR mutations is associated with potential indoor mutagens, there is no doubt that women would have a higher mutation rate than men. On the other hand, female endocrine factors could also play a role in EGFR mutations.

Histology of NSCLC

EGFR mutations are more frequent in lung adenocarcinoma than in other histological types of NSCLC (table 3). As the predominant subtype of NSCLC, adenocarcinoma usually originates from a peripheral airway compartment, either surface epithelium or bronchial mucosal glands.⁷⁷ It is likely that the specific cellular milieu in these cell types is more susceptible to the influence of mutagens that induce EGFR mutations compared with other cell types.

Clinical significance of EGFR mutations

EGFR mutations have been considered the best predictive marker for response to treatment with EGFR‐TKIs in NSCLC. A number of retrospective studies have indicated that patients with the mutations have higher objective response rates to gefitinib than patients with the wild type (table 4). The first prospective clinical trial has recently been conducted among 75 patients with chemotherapy‐naive advanced NSCLC in Japan. Among 25 patients with EGFR mutations, 16 received gefitinib monotherapy and nine received standard chemotherapy. In agreement with the retrospective studies, the group receiving gefitinib had a 75% response rate to gefitinib.⁷⁸ Another recent prospective Phase II trial of gefitinib has shown similar results.⁷⁹

Table 4 EGFR mutations, clinical tyrosine kinase inhibitor response and survival.

	Mutation/EGFR‐TKI responders (%)	ORR* (mutations vs wild type)	TTP* (months; mutations vs wild type)	Median survival (months; mutation vs wild type)
Lynch40	8/9 (89)	—	—	—
Paez39	5/5 (100)	—	—	—
Huang44	7/9 (78)	—	—	—
Cappuzzo67	8/12 (67)	53% vs 5%	9.9 vs 2.6 (p = 0.02)	20.8 vs 8.4 (p = 0.09)
Eberhard47	8/26 (31)	53% vs 18%	8 vs 5 (p<0.001)	Not reached vs 10 (p<0.001)
Mitsudomi42	24/26 (92)	83% vs 10%	—	Significantly longer (p = 0.005)
Taron50	16/22 (73)	94% vs 13%	—	Not reached vs 9.9 (p<0.001)
Shih49	20/23 (87)	69% vs 9%	9 vs. 2.2 (p = 0.001)	13.9 vs 4.8 (p = 0.013)
Takano70	32/35 (91)	82% vs 11%	12.6 vs 1.7 (p<0.001)	20.4 vs 6.9 (p<0.001)

*ORR, objective response rate (complete response or partial response); TTP, time to progression.

—, data are not available.

Sensitivity to gefitinib or erlotinib is also associated with the type of mutation and the existence of additional mutations. Patients with deletion mutation in exon 19 have higher response rates than those with L858R in exon 21.⁴²,⁸⁰ An additional mutation in exon 22 (K884E) is able to make tumour cells sensitive to gefitinib but resistant to erlotinib.⁸¹ Further laboratory and clinical studies are required to elucidate the underlying mechanisms.

Furthermore, EGFR mutations have been considered a favourable prognostic indicator in NSCLC. Patients with the mutations have improved time‐to‐progression and longer overall survival than those with the wild type (table 4).⁴²,⁴⁹,⁵⁰,⁶⁷,⁷⁰ However, two Japanese studies have found conflicting results, namely that there is no significant difference in survival between the groups with and without the mutations.⁴³,⁶² This discrepancy could reflect the fact that other prognostic factors besides EGFR mutations also play an important role in survival among patients with NSCLC.

Secondary EGFR mutations and acquired resistance to gefitinib

Gefitinib has brought significant treatment response among patients with NSCLC with EGFR mutations. However, the mean duration of response is generally 6–8 months.³⁷,³⁸ Most of these patients eventually relapse and are resistant to further treatment with EGFR‐TKIs. The acquired resistance is closely associated with the development of a secondary mutation in exon 20, that is, a substitution of methionine for threonine at amino acid position 790 (T790M).⁵²,⁵³ Pao et al⁵² have reported that three of six patients with acquired resistance to TKIs harbour the T790M mutation in the progressing tumours. T790M is believed to abrogate the binding of TKIs to the ATP‐kinase‐binding pocket and lead to the continued activation of ErbB‐3/PI3K/Akt signalling.⁵³,⁸² The scenario is analogous to the secondary mutations in BCR‐ABL and KIT that confer acquired resistance to imatinib (Gleevec) in chronic myeloid leukaemia and gastrointestinal stromal tumour.⁸³,⁸⁴ T790M has been considered to be present only in relapsed tumours and its appearance to be secondary to treatment of TKIs. Nevertheless, rare T790M mutation has been detected in untreated tumour⁵⁵ and even in germline DNA, and it is associated with family aggregation of NSCLC.⁵⁴ Therefore, T790M could play a role in lung tumorigenesis.

Association of EGFR mutations with K‐ras and p53 gene mutations

K‐ras is a key molecule in the signalling pathways, which regulate cellular proliferation and transformation. The K‐ras mutation is one of the major genetic changes detected in lung cancer. The mutations are more common in adenocarcinomas, are closely correlated with cigarette smoke and are predominantly the guanine‐to‐thymine transversion at codon 12.⁸⁵,⁸⁶ Cigarette smoke has been proposed as the causal mutagen. P53 is another well‐characterised gene in lung cancer. Most of the mutations are located in evolutionally conserved regions, and the mutation spectra are different between smokers and non‐smokers. The relationship between the type of p53 mutations and the histology of lung cancer is still unclear.⁸⁷⁸⁸

The correlation between EGFR mutations and K‐ras mutations has been intensively investigated. Generally, EGFR mutations are present only in lung adenocarcinomas which do not harbour K‐ras mutations.⁴³,⁴⁶,⁴⁷,⁴⁸,⁵⁵,⁶² This observation might imply that there are different subsets of lung adenocarcinomas, which possess different mutation spectra and causal mutagens. By contrast, mutations in the EGFR gene and the p53 gene happen independently,⁴⁸,⁵⁵ but the status of gene–gene interaction and its influence on lung carcinogenesis and treatment response remains unknown.

Summary and future research directions

The discovery of somatic EGFR mutations has been claimed as a big victory of molecular medicine.⁴¹ It has had a considerable effect on the treatment of NSCLC. The strong associations between the mutations and gefitinib responsiveness and favourable prognosis provide forceful support for an individual genotype‐based therapeutic strategy. By now, a number of genotyping methods have been developed. However, most of them are technically complicated and need substantial research resources and expertise, which make routine clinical screening difficult and impractical. Developing a set of simple and accurate genotyping methods would be of great importance for translating this bench discovery into clinical application. On the other hand, further basic studies are required to investigate whether other functional consequences are present downstream of EGFR signalling pathways and whether additional predictive biomarkers are available. For patients with acquired resistance to EGFR‐TKIs, monitoring the EGFR mutation status in recurrent tumours is crucial for revealing the molecular mechanisms of drug resistance and developing new generations of TKIs. Finally, more large prospective studies to evaluate the therapeutic and prognostic value of EGFR mutations are required in patients with EGFR‐mutation‐enriched NSCLC. Prospective study is usually superior to retrospective study in bias and confounder control and therefore can provide more accurate assessment of the significance of EGFR mutations in the clinical treatment of NSCLC and of the possibility that EGFR‐TKIs might replace conventional chemotherapy as the preferred antitumour drugs for the subset of patients with NSCLC.

Gealy R, Zhang L, Siegfried JM, et al. Comparison of mutations in the p53 and K‐ras genes in lung carcinomas from smoking and nonsmoking women. Cancer Epidemiol Biomarkers Prev 1999;8(Pt 1):297–302.

Abbreviations

EGF(R) - epidermal growth factor (receptor)

NSCLC - non‐small cell lung cancer

PCR - polymerase chain reaction

SSCP - single‐strand conformation polymorphism

TKI - tyrosine kinase inhibitor