Somatic Epidermal Growth Factor Receptor Research Articles

Detection of EGFR mutational profile by direct dideoxy sequencing in cytology and non-cytology biopsy samples

Epidermal growth factor receptor (EGFR) mutational analysis is recommended in the diagnostic work-up of non-small cell lung carcinoma. The first diagnostic biopsy is usually obtained by a minimally invasive procedure, especially in patients with unresectable disease. This paper aims to compare the types of somatic EGFR mutations detected by cytology and non-cytology samples by direct dideoxy sequencing and propose practical guidelines for handling such material. Only samples with sufficient polymerase chain reaction (PCR) product were considered, a total 310 samples (302 patients), of which 168 samples were cytology material and 142 samples were non-cytology biopsy material. All samples were assessed for tumour content and bidirectional direct sequencing was performed on exons 18, 19, 20 and 21. There were 49 cases with EGFR mutation detected (16.2%), without a significant difference in the detection of mutations between either cytology or non-cytology material. EGFR mutation was detected in most sample types including endoscopic ultrasound guided FNA, bronchial washings/brushings and pleural/peritoneal fluid samples. Cytology material can provide an adequate source of material for EGFR mutational analysis, with coordinated effort between clinicians and pathologists critical for best outcome.

A comparison of epidermal growth factor receptor mutation testing methods in different tissue types in non-small cell lung cancer.

The detection of somatic epidermal growth factor receptor (EGFR) mutations is valuable when an appropriate therapy, either EGFR-tyrosine kinase inhibitor (TKI) therapy or chemotherapy, for patients with advanced non-small cell lung cancer (NSCLC) needs to be selected. Although it is well‑understood that EGFR mutation detection is significant for the decision‑making regarding treatment, no consensus on the methodology that should be the most preferebale for detecting mutations in clinical practice has been reached. The presence of false positives due to the technique carried out for mutation analysis affects the accurate estimation of response EGFR-TKI therapy. Furthermore, false negatives directly exclude the potential application of an EGFR-TKI. In the present study, we present the results of detecting EGFR mutations in individual sample types using three different low- or high-sensitivity techniques. We suggest that the choice of the method used should be made based on the type of the sample. Our results revealed that EGFR mutations were less frequently detected in bronchoscopic biopsies, regardless of the method used. However, the amplification refractory mutation system (ARMS) was optimal owing to the small amount of DNA prepared for biopsy. The cytology sample was a valuable alternative to traditional samples, given that a sensitive method for detecting mutations was used. For surgical resections, the testing method may be selected based on the expertise of each laboratory, but direct sequencing is highly recommended. We also suggest that two methods should be used sequentially (the screening and targeted methods) in clinical practice due to the presence of non-neglected discordance between any method from its own benefits and drawbacks.

Mutation status of somatic EGFR and KRAS genes in Chinese patients with prostate cancer (PCa)

Activating mutations of the epidermal growth factor receptor (EGFR) confers sensitivity to tyrosine kinase inhibitors (TKIs). In colorectal cancer and in lung adenocarcinomas, clinical trials have shown a lack of response to anti-EGFR therapy when KRAS gene mutations are present. In this study, the mutation status of specified exons of the EGFR and KRAS genes was profiled in patients with prostate cancer (PCa). Direct Sanger sequencing was used to screen for mutations in exons 19-21 of EGFR and in exon 2 of KRAS in 88 Chinese patients diagnosed with prostate adenocarcinomas. Mutations were detected in 11 patients. In nine cases (10 %), activating mutations in the region of EGFR encoding the tyrosine kinase (TK) domain were present. Deletions in exon 19 and the L858R substitution in exon 21 were "hotspot" mutations, together accounting for five (55 %) of nine cases. Many synonymous substitutions were also detected. KRAS mutations were found in two cases (2.3 % of 88). There were no cases with mutations in both EGFR and KRAS, suggesting that mutations in the two genes might be mutually exclusive. Although prognostic relevance of EGFR expression by immunohistochemistry (IHC) was observed in PCa patients in previous studies, we found no statistically significant association between EGFR or KRAS mutations and clinicopathological features (including age, smoking status, preoperative prostate-specific antigen, Gleason scores, and tumor stage). We contend that accurate profiling of the mutation status of EGFR and KRAS could improve prognostic stratification, and we suggest a potential anti-EGFR therapy for patients with PCa with EGFR mutations.

Curcumin Lowers Erlotinib Resistance in Non-Small Cell Lung Carcinoma Cells With Mutated EGF Receptor

Non-small cell lung cancer (NSCLC) patients with activating mutations in the epidermal growth factor receptor (EGFR) are responsive to erlotinib, an EGFR-tyrosine kinase inhibitor (EGFR-TKI). However, patients with secondary somatic EGFR mutations are resistant to EGFR-TKI treatment. In this study, we investigated the effect of curcumin on the tumor growth of erlotinib-resistant NSCLC cells. Cell proliferation was determined by MTT assay. Apoptosis was examined using TUNEL staining. Protein expression of genes was determined by Western blot. Tumor growth was assessed in a xenograft mouse model. Results showed that erlotinib had a stronger effect on the induction of apoptosis in erlotinib-sensitive PC-9 cells but showed a weaker effect on erlotinib-resistant H1975 and H1650 cells than cisplatin and curcumin. Furthermore, curcumin significantly increased the cytotoxicity of erlotinib to erlotinib-resistant NSCLC cells, enhanced erlotinib-induced apoptosis, downregulated the expressions of EGFR, p-EGFR, and survivin, and inhibited the NF-κB activation in erlotinib-resistant NSCLC cells. The combination of curcumin and erlotinib exhibited the same effects on apoptosis as the combination of curcumin and cisplatin in erlotinib-resistant NSCLC cells. Moreover, the combined treatment of curcumin and erlotinib significantly inhibited tumor growth of erlotinib-resistant NSCLC cells in vivo. Our results indicate that curcumin is a potential adjuvant for NSCLC patients during erlotinib treatment.

Epidermal growth factor receptor mutations in lung adenocarcinoma

Despite tremendous progress in the last decade, lung adenocarcinoma still represents a tumor with unfavorable prognosis when detected at advanced clinical stage. High-stage tumors are not amenable to surgical resection, and therefore systemic therapies are needed to control these tumors to prolong patient survival. In the era of molecular and personalized therapeutics, the discovery of mutations in epidermal growth factor receptor (EGFR) in 15–20% of lung adenocarcinomas and the associated response to EGFR-targeting tyrosine kinase (TK) inhibitors have provided a successful avenue of attack in high-stage adenocarcinomas. In this review, we will provide an overview of the EGFR pathway, review the significant somatic EGFR alterations in lung adenocarcinoma and highlight their implications for treatment. In addition, we will examine pathways by which tumors resist EGFR TK therapy, both as primary nonresponders and by acquired resistance. In doing so, we will examine other oncogenic pathways whose status in tumor samples may impact therapeutic responses despite presence of activating EGFR mutations.

Melatonin Sensitizes H1975 Non-Small-Cell Lung Cancer Cells Harboring a T790M-Targeted Epidermal Growth Factor Receptor Mutation to the Tyrosine Kinase Inhibitor Gefitinib

Background/Aims: The use of tyrosine kinase inhibitors (TKIs) to target active epidermal growth factor receptor (EGFR)-harbouring mutations has been effective in patients with advanced non-small-cell lung cancer (NSCLC). However, the use of TKIs in NSCLS patients with somatic EGFR mutations, particularly T790M, causes drug resistance. Thus, in the present study, we investigated overcoming resistance against the TKI gefitinib by combination treatment with melatonin in H1975 NSCLC cells harbouring the T790M somatic mutation. Methods: H1975 and HCC827 cells were treated with melatonin in combination with gefitinib, and cell viability, cell cycle progression, apoptosis, and EGFR, AKT, p38, Bcl-2, Bcl-xL, caspase 3 and Bad protein levels were examined. Results: Treatment with melatonin dose-dependently decreased the viability of H1975 cells harbouring the T790M somatic mutation compared to HCC827 cells with an EGFR active mutation. Melatonin-mediated cell death resulted in decreased phosphorylation of EGFR and Akt, leading to attenuated expression of survival proteins, such as Bcl-2, Bcl-xL and survivin, and activated caspase 3 in H1975 cells, but not in HCC827 cells. However, we did not observe a significant change in expression of cell cycle proteins, such as cyclin D, cyclin A, p21 and CDK4 in H1975 cells. Surprisingly, co-treatment of gefitinib with melatonin effectively decreased the viability of H1975 cells, but not HCC827 cells. Moreover, co-treatment of H1975 cells caused consistent down-regulation of EGFR phosphorylation and induced apoptosis compared to treatment with gefitinib or melatonin alone. Conclusions: Our findings demonstrate that melatonin acts as a potent chemotherapeutic agent by sensitising to gefitinib TKI-resistant H1975 cells that harbour a EGFR T790M mutation.

Results of the First Italian External Quality Assurance Scheme for Somatic EGFR Mutation Testing in Non–Small-Cell Lung Cancer

The Italian Association of Medical Oncology (AIOM) and the Italian Society of Pathology and Cytology organized an external quality assessment (EQA) scheme for EGFR mutation testing in non-small-cell lung cancer. Ten specimens, including three small biopsies with known epidermal growth factor receptor (EGFR) mutation status, were validated in three referral laboratories and provided to 47 participating centers. The participants were requested to perform mutational analysis, using their usual method, and to submit results within a 4-week time frame. According to a predefined scoring system, two points were assigned to correct genotype and zero points to false-negative or false-positive results. The threshold to pass the EQA was set at higher than 18 of 20 points. Two rounds were preplanned. All participating centers submitted the results within the time frame. Polymerase chain reaction (PCR)/sequencing was the main methodology used (n = 37 laboratories), although a few centers did use pyrosequencing (n = 8) or real-time PCR (n = 2). A significant number of analytical errors were observed (n = 20), with a high frequency of false-positive results (n = 16). The lower scores were obtained for the small biopsies. Fourteen of 47 centers (30%) that did not pass the first round, having a score less than or equal to 18 points, used PCR/sequencing, whereas 10 of 10 laboratories, using pyrosequencing or real-time PCR, passed the first round. Eight laboratories passed the second round. Overall, 41of 47 centers (87%) passed the EQA. The results of the EQA for EGFR testing in non-small-cell lung cancer suggest that good quality EGFR mutational analysis is performed in Italian laboratories, although differences between testing methods were observed, especially for small biopsies.

Frequency of EGFR mutations in Greek non-small-cell lung cancer (NSCLC) patients.

e21141 Background: NSCLC patients harboring activating somatic mutations within the tyrosine kinase (TK) domain of the epidermal growth factor receptor (EGFR) significantly benefit from EGFR targeted therapy. Treatment with the recently approved EGFR inhibitor IRESSA (gefitinib) leads to improved response and survival outcomes, therefore screening for EGFR mutations has entered routine clinical practice. Several clinico-pathological factors correlate with these mutations including gender, smoking history, and histology. The frequency of EGFR mutations is also ethnicity-dependent, wherein the incidence in Asian populations is ~30%, while in Caucasians (Whites) it is lower, ~ 15%. However, limited data is available on intra-ethnic differences throughout Europe. Aim: The aim of this study was to determine the frequency and spectrum of EGFR mutations in an unselected group of Greek NSCLC patients and investigate technical aspects of analysis. Methods: We set up High Resolution Melting Analysis (HRMA) assays to identify mutations in exons 18-21 of the EGFR gene and validated their analytical sensitivity by making serial dilutions of samples with known mutations and tumor cell content (TCC). A total of 698 NSCLC patients were screened with HRMA for somatic EGFR mutations in exons 18-21 and mutation status was verified by bi-directional sequencing. Pathological review was obtained for all samples and macro-dissection was used to ensure a %TCC of >75% in all possible cases. Results: The sensitivity of our HRM assays was found to be ≤1.5% Using HRMA and bi-directional sequencing a frequency of 19.05% was obtained; 105 x exon 19, 21 x exon 21, 6 x exon 20 and 1 x exon 18 . Conclusions: Applying a very sensitive mutation detection technique in a large cohort of unselected Greek NSCLC patients in routine diagnostic practice, we obtained an overall mutation frequency of 19.05%. This mutation frequency is similar to that found by the SLADB and EURTAC studies in European populations. Differences in sensitivity between techniques suggest that more than one technique should be advised in routine diagnostic practice.

Epidermal growth factor receptor (EGFR) expression and mutations in the EGFR signaling pathway in correlation with anti-EGFR therapy in head and neck squamous cell carcinomas

Epidermal growth factor receptor (EGFR) is an important therapeutic target and a poor prognosis factor in head and neck squamous cell carcinoma (HNSCC). The aim of the study was to analyze EGFR expression and KRAS and EGFR mutational status and to correlate it with treatment response to anti-EGFR therapy combined with radiotherapy in 29 patients with advanced head and neck squamous cell carcinomas (HNSCC).EGFR gene expression normalized to GAPDH and EGFR variant type III (EGFRvIII) was detected in tumor tissue using real time reverse transcription -PCR. The mutational status of the EGFR and KRAS genes was investigated by real time PCR with sequence specific primers.Gene expression median values were 3.1x10(8) GAPDH gene copies per µg of RNA, and 8x10(6) EGFR gene copies per µg of RNA. The median EGFR/GADPH ratio reached 0.14. Patients, who achieved complete response after Cetuximab combined with radiotherapy, had significantly higher expression of the EGFR gene in tumors than patients with partial remission or patient without treatment response. An EGFRvIII mutation was found in 20.7 % of patients and no association was found between this mutation and treatment response. 27 patients (93.1 %) had an EGFR gene wild type tumor, and deletion in exon 19 was found in two patients with a poor clinical outcome. Most of the patients (82.8%) had a KRAS wild type tumor; a p.Gly12Cys was found in three patients and a p.Gly12Val mutation in one. Presence of a p.Gly12Val mutation in the KRAS gene was associated with an absence of response to treatment. Our data suggest that KRAS mutation (p.Gly12Val) and somatic EGFR mutation located in exon 19 may contribute to the limited clinical response to therapy with cetuximab + radiotherapy. Higher EGFR gene expression serves as an independent indicator of good clinical response to EGFR-targeted therapy + radiotherapy.

Quantitative Detection of EGFR Mutations in Circulating Tumor DNA Derived from Lung Adenocarcinomas

Examination of somatic epidermal growth factor receptor (EGFR) mutations is now a diagnostic routine for treatment of cancer using EGFR tyrosine kinase inhibitors (EGFR-TKI). Circulating tumor DNA is a promising target for noninvasive diagnostics. We evaluated its utility by quantitatively detecting activating and resistant mutations, which were measured with BEAMing (beads, emulsion, amplification, and magnetics). Twenty-three patients with lung cancer with progressive disease after EGFR-TKI treatment and 21 patients who had never been treated with EGFR-TKIs were studied. Their primary tumors were confirmed to have activating mutations. In the plasma DNA of each patient, the activating mutation found in the corresponding primary tumor and the T790M resistance mutation were quantified by BEAMing. In 32 of 44 patients, activating mutations were detected in the plasma DNA [72.7%; 95% confidence interval (CI), 58.0%-83.6%]. The T790M mutation was detected in 10 of 23 patients in the first group (43.5%; 95% CI, 25.6%-53.4%). The ratio of T790M to activating mutations ranged from 13.3% to 94.0%. The peak of the distribution of the mutation allele fraction in the plasma DNA was in the 0.1% to 1% range. The major advantage of BEAMing is its ability to calculate the fraction of T790M-positive alleles from the alleles with activating mutations. This feature enables the detection of increases and decreases in the number of T790M mutations in cancer cells, regardless of normal cell DNA contamination, which may be useful for monitoring disease progression. Circulating tumor DNA could potentially be used as an alternative method for EGFR mutation detection.

The Value of Biomarkers in Patients With Sarcomatoid Carcinoma of the Lung: Molecular Analysis of 33 Cases

BackgroundSarcomatoid carcinoma (SC) of the lung is a rare histologic group of lung cancers with a poor prognosis. To better understand the prognosis of lung SC, in this study, we evaluated the correlation between several biomarkers and clinical outcomes in patients with lung SC. Patients and MethodsA cohort of 33 patients with lung SC was studied. Protein expressions of excision repair cross-complementation group 1 (ERCC1) and epidermal growth factor receptor (EGFR) were examined by immunohistochemistry. Somatic EGFR and KRAS mutations were identified by direct sequencing. EGFR gene copy number was evaluated by fluorescence in situ hybridization. ERCC1 messenger RNA expression in paraffin-embedded tumor specimens was detected by branched DNA assay. ResultsOur analyses identified 9 patients (9/32) with EGFR mutations and only 1 patient (1/32) with a KRAS mutation. No exon 19 deletion of EGFR gene was detected. Lower messenger RNA levels of ERCC1 were detected in patients with EGFR mutations and/or fluorescence in situ hybridization amplified status. Survival analysis identified several significant factors, including performance status and clinical staging, that predicted for overall survival. ConclusionSC exhibits diverse genotypic variations. Results of our study suggest that chemotherapy could still be an optimal solution for untreated advanced SC, whereas EGFR tyrosine kinase domain inhibitors alone may not be an effective approach.

CYP1A1*2A polymorphism as a predictor of clinical outcome in advanced lung cancer patients treated with EGFR-TKI and its combined effects with EGFR intron 1 (CA)n polymorphism

BackgroundMutations in the epidermal growth factor receptor (EGFR) have been confirmed as predictors of efficacy for EGFR-tyrosine kinase inhibitors (TKIs). We investigated whether polymorphisms of the EGFR and cytochrome P450, family 1, member A1 (CYP1A1) genes were associated with clinical outcome in NSCLC patients treated with EGFR-TKI. MethodsGenotypes for the intron 1 (CA)n repeat and R497K polymorphisms in the EGFR gene and the *2A (3801 T→C) and *2C (2455 A→G) polymorphisms in CYP1A1 gene were evaluated in 115 NSCLC patients by PCR-RFLP and DNA sequencing. Genetic polymorphisms were correlated with clinical outcomes of EGFR-TKIs. From a subgroup of patients whose tumour tissues were available, associations between somatic EGFR mutations, EGFR expression, and genomic polymorphisms were also analysed. ResultsEGFR intron 1 (CA)n and CYP1A1*2A polymorphisms were independent predictive factors (p=0.046, p=0.011, respectively) and the latter was also a prognostic factor (p=0.001) for patients treated with EGFR-TKIs. We also observed a strong synergistic effect from two genotypes. Specifically, patients with both the T/T allele of the CYP1A1 gene and shorter intron 1 CA repeats (⩽16 CA) of the EGFR gene showed an improved response (p=0.002) compared with patients with the T/C or C/C allele and longer intron 1 CA repeats (both alleles >16 CA). In contrast, for R497K and CYP1A1*2C, no relationship was observed with clinical outcome for patients treated with EGFR-TKIs (p=0.573; p=0.629, respectively). Both SNPs in the CYP1A1 gene showed a correlation with EGFR somatic mutations. ConclusionsThe findings of this study suggest that the CYP1A1*2A polymorphism is a predictor for clinical outcome in NSCLC patients treated with EGFR-TKI therapy, and combining analysis of both CYP1A1*2A and EGFR intron 1 (CA)n polymorphisms may be useful for predicting treatment outcome in NSCLC patients treated with EGFR-TKIs.

Clinical relevance of epidermal growth factor receptor (EGFR) alterations in human pancreatic tumors

Pancreatic cancer is a malignant neoplasm with an extremely poor prognosis. The mechanisms of aggressive growth and metastasis are currently not well understood. Expression of epidermal growth factor receptor (EGFR) has been suggested to be associated with the malignant transformation of pancreatic cancer. In this study, we examined the EGFR status of 52 pancreatic tumors by PCR-sequencing (exons 19 and 21), immunohistochemistry and FISH probes. We subsequently investigated the relationship between EGFR status and clinicopathological factors. Somatic alterations in EGFR (R841R, T571T and R831C) were observed only in ductal adenocarcinoma (3/34). In 4 (8%) of the 52 tumors analyzed EGFR was overexpressed, 6 (12%) of the tumors showed moderate expression while 19 (32%) were weakly stained. EGFR overexpression (3+ score) was frequently found in endocrine tumors (29%) followed of ampullary tumors (13%; p < 0.01). No significant correlation was observed between the presence of a somatic EGFR mutation and clinicopathological variables. Fluorescence in situ hybridization (FISH) analysis did not demonstrate amplification in any tumors. Only three somatic mutations in the EGFR gene were detected in pancreatic ductal adenocarcinoma and no association was observed with the clinical variables. Our results suggest that EGFR mutations are rare in pancreatic tumors and not associated with clinical prognosis, and treatment response.

Mechanisms of Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Patients with Advanced Non-Small-Cell Lung Cancer: Clinical and Molecular Considerations

Non-Small-Cell Lung Cancer (NSCLC) with somatic mutations of the epidermal growth factor receptor (EGFR) is anticipated to respond to small-molecule tyrosine kinase inhibitors (TKIs) of the EGFR tyrosine kinase. There are, however, patients with EGFR mutated tumors who do not demonstrate tumor response. The most widely accepted mechanism of 'de novo' (inherent) resistance to these TKIs involves mutations of the KRAS gene. KRAS is a downstream mediator of EGFR-induced cell signaling, such mutations appear to be mutually exclusive from EGFR mutations in lung cancer. The first molecular modifier of resistance identified in patients who developed resistance (termed 'acquired resistance') to TK inhibition was a new acquired somatic EGFR mutation (T790M). Today there is an ever-growing series of molecular events that have recently come to the forefront to explain other instances of TKI resistance not attributable to T790M or KRAS. These include a number of molecules that interact with EGFR or form part of its downstream signaling pathway such as HER-2, IGFR-1, MET and B-RAF. Considering that the majority of studies carried out to date with respect to the identification of resistant clones have not used highly sensitive techniques (e.g. allelic discrimination to identify somatic mutations), coupled with the relatively low number of studies examining multiple molecular markers and the accepted molecular heterogeneity of NSCLC raise question as to the existence of 'acquired' versus 'de-novo' resistance. By examining the current knowledge base with respect to mechanisms of resistance to EGFR TKIs in NSCLC, we explore whether 'acquired' resistance is 'de-novo' resistance in disguise, and discuss the promises and limitations of molecular stratification with respect to strategies incorporating TKIs in the treatment of NSCLC.

Frequency and Type of Epidermal Growth Factor Receptor Mutations in African Americans with Non-small Cell Lung Cancer

Epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) predict response to tyrosine kinase inhibitors. Mutations occur more commonly in never smokers and East Asians, but there are conflicting reports on the frequency of EGFR mutations in tumors from African Americans. Tumors from 67 African American and 77 white participants in previous case-control studies of lung cancer were selected to determine EGFR mutational status. Mutation analysis was performed using the Sequenom mass array analyzer (Sequenom, San Diego, CA). Overall, 13.9% of the study population carried an EGFR mutation. EGFR mutations occurred in 11.9% of tumors from African Americans compared with 15.6% in whites (p = 0.53). All mutations found in African Americans were deletions in exon 19. The majority of mutations were found in nonsmokers among both African Americans (7/8) and whites (8/12). These results indicate that African Americans with NSCLC harbor somatic EGFR mutations at a frequency similar to whites with NSCLC. Thus, clinicians should not use race as a clinical decision parameter for the use of EGFR-tyrosine kinase inhibitors.

Inherited Germline T790M Mutation and Somatic Epidermal Growth Factor Receptor Mutations in Non-small Cell Lung Cancer Patients

Five cases of non-small cell lung cancer, associated with germline transmission of epidermal growth factor receptor (EGFR)-T790M mutation, have been reported1; these patients had family histories of lung cancer. The activity of gefitinib was tested in only two patients, who were both refractory to this drug.2 Herein, we describe a family of European descent in which two family members had non-small cell lung cancer associated with germline transmission of T790M mutation and who were treated with gefitinib (Figure 1A). The proband was a 72-year-old female, never-smoker, who was diagnosed with an adenocarcinoma of the lung with bilateral pulmonary lesions. This patient was initially treated with chemotherapy for four courses, resulting in stable disease for 6 months, as determined by radiological examination. When the disease progressed, treatment with oral gefitinib at the dose of 250 mg/d was initiated, resulting in a partial response lasting for 9 months; subsequently, the patient developed pleural and cerebral progression of disease and died within 5 months. A 74-year-old never-smoker sister of the proband was diagnosed with a poor differentiated locally advanced (stage IIIB for pleural effusion) carcinoma of the lung and was treated with a first-line chemotherapy, resulting in a partial response lasting for 12 months, after which new bone metastases were observed. As second-line treatment, oral gefitinib 250 mg/d was started, and a partial response was obtained that is lasting from 45 months; the patient is still alive and on treatment with gefitinib. No other members of this family developed lung cancer. DNA was isolated from peripheral blood mononuclear cells or paraffin-embedded tumor samples, using the microDNA-kit (Qiagen, Hilden, Germany). Nested polymerase chain reaction to amplify EGFR (exons 18–21) and K-RAS (exon 1) and sequencing of polymerase chain reaction products on a ABI-3100 Genetic Analyzer was performed. The EGFR-T790M mutation was found in the germline DNA of both patients (Figure 1B), whereas no mutations were detected in the EGFR exons 18, 19, and 21. In contrast, no EGFR mutations were observed in the two daughters of proband and in the two sons of individual II. In the tumor tissue of individual I, we identified the deletion of EGFR E746-A750 in exon 19 but no EGFR mutations in the exons 18, 20, and 21 (Figure 1C). In the tissue of individual II, no EGFR mutations in the exons 18, 19, and 21 were found, whereas the exon 20 could not be studied because no sufficient tumor tissue was present. No mutations were observed in codons 12 and 13 of K-RAS exon 1 in the tissues of both patients. Our data support the hypothesis that an inherited T790M mutation confers an enhanced susceptibility to develop lung tumors3; moreover, the presence of a T790M germline mutation does not necessarily predict for resistance to EGFR-tyrosine kinase inhibitors. The responses to gefitinib could possibly be due to the absence of T790M and K-RAS mutations and the occurrence of EGFR-activating mutations in the tumor tissues. However, other mechanisms, such as increased gene copy number,4 may be responsible for response to gefitinib in the case of individual II.

Mass spectrometry mapping of epidermal growth factor receptor phosphorylation related to oncogenic mutations and tyrosine kinase inhibitor sensitivity.

The epidermal growth factor receptor (EGFR) plays an important role in cancer by activating downstream signals important in growth and survival. Inhibitors of EGFR are frequently selected as treatment for cancer including lung cancer. We performed an unbiased and comprehensive search for EGFR phosphorylation events related to somatic activating mutations and EGFR inhibitor (erlotinib) sensitivity. EGFR immunoprecipitation combined with high resolution liquid chromatography-mass spectrometry and label free quantitation characterized EGFR phosphorylation. Thirty (30) phosphorylation sites were identified including 12 tyrosine (pY), 12 serine (pS), and 6 threonine (pT). Site-specific phosphorylation was monitored by comparing ion signals from the corresponding unmodified peptide. Phosphorylation sites related to activating mutations in EGFR as well as sensitivity to erlotinib were identified using 31 lung cancer cell lines. We identified three sites (pY1092, pY1110, pY1172) correlated with activating mutations and three sites (pY1110, pY1172, pY1197) correlated with erlotinib sensitivity. Five sites (pT693, pY1092, pY1110, pY1172, and pY1197) were inhibited by erlotinib in concentration-dependent manner. Erlotinib sensitivity was confirmed using liquid chromatography coupled to multiple reaction monitoring (LC-MRM) and quantitative Western blotting. This LC-MS/MS strategy can quantitatively assess site-specific EGFR phosphorylation and can identify relationships between somatic mutations or drug sensitivity and protein phosphorylation.

Favorable Response to Gefitinib Treatment of Lung Adenocarcinoma With Coexisting Germline and Somatic Epidermal Growth Factor Receptor Mutations

Favorable Response to Gefitinib Treatment of Lung Adenocarcinoma With Coexisting Germline and Somatic Epidermal Growth Factor Receptor Mutations

The different efficacy of gefitinib or erlotinib according to epidermal growth factor receptor exon 19 and exon 21 mutations in Korean non-small cell lung cancer patients

Epidermal growth factor receptor (EGFR) mutations are associated with sensitivity to gefitinib or erlotinib in non-small cell lung cancer (NSCLC). We investigated the relationships between the two most common types of somatic EGFR mutations, exon 19 deletions and L858R mutations, and clinical outcomes of Korean NSCLC patients after treatment with gefitinib or erlotinib. In Korean patients with NSCLC, EGFR exon 19 deletions and EGFR L858R mutation were identified from tumor specimens obtained before treatment with gefitinib or erlotinib. The response rates, progression-free survival (PFS), and overall survival (OS) were compared between the two groups. A total of 77 patients with either an exon 19 deletion (n = 58) or L858R mutation (n = 19) were treated with gefitinib or erlotinib. The overall response rate was 69%. Patients with an exon 19 deletion had a significantly longer PFS compared with patients with L858R mutation (9.5 vs. 7.7 months; P = 0.029). The L858R mutation was independently associated with a shorter PFS compared with an exon 19 deletion, even after adjusting for other clinical factors (hazard ratio 2.72; 95% CI 1.38-5.38). However, there were no significant differences in response rate (71 vs. 63%) and OS (21.4 vs. 30.7 months) between subjects with exon 19 deletions and L858R mutations, respectively. In Korean NSCLC patients, EGFR exon 19 deletions are associated with longer PFS compared with EGFR L858R mutations. These observations need to be confirmed by large-scale studies of patients.

A multicenter phase II trial of adjuvant erlotinib in patients with resected non-small cell lung cancer (NSCLC) and mutations in the epidermal growth factor receptor (EGFR): Toxicity evaluation.

7078 Background: Adjuvant chemotherapy for patients (pts) with resected NSCLC provides a modest survival benefit, but a large proportion of pts still relapse, presumably due to inherent chemoresistance. Tumors with activating EGFR mutations are exquisitely sensitive to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib; hence the use of adjuvant TKIs may more effectively prevent recurrences. This trial investigates the safety and efficacy of adjuvant erlotinib in pts with stage I-IIIA NSCLC and somatic EGFR mutations. Methods: Eligible pts have surgically resected stage IA-IIIA NSCLC and activating EGFR mutations. Within 6 months of surgery, and following the completion of routine adjuvant treatment, pts are treated with 150 mg/day of erlotinib for a total of 2 years, with surveillance scans every 6 months. The primary endpoint is disease-free survival at 2 years. Results: Thirty-six pts were enrolled at five sites between 1/08 and 11/09. Demographics include: stage I 53%; stage II 19%; stage IIIA 28%; female 75%; never-smokers 56%; exon 19 deletion 61%, L858R 36%. Significant toxicities are listed below (Table). 6 pts have been dose reduced to 100 mg/day and 4 additional pts to 50 mg/day for toxicities including rash, fatigue, diarrhea, transaminitis, and hyperbilirubinemia. 5 pts have withdrawn as a result of toxicity (n=3) or patient preference (n=2), mostly within the first month of treatment (n=4). Current average follow-up time is 8 months. Conclusions: Chronic treatment with adjuvant erlotinib in NSCLC pts with EGFR mutations is feasible. Major observed toxicities are consistent with those previously reported for erlotinib. Based the demonstrated role of EGFR TKIs in advanced NSCLC with EGFR mutations, this trial has been expanded to accrue 100 total patients. Toxicities Event Grade and frequency (%) I II III IV Total Rash 33 36 14 - 83 Diarrhea 58 3 3 - 64 Fatigue 31 17 6 - 53 Alopecia 19 11 - - 31 Nausea/vomiting 22 8 - - 31 Dry skin 25 - - - 25 Mucositis 17 3 - - 19 Pruritis 8 6 3 - 17 Skin erythema 11 6 - - 17 Ocular: dry eye, conjunctivitis 8 8 - - 17 Nail changes 14 - - - 14 AST/ALT 6 - 3 - 8 Bilirubin 3 3 - - 6 Pneumonitis - - - - 0 Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Genentech, GlaxoSmithKline, OSI, Roche Genentech Genzyme