Identification of non-small-cell lung cancer with activating EGFR mutations in malignant effusion and cerebrospinal fluid: rapid and sensitive detection of exon 19 deletion E746-A750 and exon 21 L858R mutation by immunocytochemistry

Background

Recently, we have reported that EGFR mutation-specific antibodies performed well in immunohistochemical analysis, with good sensitivity. We investigated whether this method could detect non-small-cell lung cancer (NSCLC) carrying EGFR mutations in malignant effusions and cerebrospinal fluid (CSF), comparable to the peptide nucleic acid–locked nucleic acid (PNA–LNA) PCR clamp assay. Furthermore, we compared activating EGFR mutations between primary and recurrent NSCLC.

Patients and methods

Twenty-four patients with NSCLC effusions and CSF were examined by immunocytochemistry using antibodies specific for the E746-A750 deletion mutation in exon 19 and the L858R point mutation in exon 21. The PNA–LNA PCR clamp assay was used to detect the E746-A750 deletion at exon 19, L858R mutation at exon 21, and T790M mutation at exon 20.

Results

We were able to identify EGFR mutations in NSCLC effusion and CSF with a sensitivity of 100% (5/5) using the anti-delE746-A750 antibody and 100% (8/8) using the anti-L858R antibody. Furthermore, in samples without these EGFR mutations, immunocytochemistry with the two specific antibodies identified 91% (10/11) as negative for both the deletion and the point mutations in EGFR. Activating EGFR mutations decreased in recurrent NSCLC compared with primary NSCLC, and the T790M mutation was detected in recurrent NSCLC of patients receiving gefitinib treatment.

Conclusions

Identification of EGFR mutations is important for patients with primary and recurrent NSCLC. Rapid and sensitive immunocytochemistry using mutation-specific antibodies to detect EGFR mutations will be useful for diagnosing responsiveness to EGFR-targeted drugs.     

Immunohistochemistry for EGFR Mutation Detection in Non–Small-Cell Lung Cancer

Introduction

The sensitivity and specificity of immunohistochemistry (IHC) was compared with the standard polymerase chain reaction (PCR)-based method for detecting common activating epidermal growth factor receptor (EGFR) mutations in non–small-cell lung cancer (NSCLC). Additionally, we evaluated predictive value of IHC EGFR mutation–positive status for EGFR tyrosine kinase inhibitor (TKI) treatment outcome and estimated cost-effectiveness for the upfront IHC testing.

The usefulness of mutation-specific antibodies in detecting epidermal growth factor receptor mutations and in predicting response to tyrosine kinase inhibitor therapy in lung adenocarcinoma

Introduction

Among the mutations of epidermal growth factor receptor (EGFR), deletions in exon 19 (DEL), and point mutations in exon 21 (L858R) predict the response to EGFR-tyrosine kinase inhibitors (TKIs) in primary lung adenocarcinoma. The ability to detecting such mutations using immunohistochemistry (IHC) would be advantageous.

Methods

The molecular-based and IHC-based EGFR mutations were analyzed in 577 lung adenocarcinomas using high resolution melting analysis (HRMA) and 2 mutation-specific antibodies, respectively.

Results

In the molecular-based analyses, DEL was detected in 135 cases (23%), and L858R was detected in 172 cases (30%). In the IHC-based analyses, a positive reaction was detected in 59 cases (10%) for the DEL-specific antibody, and in 139 cases (24%) for the L858R-specific antibody. With the molecular-based results set as the gold standard, the sensitivity and specificity of the DEL-specific antibody were 42.2% and 99.5%, respectively, while the sensitivity and specificity of the L858R-specific antibody were 75.6% and 97.8%, respectively. The antibody specificities improved when the threshold for the mutation-positive reactions was set as >50% of immunopositive tumor cells. The significant predictors of the clinical response to EGFR-TKI were molecular-based EGFR mutations (p < 0.001) and IHC-based EGFR mutations (p = 0.001). However, a multivariate analysis revealed that only molecular-based EGFR mutations were significantly correlated with the clinical response (p < 0.001).

Conclusions

Mutation-specific antibodies demonstrated extremely high specificities, but their sensitivities were not higher than those of molecular-based analyses. However, IHC should be performed before a molecular-based analysis, because it is more cost-effective and can effectively select candidates for EGFR-TKI therapy.     

Possible differential EGFR-TKI efficacy among exon 19 deletional locations in EGFR-mutant non-small cell lung cancer

Background

Exon 19 deletion mutations (Del-19s) and the exon 21 L858R point mutation are the most common epidermal growth factor receptor (EGFR) mutations. In Del-19, several subtypes actually exist, consisting of the deletional location with or without amino acid insertion/substitution. Little evidence has been described whether the Del-19 subtype affects EGFR-tyrosine kinase inhibitor (TKI) efficacy.

Methods

Between December 2005 and July 2012, we investigated 105 patients harboring a Del-19 who had received EGFR-TKIs. Efficacies of EGFR-TKIs such as response rate (RR), progression-free survival (PFS), and overall survival (OS) were retrospectively evaluated among various patient characteristics.

Results

Among these 105 patients with Del-19s, 78 (74%) patients had a deletion from E746 (Del-E746), and 27 (26%) exhibited a deletion from L747 (Del-L747). Median PFS of Del-E746 (11.7 months, 95% confidence interval [CI]: 9.3–15.6) was significantly longer than Del-L747 (10.0 months, 95% CI: 6.4–12.7) (p = 0.022). Insertions/substitutions were found in 19 patients (18%), and 91 patients (82%) were without insertions/substitutions. Median PFS without insertions/substitutions (11.7 months, 95% CI 9.3–15.2) was significantly longer than with insertions/substitutions (10.0 months, 95% CI: 4.0–10.6) (p = 0.024). No relationships were found for RR among all patient characteristics. In multivariate analysis, performance status (PS) (0/1 vs 2/3) and initial deletion site (Del-E746 vs Del-L747) were significant factors for longer PFS, whereas PS, gender (male vs female) and histology (adeno vs squamous) for longer OS.

Conclusions

Our data indicated better efficacy of EGFR-TKI in Del-E746 than Del-L747. Deletional locations may affect EGFR-TKI efficacy.     

A novel method for detection of mutation in epidermal growth factor receptor

Background

For the rapid and sensitive screening of epidermal growth factor receptor (EGFR) hot-spot mutations, we developed a novel method combining mutant-enriched PCR with amplification refractory mutation system (ARMS) TaqMan real-time PCR in a one-step reaction tube.

Methods and results

We designed two pairs of primers to enrich and genotyping each mutation (E746_A750del and L858R): nest primers and ARMS primers. Before the PCR assays were carried out, the restriction enzymes were used to cut wild alleles. The results showed that this method could detect mutant alleles mixed samples containing 0.1% with a cutoff ΔCt value of 12. We used this method in a survey of 73 non-small cell lung cancer (NSCLC) samples, detecting 14 mutant samples of E746_A750del and 12 mutant samples of L858R. The results well agreed with the results of DxS. All unmatched samples were identified by sequencing and the results showed that our method has high specificty.

Conclusion

The mutant-enriched ARMS TaqMan PCR could be useful in the detection of mutation in clinical samples containing only a small number of mutant alleles.     

Epidermal Growth Factor Receptor Gene Mutation Detection in Histology and Cytology Specimens of Primary Lung Adenocarcinoma: Immunohistochemistry Versus the Molecular Method

Background: Epidermal growth factor receptor (EGFR) gene in lung adenocarcinoma is associated with good clinical response to EGFR-tyrosine kinase therapy. The two most common EGFR gene mutations, representing 80 to 90%, are the E746-A750 deletion in exon 19 and the L858R point mutation in exon 21. Materials and Methods: We have conducted the study to evaluate immunohistochemistry’s performance in detecting the E746-A750 deletion in exon 19 of the EGFR gene in primary lung adenocarcinoma cases. This study examined 133 cases of primary lung adenocarcinoma for three years duration. The selected cases were tested for EGFR gene mutations by real-time PCR by a reference laboratory. Most cases (124) were diagnosed by tissue biopsy, though nine used cell block cytology. We performed an immunohistochemistry test on 75 cases that contained adequate diagnostic material in the paraffin block. Results: The test result was scored as 0 to 3+, based on the staining intensity and percentage of positive tumor cells. We evaluated the immunohistochemistry test’s sensitivity and specificity compared to the EGFR gene mutations by real-time PCR. There was a significant association between gender, smoking status, and the EGFR gene mutations (P < 0.001). The overall sensitivity and specificity of the immunohistochemistry test were 40% and 100%, respectively. The positive predictive value and negative predictive values were 100% and 76.9%, each. Conclusions: The immunohistochemistry has high specificity but low sensitivity in the detection of E746-A750 deletion in exon 19 of the EGFR gene. The mutation-specific antibody used in this study was unable to detect other uncommon variants of exon 19 deletions. With high specificity value, immunohistochemistry may provide an adjunct to molecular testing for detecting the most common EGFR gene mutations in cases of a low cellularity sample, financially-limited situations, or in critically ill cases where urgent targeted therapy is needed.     

Impact of Exon 19 Deletion Subtypes in EGFR-Mutant Metastatic Non–Small-Cell Lung Cancer Treated With First-Line Tyrosine Kinase Inhibitors

BackgroundCommon epidermal growth factor receptor (EGFR) mutations in non–small-cell lung cancer (NSCLC) predict sensitivity to EGFR tyrosine kinase inhibitors (TKIs), with exon 19 deletions being associated with better outcome compared to L858R mutations. We aimed to investigate the impact of different exon 19 deletions on patient outcome in EGFR-mutant NSCLC treated with first-line TKIs.Patients and MethodsIn this retrospective analysis, 106 patients with metastatic NSCLC harboring EGFR exon 19 deletions and treated with first-line TKIs were included. The primary end point was overall survival (OS), the secondary end point progression-free survival (PFS). Analyses were performed by grouping exon 19 deletions according to 2 models: we compared different type of deletion (delE746_A750 vs. deletions other than delE746-A750, defined as “uncommon”) or different starting codon of deletion (E746 vs. L747).ResultsThe frequency of uncommon deletions of exon 19 was 36%. When delE746_A750 (n = 68) was compared to the other deletions in exon 19 (n = 38), no differences were found, either in terms of OS (P = .65) or PFS (P = .65). Similarly, no difference in OS (P = .74) or PFS (P = .99) emerged when comparing the E746 group (n = 81) to the L747 group (n = 25). On multivariate analysis including clinical characteristics and type of deletions (delE746_A750 vs. uncommon deletions or E746 vs. L747), only the presence of brain metastases at diagnosis or during TKI treatment was associated with shorter PFS but not with worse OS.ConclusionDifferent exon 19 deletions are equally sensitive to first-line EGFR-TKIs in EGFR-mutant NSCLC.     

EGFR mutation frequency and effectiveness of erlotinib: A prospective observational study in Danish patients with non-small cell lung cancer

Objectives

In 2008, we initiated a prospective study to explore the frequency and predictive value of epidermal growth factor receptor (EGFR) mutations in an unselected population of Danish patients with non-small cell lung cancer offered treatment with erlotinib, mainly in second-line.

Materials and methods

Four hundred and eighty eight patients with advanced NSCLC were included. The mutation status was assessed using the cobas^® EGFR Mutation Test. Erlotinib was administrated (150 mg/d) until disease progression or unacceptable toxicities occurred. The primary endpoint was progression-free survival. Secondary endpoints were overall survival and response.

Results

Biopsies were retrieved from 467 patients, and mutation results obtained for 462. We identified 57 (12%) patients with EGFR mutations: 33 exon 19 deletions, 13 exon 21 mutations, 5 exon 18 mutations, 3 exon 20 insertions, 1 exon 20 point mutation (S768I), and two complex mutations. Seven percent of the patients were never smokers. The differences in median progression-free survival and overall survival between the mutated group and the wild-type group were 8.0 vs. 2.5 months, p < 0.001 and 12.1 vs. 3.9 months, p < 0.001. Performance status (0–1 vs. 2–3) and line of treatment (1st vs. 2nd and 3rd) had no influence on outcome in EGFR-mutated patients.

Conclusion

We found a higher frequency of EGFR mutations than expected in a cohort with less than 10% never smokers. The outcome after treatment with erlotinib was much better in patients with EGFR mutations than in patients with wild-type EGFR and was independent of performance status and treatment line in EGFR-mutated patients.     

Comparative study of IDH1 mutations in gliomas by immunohistochemistry and DNA sequencing

Background

Mutations involving isocitrate dehydrogenase 1 (IDH 1) occur in a high proportion of diffuse gliomas, with implications on diagnosis and prognosis. About 90% involve exon 4 at codon 132, replacing amino acid arginine with histidine (R132H). Rarer ones include R132C, R132S, R132G, R132L, R132V, and R132P. Most authors have used DNA-based methods to assess IDH1 status. Preliminary studies comparing imunohistochemistry (IHC) with IDH1-R132H mutation-specific antibodies have shown concordance with DNA sequencing and no cross-reactivity with wild-type IDH1 or other mutant proteins. The present study compares results of IHC with DNA sequencing in diffuse gliomas.

Materials and methods

Fifty diffuse gliomas with frozen tissue samples for DNA sequencing and adequate tissue in paraffin blocks for IHC using IDH1-R132H specific antibody were assessed for IDH1 mutations.

Results

Concordance of findings between IHC and DNA sequencing was noted in 88% (44/50) cases. All 6 cases with discrepancy were immunopositive with DIA-H09 antibody. While in 3 of these 6 cases, DNA sequencing failed to reveal any mutations, R132L (arginine replaced by leucine) mutation was found in the rest 3 cases. Interestingly, of the immunopositive cases, 46.6% (14/30) showed immunostaining in only a fraction of tumor cells.

Conclusions

IHC is an easy and quick method of detecting IDH1-R132H mutations, but there may be some discrepancies between IHC and DNA sequencing. Although there were no false-negative cases, cross-reactivity with IDH1-R132L was seen in 3, a finding not reported thus far. Because of more universal availability of IHC over genetic testing, cross-reactivity and staining heterogeneity may have bearing over its use in detecting IDH1-R132H mutation in gliomas.     

Usefulness of immunohistochemistry for the detection of the BRAF V600E mutation in Japanese lung adenocarcinoma

Purpose

Mutations in components of the mitogen-activated protein kinase (MAPK) cascade may be a new candidate for target for lung cancer. The usefulness of immunohistochemistry (IHC) as a new approach for the detection of BRAF V600E in cancer patients has been recently reported.

Methods

To increase the sensitivity, we modified BRAF V600E expression detection assay by IHC using mutation specific antibody. From the screening step, we found a novel 599 insertion T BRAF mutation in lung adenocarcinoma. In this study included 26 surgically removed cases with EGFR, Kras, erbB2, EML4-ALK and KIF5B-RET wild-type (wt) lung adenocarcinomas, including 7 BRAF mutants (5 V600E, 1 N581I, and 1 novel 599 insertion T mutation) analyzed by DNA sequencing. Detection of the BRAF V600E mutation was carried out by the Dako EnVision™ FLEX detection system using the VE1 clone antibody and compared with the results of direct sequencing.

Results

The autostainer IHC VE1 assay was positive in 5 of 5 (100%) BRAF V600E-mutated tumors and negative in 20 of 21 (95.2%) BRAF non-V600E tumors, except for a novel 599 insertion T case.

Conclusion

IHC using the VE1 clone and FLEX linker is a specific method for the detection BRAF V600E and may be an alternative to molecular biology for the detection of mutations in lung adenocarcinomas. This method might be useful for screening to use molecular target therapy for lung adenocarcinomas.     

Identification of EGFR mutations in esophageal cancer

Background

It is well known that the prognosis for esophageal cancer is worse than for other digestive cancers in spite of multimodality treatment, and there is an urgent need to improve this situation. The epidermal growth factor receptor (EGFR) inhibitor, gefitinib, was approved in Japan to treat advanced non-small cell lung cancer patients and several papers have since reported that the successfully treated patients had genetic mutations in EGFR.

Purpose

The aim of this study was to investigate the existence of EGFR mutations in esophageal cancer cell lines and primary lesions, and also to explore the possibility of treating esophageal cancer using gefitinib.

Materials and Methods

Nineteen esophageal cancer cell lines were cultured and DNA was extracted using an ultracentrifugation method. Fifty cases of primary cancer and corresponding normal tissue samples were obtained and DNA was extracted using the same protocol. Nested PCR and DNA sequencing targeting exons 18, 19, 20 and 21 of EGFR were performed to investigate the presence of mutations in esophageal cancer cell lines and primary tumors.

Results

Three of the 19 cell lines had the same silent mutation at nucleotide 2607, a G-to-A substitution in exon 20. One of the 50 patients had an EGFR mutation in codon 719, resulting in an amino acid substitution from glycine to aspartic acid.

Conclusion

EGFR mutations in esophageal carcinoma are rare but do exist, and thus gefitinib could be included in esophageal cancer treatment regimens by selecting those patients who possess such mutations.     

免疫组织化学法检测肺癌患者EGFR敏感突变的临床研究

目的 探讨免疫组织化学法（immunohistochemistry, IHC）检测EGFR突变的应用价值。方法 通过针对delE746-A750、L858R突变的特异性抗体检测肺癌患者EGFR突变状态,并且与DNA直接测序法进行对照。结果 136例标本中IHC评分0分的48例,其中DNA直接测序法检测突变的3例、评分1+的53例中突变的11例、2+的27例中突变的22例、 3+者中的8例均存在EGFR突变。以≤1+为阴性,≥2+为阳性,敏感度为71.43%,特异性为94.68%,阳性预测值（positive predictive value, PPV）为85.71%,阴性预测值（negative predictive value, NPV）为88.12%,κ值为0.683。结论 免疫组织化学检测EGFR突变应以评分≤1+为阴性,≥2+为阳性来评估EGFR突变状态。     

EGFR mutation status in tumour-derived DNA from pleural effusion fluid is a practical basis for predicting the response to gefitinib

Epidermal growth factor receptor (EGFR) mutations are strong determinants of tumour response to EGFR tyrosine kinase inhibitors in non-small-cell lung cancer (NSCLC). Pleural effusion is a common complication of lung cancer. In this study, we assessed the feasibility of detection of EGFR mutations in samples of pleural effusion fluid. We obtained 43 samples, which was the cell-free supernatant of pleural fluid, from Japanese NSCLC patients, and examined them for EGFR mutations. The epidermal growth factor receptor mutation status was determined by a direct sequencing method (exons 18-21 in EGFR). EGFR mutations were detected in 11 cases (E746_A750del in seven cases, E746_T751del insA in one case, L747_T751del in one case, and L858R in two cases). The EGFR mutations were observed more frequently in women and non-smokers. A comparison between the EGFR mutant status and the response to gefitinib in the 27 patients who received gefitinib revealed that all seven patients with partial response and one of the seven patients with stable disease had an EGFR mutation. No EGFR mutations were detected in the patients with progressive disease. The results suggest that DNA in pleural effusion fluid can be used to detect EGFR mutations and that the EGFR mutation status may be useful as a predictor of the response to gefitinib.     

HLA Class I Binding of Mutant EGFR Peptides in NSCLC Is Associated With Improved Survival

IntroductionCancer-associated mutations have the potential to generate neoantigens and elicit CD8-positive T-cell–dependent adaptive immune responses. There are currently no reports of CD8-positive T-cells with specificity for neoepitopes generated by EGFR mutations, which are driver oncogenes in a subset of patients with lung cancer.MethodsWe used NETMHCpan 4.0 to identify putative protective human leukocyte antigen (HLA) class I allotypes that are predicted in silico to bind and present mutant EGFR-generated peptides on the basis of predefined criteria. We associated the presence or absence of these alleles with clinical outcomes in patients from The Cancer Genome Atlas with lung adenocarcinoma.ResultsWe identified 12 HLA class I alleles that fulfilled the predefined criteria for being protective for EGFR p.L858R and six for EGFR p.E746_A750del, the two most common EGFR mutations in lung cancer. We validated the in silico predictions for peptide-HLA allele binding in vitro. A third (12 of 36) of patients with mostly early stage lung adenocarcinoma in The Cancer Genome Atlas with either EGFR p.L858R or EGFR p.E746_A750del had at least one protective allele in their host genomes. More importantly, patients with protective alleles exhibited better disease-free (hazard ratio: 0.20, 95% confidence interval: 0.05–0.78) and overall survival (hazard ratio: 0.13, 95% confidence interval: 0.02–0.64), and this effect was independent of the EGFR mutation type, stage, age, and sex.ConclusionsOur data revealed that clinical outcomes were improved in patients with EGFR mutation–positive lung adenocarcinoma who harbored protective HLA class I alleles. Thus, immunity with specificity for mutant EGFR is possible in a subset of patients with early stage lung cancer and portends a better prognosis.     

Efficacy of first-line systemic treatment in correlation with BRAF V600E and different KRAS mutations in metastatic colorectal cancer - a single institution retrospective analysis

Background

KRAS mutation status in codons 12 and 13 is recognized as a predictive factor for resistance to anti-EGFR monoclonal antibodies. Despite having a wild type KRAS (wt-KRAS), not all patients with wt-KRAS respond to anti-EGFR antibody treatment. Additional mechanisms of resistance may activate mutations of the other main EGFR effectors pathway. Consequently, other molecular markers in colorectal cancer are needed to be evaluated to predict the response to therapy.

Patients and methods

In this retrospective study, objective responses (OR), time to progression (TTP), overall survival (OS) were analyzed in 176 metastatic colorectal cancer (mCRC) patients treated with first-line chemotherapy in combination with monoclonal antibodies in respect of KRAS status in codons 12 and 13 and BRAF mutational status.

Results

The KRAS mutations were found in 63 patients (35.8 %), the KRAS mutation in codon 12 in 53 patients (30.1%) and the KRAS mutation in codon 13 in 10 patients (5.7%). The BRAF V600E mutation was detected in 13 of 176 patients (7.4%). In the subgroup of mCRC patients having wt-KRAS and wild type BRAF (wt-BRAF), the objective response rates were higher (OR 54.0% ,CR 14.7%, PR 39.3%) than in the patients with wt-KRAS and mt-BRAF (OR 38.5%,CR 15.4%, PR 23.1%), the difference was not statistically significant (p= 0.378). Median OS in patients with wt-KRAS wt-BRAF, and in patients with wt-KRAS mt-BRAF, was 107.4 months and 45 months, respectively. The difference was statistically significant (p= 0.042). TTP in patients with wt-KRAS wt-BRAF, and in patients with wt-KRAS mt-BRAF, was 16 months and 12 months, respectively. The difference was not statistically significant (p= 0.558).

Conclusions

Patients with BRAF V600E mutation have statistically significantly worse prognosis than the patients with wt-BRAF and progress earlier during treatment. The definitive role of the BRAF V600E mutation as a prognostic and predictive factor for the response to anti-EGFR monoclonal antibodies needs to be analyzed in large prospective clinical studies.     

A comparison of ARMS and DNA sequencing for mutation analysis in clinical biopsy samples

Background

We have compared mutation analysis by DNA sequencing and Amplification Refractory Mutation System™ (ARMS™) for their ability to detect mutations in clinical biopsy specimens.

Methods

We have evaluated five real-time ARMS assays: BRAF 1799T>A, [this includes V600E and V600K] and NRAS 182A>G [Q61R] and 181C>A [Q61K] in melanoma, EGFR 2573T>G [L858R], 2235-2249del15 [E746-A750del] in non-small-cell lung cancer, and compared the results to DNA sequencing of the mutation ''hot-spots'' in these genes in formalin-fixed paraffin-embedded tumour (FF-PET) DNA.

Results

The ARMS assays maximised the number of samples that could be analysed when both the quality and quantity of DNA was low, and improved both the sensitivity and speed of analysis compared with sequencing. ARMS was more robust with fewer reaction failures compared with sequencing and was more sensitive as it was able to detect functional mutations that were not detected by DNA sequencing. DNA sequencing was able to detect a small number of lower frequency recurrent mutations across the exons screened that were not interrogated using the specific ARMS assays in these studies.

Conclusions

ARMS was more sensitive and robust at detecting defined somatic mutations than DNA sequencing on clinical samples where the predominant sample type was FF-PET.     

Functional analysis of cancer-associated EGFR mutants using a cellular assay with YFP-tagged EGFR intracellular domain

Background

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.

Results

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.

Conclusion

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.

     

Pyrosequencing,a method approved to detect the two major EGFR mutations for anti EGFR therapy in NSCLC

Background

Epidermal Growth Factor Receptor (EGFR) mutations, especially in-frame deletions in exon 19 (ΔLRE) and a point mutation in exon 21 (L858R) predict gefitinib sensitivity in patients with non-small cell lung cancer. Several methods are currently described for their detection but the gold standard for tissue samples remains direct DNA sequencing, which requires samples containing at least 50% of tumor cells.

Methods

We designed a pyrosequencing assay based on nested PCR for the characterization of theses mutations on formalin-fixed and paraffin-embedded tumor tissue.

Results

This method is highly specific and permits precise characterization of all the exon 19 deletions. Its sensitivity is higher than that of "BigDye terminator" sequencing and enabled detection of 3 additional mutations in the 58 NSCLC tested. The concordance between the two methods was very good (97.4%). In the prospective analysis of 213 samples, 7 (3.3%) samples were not analyzed and EGFR mutations were detected in 18 (8.7%) patients. However, we observed a deficit of mutation detection when the samples were very poor in tumor cells.

Conclusions

pyrosequencing is then a highly accurate method for detecting ΔLRE and L858R EGFR mutations in patients with NSCLC when the samples contain at least 20% of tumor cells.     

Somatic mutations of EGFR gene in squamous cell carcinoma of the head and neck.

PURPOSE: Recently, the kinase domain mutations of epidermal growth factor receptor (EGFR) gene have been identified in non-small-cell lung cancer, and these mutations have been related to the clinical response to the tyrosine kinase inhibitor gefitinib. Gefitinib treatment has also shown clinical benefits in squamous cell carcinoma of the head and neck (SCCHN). The aim of this study was to explore the possibility that SCCHN harbored the EGFR mutations. EXPERIMENTAL DESIGN: In this study, we analyzed EGFR gene in 41 SCCHN for the detection of the somatic mutations by PCR-single-strand conformational polymorphism analysis. RESULTS: Overall, we detected three EGFR mutations (7.3%), and all of the mutations were the same in-frame deletion mutation in exon 19 (E746_A750del). CONCLUSION: These data indicated that in addition to non-small-cell lung cancer, SCCHN harbors the EGFR gene mutations, and suggested the rationale for the clinical applicability of gefinitib to SCCHN patients.