Epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) have been reported to be related to certain clinical characteristics (i.e., female, non-smokers with adenocarcinoma) and gefitinib responsiveness. This exploratory analysis was performed to determine the incidence of EGFR mutations in male smokers with squamous cell carcinoma, who were treated with EGFR tyrosine kinase inhibitor, gefitinib. Sixty-nine Korean NSCLC patients were treated with gefitinib in a prospective study. For a subset of 20 male patients with squamous cell carcinoma and a history of smoking, pretreatment tumor tissue samples were obtained and analyzed for EGFR mutations (exons 18 to 21). EGFR mutations were found in 3 (15%) patients, including in-frame deletions within exon 19 (n=2) and L858R missence mutation in exon 21 (n=1). These 3 patients with EGFR mutations responded to gefitinib, whereas only one of remaining 17 patients with wild-type EGFR achieved clinical response. Trend toward longer progression-free (5.8 vs. 2.4 months; P=0.07) was noted in patients with EGFR mutations compared to those with wild-type EGFR. Although male smokers with squamous cell carcinoma have not been considered ideal candidates for gefitinib treatment, significant incidence of EGFR mutations was observed. The molecular markers should be considered to predict clinical benefits from gefitinib.
Lung Neoplasms; Receptor, Epidermal Growth Factor; Mutation
This study was designed to determine the relationship of cigarette smoking to the frequency and qualitative differences among KRAS mutations in lung adenocarcinomas from Korean patients.
Materials and Methods
Detailed smoking histories were obtained from 200 consecutively enrolled patients with lung adenocarcinoma according to a standard protocol. EGFR (exons 18 to 21) and KRAS (codons 12/13) mutations were determined via direct-sequencing.
The incidence of KRAS mutations was 8% (16 of 200) in patients with lung adenocarcinoma. KRAS mutations were found in 5.8% (7 of 120) of tumors from never-smokers, 15% (6 of 40) from former-smokers, and 7.5% (3 of 40) from current-smokers. The frequency of KRAS mutations did not differ significantly according to smoking history (p=0.435). Never-smokers were significantly more likely than former or current smokers to have a transition mutation (G→A or C→T) rather than a transversion mutation (G→T or G→C) that is known to be smoking-related (p=0.011). In a Cox regression model, the adjusted hazard ratios for the risk of progression with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) were 0.24 (95% CI, 0.14-0.42; p<0.001) for the EGFR mutation and 1.27 (95% CI, 0.58-2.79; p=0.537) for the KRAS mutation.
Cigarette smoking did not influence the frequency of KRAS mutations in lung adenocarcinomas in Korean patients, but influenced qualitative differences in the KRAS mutations.
EGFR; KRAS; pulmonary adenocarcinoma; cigarette smoking; EGFR-tyrosine kinase inhibitors
Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are an effective treatment for advanced non-small cell lung cancer. The objective of the present study was to compare the efficacy of gefitinib and erlotinib in patients with pulmonary adenocarcinoma, whose tumor EGFR mutation status was known. Pulmonary adenocarcinoma patients who began receiving gefitinib or erlotinib treatment from January 2005 to December 2010, and whose tumor EGFR mutation status had been determined, were included. Clinical data, type of treatment response and survival time data were collected. Of the 224 patients enrolled, 124 received gefitinib treatment and 100 received erlotinib treatment. Of these patients, 146 individuals had tumors with EGFR-activating mutations (exon 19 deletions and/point mutation of L858R in exon 21) and 78 did not. There was no difference in treatment response whether or not the patients had tumors with EGFR-activating mutations at the time they received gefitinib or erlotinib treatment. The median progression-free survival (PFS) of the gefitinib and erlotinib groups was 7.6 and 7.9 months, respectively (p=0.4731). PFS was significantly longer for patients without EGFR-activating mutations who received erlotinib treatment (n=48; median, 4.5 months) than for those who received gefitinib treatment (n=30; median, 2.3 months), with a hazard ratio of 0.58 (95% CI, 0.35–0.96; p=0.0339). Patients whose tumors had EGFR-activating mutations displayed no difference in PFS with either gefitinib (n=94; median, 10.5 months) or erlotinib treatment (n=52; median, 10.4 months). In conclusion, PFS showed no difference with either agent in patients whose tumors had EGFR-activating mutations, but was significantly longer in patients whose tumors did not have EGFR-activating mutations when receiving erlotinib treatment.
epidermal growth factor receptor; tyrosine kinase inhibitors; non-small cell lung cancer
Epidermal growth factor receptor (EGFR) mutations are a potential predictor of the effectiveness of EGFR inhibitors for the treatment of lung cancer. Although EGFR mutations were reported to occur with high frequency in nonsmoking Japanese adenocarcinoma patients, the exact nature has not been fully elucidated. We examined EGFR gene mutations within exons 18–21 and their correlations to clinico-pathological factors and other genetic alterations in tumour specimens from 154 patients who underwent resection for lung cancer at Kyoto University Hospital. Epidermal growth factor receptor mutations were observed in 60 tumours (39.0%), all of which were adenocarcinoma. Among the patients with adenocarcinoma (n=108), EGFR mutations were more frequently observed in nonsmokers than former smokers or current smokers (83.0, 50.0, 15.2%, respectively), in women than men (76.3 vs 34.0%), in tumours with bronchio-alveolar component than those without bronchio-alveolar component (78.9 vs 42.9%), and in well or moderately differentiated tumours than poorly differentiated tumours (72.0, 64.4, 34.2%). No tumours with EGFR mutations had any K-ras codon 12 mutations, which were well-known smoking-related gene mutations. In conclusion, adenocarcinomas with EGFR mutation had a distinctive clinico-pathological feature unrelated to smoking. Epidermal growth factor receptor mutations may play a key role in the development of smoking-independent adenocarcinoma.
epidermal growth factor receptor; lung adenocarcinoma; smoking; p53; K-ras; single-strand conformation polymorphism
The detection of mutations in the epidermal growth factor receptor (EGFR) gene, which predict sensitivity to treatment with EGFR tyrosine kinase inhibitors (TKIs), represents a major advance in the treatment of lung adenocarcinoma. KRAS mutations confer resistance to EGFR -TKIs. The prevalence of these mutations in African-American patients has not been thoroughly investigated.
We collected formalin-fixed, paraffin-embedded material from resected lung adenocarcinomas from African-American patients at three institutions for DNA extraction. The frequencies of EGFR exon 19 deletions, exon 21 L858R substitutions and KRAS mutations in tumor specimens from African-American patients were compared to data in Caucasian patients (n=476).
EGFR mutations were detected in 23 of the 121 specimens from African-American patients (19%, 95% CI 13–27%), while KRAS mutations were found in 21 (17%, 95% CI 12−25%). There was no significant difference between frequencies of EGFR mutations comparing African-American and Caucasian patients, 19% vs. 13% (61/476, 95% CI 10–16%) (p=0.11). KRAS mutations were more likely among Caucasians, 26% (125/476, 95% CI 23−30%) (p=0.04).
This is the largest study to date examining the frequency of mutations in lung adenocarcinomas in African-Americans. Although KRAS mutations were somewhat less likely, there was no difference between the frequencies of EGFR mutations in African-American patients as compared to Caucasians. These results suggest that all patients with advanced lung adenocarcinomas should undergo mutational analysis prior to initiation of therapy.
EGFR mutation; KRAS; African-Americans; racial differences
Most lung cancers with activating epidermal growth factor receptor (EGFR) mutations respond to gefitinib, however resistance to this tyrosine kinase inhibitor (TKI) invariably ensues. The T790M mutation occurs in 50% and MET amplification in 20% of TKI-resistant tumors. Other secondary mutations (D761Y, L747S) are rare. Our goal was to determine the effects of erlotinib 150mg/day in EGFR mutated patients resistant to gefitinib 250mg/day, since the EGFR TKI erlotinib is given at a higher biologically active dose than gefitinib.
Retrospective review of 18 EGFR mutated (exon 19 deletions, L858R, L861Q) patients that were given gefitinib and subsequently erlotinib. 7 patients had tumor re-sampling after TKI therapy, and were analyzed for secondary EGFR mutations and MET amplification.
Most patients (14/18) responded to gefitinib with median progression-free survival (PFS) of 11 months (95%CI,4-16). After gefitinib resistance (de novo or acquired), 78% (14/18) of these patients displayed progressive disease while on erlotinib with PFS of 2 months (95%CI,2-3). 6/7 re-sampled patients acquired the T790M mutation, and 0/3 had MET amplification. Only 1 gefitinib-resistant patient with the acquired L858R-L747S EGFR, which in vitro is sensitive to achievable serum concentrations of erlotinib 150mg/day, achieved a partial response to erlotinib.
In EGFR mutated tumors resistant to gefitinib 250mg/day, a switch to erlotinib 150mg/day does not lead to responses in most patients. These findings are consistent with pre-clinical models, since the common mechanisms of TKI-resistance (T790M and MET amplification) in vitro are not inhibited by clinically achievable doses of gefitinib or erlotinib. Alternative strategies to overcome TKI resistance must be evaluated.
Epidermal growth factor receptor; EGFR; mutation; tyrosine kinase inhibitors; gefitinib; erlotinib; L858R; exon 19 deletions; T790M; lung cancer; non-small cell lung cancer
EGFR genotyping is now standard in the management of advanced lung adenocarcinoma, as this biomarker predicts marked benefit from treatment with EGFR tyrosine kinase inhibitors (TKIs). EGFR exon 19 insertions are a poorly described family of EGFR mutations, and their association with EGFR TKI-sensitivity in lung adenocarcinoma is uncertain.
Patients with lung cancers harboring EGFR exon 19 insertions were studied. The predicted effects of the insertions on the structure of the EGFR protein were examined, and EGFR exon 19 insertions were introduced into Ba/F3 cells to assess oncogenicity and in vitro sensitivity to EGFR TKIs. In patients receiving TKI, response magnitude was assessed with serial computed tomography (CT) measurement.
Twelve tumors harboring EGFR exon 19 insertions were identified; patients were predominately female (92%) and never-smokers (75%). The 11 specimens available for full sequencing all demonstrated an 18 bp insertion that resulted in the substitution of a Pro for Leu at residue 747. The mutant EGFR transformed the Ba/F3 cells, which were then sensitive to EGFR TKI. Six patients with measurable disease received TKI and 5 had a response on serial CT.
EGFR exon 19 insertions are a newly appreciated family of EGFR TKI-sensitizing mutations, and patients with tumors harboring these mutations should be treated with EGFR-TKI. While these mutations may be missed through the use of some mutation-specific assays, the addition of PCR product size analysis to multi-gene assays allows sensitive detection of both exon 19 insertion and deletion mutations.
KRAS mutations are found in ~ 25% of lung adenocarcinomas in Western countries and, as a group, have been strongly associated with cigarette smoking. These mutations are predictive of poor prognosis in resected disease as well as resistance to treatment with erlotinib or gefitinib.
We determined the frequency and type of KRAS codon 12 and 13 mutations and characterized their association with cigarette smoking history in patients with lung adenocarcinomas.
KRAS mutational analysis was performed on 482 lung adenocarcinomas, 81 (17%) of which were obtained from patients who had never smoked cigarettes. KRAS mutations were found in 15% (12/81; 95% CI 8%-24%) of tumors from never smokers. Similarly, 22% (69/316; 95% CI 17%-27%) of tumors from former smokers, and 25% (21/85; 95% CI 16%-35%) of tumors from current smokers had KRAS mutations. The frequency of KRAS mutation was not associated with age, gender, or smoking history. The number of pack years of cigarette smoking did not predict an increased likelihood of KRAS mutations. Never smokers were significantly more likely than former or current smokers to have a transition mutation (G→A) rather than the transversion mutations known to be smoking related (G→T or G→C; p<0.0001).
Based upon our data, KRAS mutations are not rare among never smokers with lung adenocarcinoma and such patients have a distinct KRAS mutation profile. The etiologic and biological heterogeneity of KRAS mutant lung adenocarcinomas is worthy of further study.
The presence of EGFR kinase domain mutations in a subset of NSCLC patients correlates with the response to treatment with the EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Although most EGFR mutations detected are short deletions in exon 19 or the L858R point mutation in exon 21, more than 75 different EGFR kinase domain residues have been reported to be altered in NSCLC patients. The phenotypical consequences of different EGFR mutations may vary dramatically, but the majority of uncommon EGFR mutations have never been functionally evaluated.
We demonstrate that the relative kinase activity and erlotinib sensitivity of different EGFR mutants can be readily evaluated using transfection of an YFP-tagged fragment of the EGFR intracellular domain (YFP-EGFR-ICD), followed by immunofluorescence microscopy analysis. Using this assay, we show that the exon 20 insertions Ins770SVD and Ins774HV confer increased kinase activity, but no erlotinib sensitivity. We also show that, in contrast to the common L858R mutation, the uncommon exon 21 point mutations P848L and A859T appear to behave like functionally silent polymorphisms.
The ability to rapidly obtain functional information on EGFR variants of unknown relevance using the YFP-EGFR-ICD assay might prove important in the future for the management of NSCLC patients bearing uncommon EGFR mutations. In addition, our assay may be used to determine the response of resistant EGFR mutants to novel second-generation TKIs.
The epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs), gefitinib and erlotinib, are reversible competitive inhibitors of the tyrosine kinase domain of EGFR that bind to its adenosine-5′ triphosphate-binding site. Somatic activating mutations of the EGFR gene, increased gene copy number and certain clinical and pathological features have been associated with dramatic tumor responses and favorable clinical outcomes with these agents in patients with non-small-cell lung cancer (NSCLC). The specific types of activating mutations that confer sensitivity to EGFR TKIs are present in the tyrosine kinase (TK) domain of the EGFR gene. Exon 19 deletion mutations and the single-point substitution mutation L858R in exon 21 are the most frequent in NSCLC and are termed ‘classical’ mutations. The NSCLC tumors insensitive to EGFR TKIs include those driven by the KRAS and MET oncogenes. Most patients who initially respond to gefitinib and erlotinib eventually become resistant and experience progressive disease. The point mutation T790M accounts for about one half of these cases of acquired resistance. Various second-generation EGFR TKIs are currently being evaluated and may have the potential to overcome T790M-mediated resistance by virtue of their irreversible inhibition of the receptor TK domain.
epidermal growth factor receptor; mutation; non-small-cell lung cancer; tyrosine kinase inhibitor; tyrosine kinase
Most advanced non–small-cell lung cancers (NSCLCs) with activating epidermal growth factor receptor (EGFR) mutations (exon 19 deletions or L858R) initially respond to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib. However, over time (median of 6−12 months), most tumors develop acquired resistance to EGFR TKIs. Intense research in these NSCLCs has identified two major mechanisms of resistance to gefitinib/erlotinib: secondary resistance mutations and “oncogene kinase switch” systems. The secondary T790M mutation occurs in 50% of EGFR-mutated patients with TKI resistance, and in vitro, this mutation negates the hypersensitivity of activating EGFR mutations. Sensitive detection methods have identified a proportion of TKI-naive tumors that carry T790M, and these resistant clones may be selected after exposure to gefitinib or erlotinib. Other secondary resistance mutations (D761Y, L747S, T854A) seem to be rare. The amplification of the MET oncogene is present in 20% of TKI-resistant tumors; however, in half of the cases with this “oncogene kinase switch” mechanism the T790M is coexistent. It is possible that other kinases (such as insulin-like growth factor-1 receptor [IGF-1R]) might also be selected to bypass EGFR pathways in resistant tumors. The growing preclinical data in EGFR-mutated NSCLCs with acquired resistance to gefitinib or erlotinib has spawned the initiation or conception of clinical trials testing novel EGFR inhibitors that in vitro inhibit T790M (neratinib, XL647, BIBW 2992, and PF-00299804), MET, or IGF-1R inhibitors in combination with EGFR TKIs, and heat shock protein 90 inhibitors. Ongoing preclinical and clinical research in EGFR-mutated NSCLC has the potential to significantly improve the outcomes of patients with these somatic mutations.
BIBW 2992; D761Y; Gefitinib; ErbB3; Erlotinib; HKI-272; L747S; MET; PF-00299804; T790M; XL647
Somatic mutations in the tyrosine kinase domain of EGFR are associated with sensitivity of lung adenocarcinomas to the EGFR tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib. Acquired drug resistance is frequently associated with a secondary somatic mutation that leads to substitution of methionine for threonine at position 790 (T790M). We aimed to identify additional second-site alterations associated with acquired resistance.
Tumor samples were obtained from 48 patients with acquired resistance. Tumor cell DNA was analyzed for EGFR kinase domain mutations. Molecular analyses were then performed to characterize biological properties of a novel mutant EGFR allele.
A previously unreported mutation in exon 21 of EGFR, which leads to substitution of alanine for threonine at position 854 (T854A), was identified in one patient with a drug-sensitive EGFR L858R-mutant lung adenocarcinoma after long-term treatment with TKIs. The T854A mutation was not detected in a pretreatment tumor sample. Crystal structure analyses of EGFR suggest that the T854 side chain is within contact distance of gefitinib and erlotinib. Surrogate kinase assays demonstrate that the EGFR T854A mutation abrogates inhibition of tyrosine phosphorylation by erlotinib. Such resistance appears to be overcome by a new irreversible dual EGFR/HER2 inhibitor, BIBW 2992.
The T854A mutation is the second reported second-site acquired resistance mutation that is within contact distance of gefitinib and erlotinib. These data suggest that acquired resistance to ATP-mimetic EGFR kinase inhibitors may often be associated with amino acid substitutions that alter drug contact residues in the EGFR ATP-binding pocket.
Erlotinib is clinically effective in patients with non–small-cell lung cancer (NSCLC) who have adenocarcinoma, are never or limited former smokers, or have EGFR mutant tumors. We investigated the efficacy of erlotinib alone or in combination with chemotherapy in patients with these characteristics.
Patients and Methods
Patients with advanced NSCLC (adenocarcinoma) who were epidermal growth factor receptor tyrosine kinase inhibitor and chemotherapy naive never or light former smokers (smokers of > 100 cigarettes and ≤ 10 pack years and quit ≥ 1 year ago) were randomly assigned to continuous erlotinib or in combination with carboplatin and paclitaxel (ECP) for six cycles followed by erlotinib alone. The primary end point was progression-free survival (PFS). Tissue collection was mandatory.
PFS was similar (5.0 v 6.6 months; P = .1988) in patients randomly assigned to erlotinib alone (arm A; n = 81) or to ECP (arm B; n = 100). EGFR mutation analysis was possible in 91% (164 of 181) of patients, and EGFR mutations were detected in 40% (51 of 128) of never smokers and in 42% (15 of 36) of light former smokers. In arm A, response rate (70% v 9%), PFS (14.1 v 2.6 months), and overall survival (OS; 31.3 v 18.1 month) favored EGFR-mutant patients. In arm B, response rate (73% v 30%), PFS (17.2 v 4.8 months), and OS (38.1 v 14.4 months) favored EGFR-mutant patients. Incidence of grades 3 to 4 hematologic (2% v 49%; P < .001) and nonhematologic (24% v 52%; P < .001) toxicity was greater in patients treated with ECP.
Erlotinib and erlotinib plus chemotherapy have similar efficacy in clinically selected populations of patients with advanced NSCLC. EGFR mutations identify patients most likely to benefit.
Activating mutations in Epidermal Growth Factor Receptor (EGFR) are common in lung adenocarcinoma of never smokers but are rare in other types of cancer. Here we have analysed mutations in exons 19 to 21 of EGFR and in exons 19 and 20 of the EGFR homolog HER2 in 54 cases of Esophageal Squamous Cell Carcinomas (ESCC) from patients recruited in Kashmir, India, a region of high incidence for this cancer. We report the detection of 3 mutations (6%) in the ATP-binding regulatory loops of the tyrosine kinase domain of EGFR (deletion 746–750, P753L, G719D). No mutation was found in HER2. This is the first report of activating EGFR mutations in ESCC, of the same type as those detected in lung adenocarcinoma of never-smokers. This suggests that a small proportion of ESCC patients in this high incidence area may benefit from treatment with EGFR tyrosine kinase inhibitors.
EGFR; Mutations; Esophageal Cancer; Kashmir
Lung cancer patients with mutations in EGFR tyrosine kinase have improved prognosis when treated with EGFR inhibitors. We hypothesized that EGFR mutations may be related to residential radon or passive tobacco smoke.
This hypothesis was investigated by analyzing EGFR mutations in seventy lung tumors from a population of never and long-term former female smokers from Missouri with detailed exposure assessments. The relationship with passive-smoking was also examined in never-smoking female lung cancer cases from the Mayo clinic.
Overall, the frequency of EGFR mutation was 41% [95% Confidence Interval (CI): 32-49%]. Neither radon nor passive-smoking exposure was consistently associated with EGFR mutations in lung tumors.
The results suggest that EGFR mutations are common in female, never-smoking, lung cancer cases from the U.S, and EGFR mutations are unlikely due to exposure to radon or passive-smoking.
EGFR mutations; never-smokers; lung cancer; radon; passive-smoking; second hand smoke; tobacco smoke
Epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) predict better outcome to EGFR tyrosine kinase inhibitors (TKIs). The most common mutations are exon 19 deletions (most frequently E746-A750) and L858R point mutation in exon 21. Here, we evaluated the accuracy of novel EGFR mutation specific antibodies in a Japanese cohort with NSCLC and compared to direct DNA sequencing and clinical outcome.
Materials and methods
Immunohistochemistry (IHC) using antibodies specific for the E746-A750 and L858R mutations in EGFR was performed on tissue microarrays of tumors from 70 gefitinib treated NSCLC patients. Extracted DNA was sequenced for mutational analysis of EGFR exons 18 to 21.
DNA sequencing showed EGFR mutations in 41 patients (58.6%), and exon 19 deletions in 18 patients (25.7%), 61% (11/18) had a deletion in the range of E746-A750) and 12 (17.1%) had exon 21 mutations (L858R). IHC showed, for the E746-A750 and L858R mutations, sensitivity (81.8% and 75%), specificity (100%, 96.6%), PPV (100%, 81.8%) and NPV (96.7%, 94.9%). Analysis for objective response rates (ORR) and survival were not correlated to IHC staining, although the combined staining showed non-significant trends towards better overall survival for patients with EGFR mutations.
The mutation specific IHC antibodies have high sensitivity and specificity for pre-defined EFGR mutations and may be suitable for screening for these pre-defined mutations. However, negative IHC results require further mutation analyses prior to excluding EGFR-targeted therapy.
EGFR; Biomarkers; Lung Cancer; NSCLC; Mutation
Our previous study revealed that 90% (47 of 52; 95% CI: 0.79–0.96) of Chinese never-smokers with lung adenocarcinoma harbor known oncogenic driver mutations in just four genes: EGFR, ALK, HER2, and KRAS. Here, we examined the status of known driver mutations specifically in female never-smokers with lung adenocarcinoma.
Tumors were genotyped for mutations in EGFR, KRAS, ALK, HER2, and BRAF. Data on age, stage, tumor differentiation, histological subtypes, and molecular alterations were recorded from 349 resected lung adenocarcinomas from female never-smokers. We further compared the clinicopathological parameters according to mutational status of these genes.
Two hundred and sixty-six (76.2%) tumors harbored EGFR mutations, 16 (4.6%) HER2 mutations, 15 (4.3%) EML4-ALK fusions, seven (2.0%) KRAS mutations, and two (0.6%) BRAF mutations. In univariate analysis, patients harboring EGFR mutations were significantly older (p<0.001), whereas patients harboring HER2 mutations were significantly younger (p=0.036). Higher prevalence of KRAS (p=0.028) and HER2 (p=0.021) mutations was found in invasive mucinous adenocarcinoma (IMA). The frequency of EGFR mutations was positively correlated with acinar predominant tumors (p=0.002). Multivariate analysis revealed that older age at diagnosis (p=0.013) and acinar predominant subtype (p=0.005) were independent predictors of EGFR mutations. Independent predictors of HER2 mutations included younger age (p=0.030) and IMA (p=0.017). IMA (p=0.006) and poor differentiation (p=0.028) were independently associated with KRAS mutations.
The frequency of driver mutations in never-smoking female lung adenocarcinoma varies with histological subtypes and age at diagnosis. These data have implications for both clinical trial design and therapeutic strategies.
Lung adenocarcinoma; Female; Never smoker; EGFR mutation; HER2 mutation; Acinar; Mucinous; Age
Targeted therapy against epidermal growth factor receptor (EGFR) represents a major therapeutic advance in lung cancer treatment. Somatic mutations of the EGFR gene, most commonly L858R (exon 21) and short in-frame exon 19 deletions, have been found to confer enhanced sensitivity towards the inhibitors gefitinib and erlotinib. We have recently identified an EGFR mutation E884K, in combination with L858R, in a patient with advanced lung cancer who progressed on erlotinib maintenance therapy, and subsequently had leptomeningeal metastases that responded to gefitinib. The somatic E884K substitution appears to be relatively infrequent, and resulted in a mutant lysine residue that disrupts an ion pair with residue R958 in the EGFR kinase domain C-lobe, an interaction that is highly conserved within the human kinome as demonstrated by our sequence analysis and structure analysis. Our studies here, using COS-7 transfection model system, show that E884K works in concert with L858R in-cis, in a dominant fashion, to change downstream signaling, differentially induce MAPK-ERK1/2 signaling and associated cell proliferation, and differentially alter sensitivity of EGFR phosphorylation inhibition by ERBB family inhibitors in an inhibitor-specific fashion. Mutations of the conserved ion pair E884-R958 may result in conformational changes that alter kinase substrate recognition. The analogous E1271K-MET mutation conferred differential sensitivity towards preclinical MET inhibitors SU11274 (unchanged), and PHA665752 (more sensitive). Systematic bioinformatics analysis of the mutation catalog in the human kinome (COSMIC) revealed the presence of cancer-associated mutations involving the conserved E884 homologous residue, and adjacent residues at the ion pair, in known proto-oncogenes (KIT, RET, MET, FAK) and tumor suppressor gene (LKB1). Targeted therapy using small molecule inhibitors should take into account potential cooperative effects of multiple kinase mutations, and their specific effects on downstream signaling and inhibitor sensitivity. Improved efficacy of targeted kinase inhibitors may be achieved by targeting the dominant activating mutations present.
EGFR; MET; mutation; tyrosine kinase inhibitor; sensitivity; resistance; structure; kinome
To determine the proportion of lung adenocarcinomas from East Asian never-smokers who harbor known oncogenic driver mutations.
Patients and Methods
In this surgical series, 52 resected lung adenocarcinomas from never-smokers (< 100 cigarettes in a lifetime) at a single institution (Fudan University, Shanghai, China) were analyzed concurrently for mutations in EGFR, KRAS, NRAS, HRAS, HER2, BRAF, ALK, PIK3CA, TP53 and LKB1.
Forty-one tumors harbored EGFR mutations, three harbored EML4-ALK fusions, two harbored HER2 insertions, and one harbored a KRAS mutation. All mutations were mutually exclusive. Thus, 90% (47 of 52; 95% CI, 0.7896 to 0.9625) of lung adenocarcinomas from never-smokers were found to harbor well-known oncogenic mutations in just four genes. No BRAF, NRAS, HRAS, or LKB1 mutations were detected, while 15 had TP53 mutations. Four tumors contained PIK3CA mutations, always together with EGFR mutations.
To our knowledge, this study represents the first comprehensive and concurrent analysis of major recurrent oncogenic mutations found in a large cohort of lung adenocarcinomas from East Asian never-smokers. Since drugs are now available that target mutant EGFR, HER2, and ALK, respectively, this result indicates that prospective mutation testing in these patients should successfully assign a targeted therapy in the majority of cases.
Tumor epidermal growth factor receptor (EGFR) mutation analysis is significant for making treatment decisions for metastatic pulmonary adenocarcinoma. However, less than half of patients have adequate tumor samples for mutation analysis. Patients with adenocarcinoma of the lungs who were due to receive erlotinib treatment were included in the present study. Tumor EGFR mutation status was analyzed using DNA sequencing. Plasma specimens from the patients were collected prior to erlotinib treatment. The plasma-free DNA EGFR mutation status was analyzed using the PCR clamp method. A total of 54 consecutive patients were included in the study. The plasma-free DNA EGFR mutation status of the 54 patients was analyzed. Only 30 patients had adequate tumor samples for EGFR analysis, including 15 with activating mutations (exon 19 deletions or L858R). EGFR-activating mutations were detected in the plasma-free DNA in 25 of 54 patients. The response rate was 86.7 and 33.3% in patients with and without tumor activating mutations, respectively (p=0.002). The response rate was 68 and 31% based on the patients’ plasma-free DNA EGFR mutation status, respectively (p=0.013). No significant difference in progression-free survival (PFS) was observed between patients with and without EGFR-activating mutations, according to data from tumor tissue or plasma-free DNA analysis, although the median PFS time was longer for those patients with EGFR-activating mutations in plasma samples. Plasma EGFR mutation analysis is useful for adenocarcinoma patients who have no or inadequate tumor samples available for EGFR examination. Patients with plasma EGFR-activating mutations had an improved response rate and a statistically insignificant longer PFS.
epidermal growth factor receptor; erlotinib; targeted therapy; adenocarcinoma
Somatically acquired mutations in the epidermal growth factor receptor (EGFR) gene in lung cancer are associated with significant clinical responses to gefitinib, a tyrosine kinase inhibitor that targets EGFR. We screened the EGFR in 469 resected tumours of patients with lung cancer, which included 322 adenocarcinomas, 102 squamous cell carcinomas, 27 large cell carcinomas, 13 small cell carcinomas, and five other cell types. PCR with a specific condition was performed to identify any deletion in exon 19, while mutant-allele-specific amplification was performed to identify a mutation in codon 858 of exon 21. EGFR mutations were found in 136 cases (42.2%) with adenocarcinoma, in one case with large cell carcinoma, and in one case with pleomorphic carcinoma. An in-frame deletion in exon 19 was found in 62 cases while an L858R mutation was found in 77 cases. In the 322 cases with adenocarcinoma, these mutations were more frequently found in women than in men (P=0.0004), in well differentiated tumours than in poorly differentiated tumours (P=0.0014), and in patients who were never smokers than in patients who were current/former smokers (P<0.0001). The mutation was more frequently observed in patients who smoked ⩽20 pack-year, and in patients who quit at least 20 years before the date of diagnosis for lung cancer. The K-ras mutations were more frequently found in smokers than in never smokers, and in high-dose smokers than in low-dose smokers. In conclusion, the mutations within the tyrosine kinase domain of EGFR were found to specifically occur in lung adenocarcinoma patients with a low exposure of tobacco smoking.
EGFR; mutation; lung cancer; adenocarcinoma; smoking; screening; K-ras
EGFR is a major anticancer drug target in human epithelial tumors. One effective class of agents is the tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. These drugs induce dramatic responses in individuals with lung adenocarcinomas characterized by mutations in exons encoding the EGFR tyrosine kinase domain, but disease progression invariably occurs. A major reason for such acquired resistance is the outgrowth of tumor cells with additional TKI-resistant EGFR mutations. Here we used relevant transgenic mouse lung tumor models to evaluate strategies to overcome the most common EGFR TKI resistance mutation, T790M. We treated mice bearing tumors harboring EGFR mutations with a variety of anticancer agents, including a new irreversible EGFR TKI that is under development (BIBW-2992) and the EGFR-specific antibody cetuximab. Surprisingly, we found that only the combination of both agents together induced dramatic shrinkage of erlotinib-resistant tumors harboring the T790M mutation, because together they efficiently depleted both phosphorylated and total EGFR. We suggest that these studies have immediate therapeutic implications for lung cancer patients, as dual targeting with cetuximab and a second-generation EGFR TKI may be an effective strategy to overcome T790M-mediated drug resistance. Moreover, this approach could serve as an important model for targeting other receptor tyrosine kinases activated in human cancers.
Somatic mutations in the kinase domain of the epidermal growth factor receptor tyrosine kinase gene EGFR are common in lung adenocarcinoma. The presence of mutations correlates with tumor sensitivity to the EGFR inhibitors erlotinib and gefitinib, but the transforming potential of specific mutations and their relationship to drug sensitivity have not been described.
Methods and Findings
Here, we demonstrate that EGFR active site mutants are oncogenic. Mutant EGFR can transform both fibroblasts and lung epithelial cells in the absence of exogenous epidermal growth factor, as evidenced by anchorage-independent growth, focus formation, and tumor formation in immunocompromised mice. Transformation is associated with constitutive autophosphorylation of EGFR, Shc phosphorylation, and STAT pathway activation. Whereas transformation by most EGFR mutants confers on cells sensitivity to erlotinib and gefitinib, transformation by an exon 20 insertion makes cells resistant to these inhibitors but more sensitive to the irreversible inhibitor CL-387,785.
Oncogenic transformation of cells by different EGFR mutants causes differential sensitivity to gefitinib and erlotinib. Treatment of lung cancers harboring EGFR exon 20 insertions may therefore require the development of alternative kinase inhibition strategies.
Different EGFR mutations are associated with lung cancer. All of the classes can transform fibroblasts and lung epithelial cells, most are sensitive to erlotinib and gefininib, but exon 20 mutations are only sensitive to an irreversible EGFR inhibitor.
Sensitivity to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) and frequency of activation mutations in EGFR is lower in Caucasian than Asian non small-cell lung cancer (NSCLC) patients. Increased EGFR gene copy numbers evaluated by fluorescence in situ hybridization (FISH) has been reported as predictor of clinical benefit from EGFR-TKIs in Caucasian NSCLC patients. This study was carried out to verify whether EGFR FISH had similar performance in Japanese patients.
A cohort of 44 Japanese patients with recurrent NSCLC after surgery was treated with gefitinib 250 mg daily. The cohort included 48% females and 52% never-smokers; 73% had prior chemotherapy and 57% had stage III-IV at the time of surgery. Adenocarcinoma was the most common histology (86%). FISH was performed using the EGFR/Chromosome Enumeration Probe 7 and PathVysion DNA probes (Abbott Molecular). Specimens were classified as FISH positive when showing gene amplification or high polysomy (≥4 copies of the gene in ≥40% of tumor cells). Tumor response to gefitinib was assessed by RECIST for 33 patients with measurable diseases.
Twenty-nine tumors (66%) were EGFR FISH+ and 23 (53%) were HER2 FISH+. Overall response rate was 52%, representing 65% of EGFR FISH+ patients and 29% of EGFR FISH+ patients (p = 0.0777). Survival was not impacted by the EGFR FISH (p = 0.9395) or the HER2 FISH (p = 0.0671) status. EGFR FISH= was significantly associated with HER2 FISH+ (p = 0.015) and presence of EGFR mutation (p = 0.0060). EGFR mutation significantly correlated with response (p < 0.0001) and survival after gefitinib (p = 0.0204). EGFR and HER2 FISH status were not associated with KRAS mutation.
Frequency of EGFR FISH+ status was higher and its predictive power for TKI sensitivity was lower in this Japanese cohort than in Western NSCLC cohorts. These findings support differences in the mechanisms of EGFR pathway activation in NSCLC between Asian and Caucasian populations. Confirmation of these results in larger cohorts is warranted.
FISH; EGFR; HER2; KRAS; Biomarkers; NSCLC; Tyrosine inhibitors
EGFR mutations now guide the clinical use of EGFR-targeted therapy in lung cancer. However, standard EGFR mutation analysis requires a minimum amount of tumor tissue, which may not be available in certain situations. In this study, we combined a mass spectrometry genotyping assay (Sequenom) with a mutant-enriched PCR (ME-PCR) to detect EGFR mutations in free plasma DNA from patients with lung cancer.
DNAs were extracted from 31 plasma samples from 31 patients and analyzed by both methods for EGFR exon 19 deletion and EGFR L858R mutation. Results in plasma DNA samples were compared with EGFR mutation status obtained in tumor DNA (18/31 EGFR mutant). The relationship of EGFR mutation status in tumor and/or plasma samples to overall survival was assessed.
The EGFR mutation status in plasma DNA was identical to the primary tumor in 61% of patients (19/31). By mass spectrometry genotyping, the plasma samples contained mutant DNA corresponding to 5/14 EGFR exon 19 deletions and 3/4 EGFR L858R mutations previously diagnosed in the matched tumors. Two samples were positive in plasma DNA but negative in primary tumor tissue. Results were similar for ME-PCR. For patients treated with erlotinib, overall survival was correlated with the presence of EGFR mutation in plasma and/or tumor tissue (p=0.002), with the two patients positive only in plasma DNA showing responses and favorable outcomes.
The detection of EGFR mutations in plasma DNA samples by mass spectrometry genotyping and ME-PCR is feasible. A positive EGFR result in plasma DNA has a high predictive value for tumor EGFR status and for favorable clinical course on EGFR-targeted therapy and could therefore be useful in guiding clinical decisions in patients with insufficient or unavailable tumor specimens.
Lung cancer; EGFR; Plasma; Mass spectrometry; Mutant-enriched PCR; Adenocarcinoma