Epidermal Growth Factor Receptor Mutation Analysis Research Articles

Epidermal growth factor receptor (EGFR) mutation and p-EGFR expression in resected non-small cell lung cancer

ABSTRACTLung cancer, specifically non-small cell lung cancer (NSCLC), is a leading cause of mortality worldwide. In China, a dramatic increase in the incidence of NSCLC is expected in the next 20 years (Molina et al. Mayo Clin Proc. 2008;83:584–594). Mutated epidermal growth factor receptor (EGFR) status is a known predictor of response to tyrosine kinase inhibitors (TKIs), and immunohistochemistry may be a less costly way of predicting presence of mutation. In this study, mutation analysis of EGFR in 218 cases of NSCLC was performed. One hundred thirty tissue samples were examined via immunohistochemistry of p-EGFR (Y1045 and Y1068) and correlated with mutation status. Mutations were seen in 29% of patients, and were correlated with female sex, nonsmoking history, and adenocarcinoma histology. Phosphorylation at Y1045 was noted in 52% of cases, but in 71% of cases with EGFR mutation (P = .003). Phosphorylation of Y1068 was seen in 55% of cases but in 73% of cases with EGFR mutation (P = .006). This study correlating EGFR mutation with p-EGFR expression in resected NSCLC is one of the largest to date, although TKI response could not be assessed. The data show that, among Chinese patients, detection of p-1045 and p-1068 expression with immunohistochemistry predicts EGFR mutations. Immunohistochemical analysis of p-EGFR may be useful to predict responses to TKI therapy, although future studies are necessary.

EGFR expression variance in paired colorectal cancer primary and metastatic tumors

Background: Previous studies indicate that drugs targeting the Epidermal Growth Factor Receptor (EGFR) signaling pathways can induce objective responses, prolong time to progression and improve survival of patients with metastatic colorectal cancer (mCRC). EGFR expression in the primary tumour may not predict response to these agents and data is conflicting regarding the correlation of EGFR expression in the primary tumour with the metastatic site. In other tumour sites, the presence of EGFR mutations was associated with efficacy in a subset of patients.Objectives: The goal of this study is to correlate tumour EGFR expression between primary and liver metastatic sites, and to assess the mutational status in the EGFR kinase domain.Methods: This is a single center retrospective study of patients who underwent surgical resection of CRC, for whom paired paraffin-embedded tissue blocks of primary tumours and resected liver metastases were available. EGFR immunostaining and mutation analyses were preformed.Results: Fifty eight paired colorectal primaries and metastases were available for analysis. EGFR was detectable in 96.6% of the primary samples and in 89.7% of the metastatic samples. Perfect concordance in the intensity score between the primary and the metastases was found in 46.5% of the cases. While individual pairs were poorly concordant for intensity, the proportion of primaries with intense staining was similar to the proportion with intense staining in the metastatic samples. Overall survival did not correlate with either EGFR expression in the primary tumour, or with EGFR expression in the metastasis. There were 2 cases with mutations in the EGFR kinase domain. Both mutations were found in exon21 C>T.Conclusions: In this analysis, EGFR expression in the primary tumor site was not predictive of its level in the metastasis. EGFR expression levels in the primaries and in the metastases do not appear to be useful prognostic markers.

A phase III randomized, double-blind, placebo-controlled trial of the epidermal growth factor receptor inhibitor gefitinb in completely resected stage IB-IIIA non-small cell lung cancer (NSCLC): NCIC CTG BR.19.

LBA7005 Background: In meta-analyses, platinum-based adjuvant (adj) chemotherapy (CT) in completely resected NSCLC increased cure rate by ∼5%. At BR.19 initiation, adj study results with third-generation agents were unavailable. Gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, showed activity in monotherapy trials. We report a trial of adj gefitinib versus placebo after complete resection of NSCLC. Methods: Patients (pts) with stage IB-IIIA NSCLC were stratified by sex, stage, histology, post-op radiation, and after Jan 2003, adj CT. Pts were randomized to gefitinib 250 mg or placebo daily x 2 years. Study endpoints included overall survival (OS), disease free survival (DFS), toxicity, preplanned correlative studies. Study closed prematurely in Apr. 2005. Trial committee and NCIC CTG staff remained blinded to study drug. Results: 503 pts randomized (Sep 2002-Apr 2005); median age 67, males 54%, PS 0 54%; stage IB 49%, II 38%, III 13%; adenocarcinoma 59%, squamous 28%; ever smokers 89%; adj CT 17%; lobectomy 82%. Commonest grade 3/4 toxicities = fatigue 5%, rash 4% and diarrhea 5%. grade 3+ pneumonitis in 7 (1.4%) pts and led to death in 1. Median follow-up is 4.7 yrs; median treatment time is 4.8 mos. For gefitinib versus placebo, median DFS is 4.2 yrs versus not yet reached (NYR) HR1.22 (95% C.I. 0.93-1.61), p=0.15 and median OS 5.1 yrs versus NYR HR 1.24 (95% C.I. 0.94-1.64), p=0.14. In multivariate analysis, tumor size >4cm was predictive of poor DFS (p<0.0001) and never smoking for better OS with gefitinib (p=0.02). Results for KRAS and EGFR copy are available on 350 and 348 pts respectively. KRAS mutations were neither prognostic HR 1.12 (95% C.I. 0.67-1.86), p=0.66 nor predictive of gefitinib benefit (p = 0.16) on OS. EGFRcopy whether low/high polysomy or amplification was neither prognostic (p=0.77) nor predictive of OS benefit from gefitinib. Conclusions: Adjuvant gefitinib after complete resection of early stage NSCLC did not confer DFS or OS advantage in overall population. KRAS and EGFR copy were neither prognostic nor predictive of benefit from gefitinib. EGFR mutational analysis will be presented. [Table: see text]

Induction erlotinib therapy in stage IIIA-N2 non-small cell lung cancer.

TPS284 Background: Stage IIIA non-small cell lung cancer (NSCLC) is seen in a relatively heterogeneous group of patients with ipsilateral mediastinal (N2) lymph node involvement. The relative roles of different treatment modalities are not clear. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) may result in dramatic responses in patients with pulmonary adenocarcinoma carrying EGFR activating mutations. In case reports, the efficacy of perioperative EGFR-TKI therapy in patients with locally advanced NSCLC harboring EGFR gene mutations was satisfactory. Although no prospective data support the use of EGFR-TKIs as induction therapy in stage IIIA-N2 NSCLC, their low toxicity profile and the possibility of a rapid tumor response suggests that prospective trials are required. Therefore, this study evaluated the value of induction erlotinib therapy in IIIA-N2 NSCLC selected by EGFR gene analysis and explored a new treatment strategy for this subset. Erlotinib specifically targets the EGFR TK domain, which is highly expressed and occasionally mutated in various forms of cancer. It binds in a reversible fashion to the adenosine triphosphate (ATP) binding site of the receptor. Methods: A two-stage phase II trial of induction EGFR-TKI therapy is currently recruiting participants (ClinicalTrials.gov identifier: NCT00600587). Patients with resectable stage IIIA-N2 NSCLC confirmed by mediastinoscopy or PET/CT were assigned to the induction erlotinib therapy arm (150 mg erlotinib taken once daily and continued uninterrupted for 42 d before evaluation and thoracotomy/radiotherapy) or the induction gemcitabine/carboplatin (GC) arm based on the EGFR mutation analysis. Study type: Interventional. Study Design: Active control, factorial assignment, nonrandomized, open label, safety/efficacy study, treatment. Primary outcome measures: Response to induction therapy, disease-free survival. Estimated enrollment: Fifteen cases in the erlotinib arm are needed in the first stage, and 46 cases in the second stage. At present, six patients have been assigned to the induction erlotinib therapy arm and seven to the induction GC arm. No significant financial relationships to disclose.

Immunohistochemistry detection of epidermal growth factor receptor (EGFR) exons 19 and 21 mutations in non-small cell lung cancer (NSCLC) using novel mutation specific antibodies.

10542 Background: Activating EGFR mutations have become a backbone in the therapy alogrithm for NSCLC as mutations predict significantly better response and survival to EGFR TKIs. The most common EGFR mutations in NSCLC are exon 19 deletions and the exon 21 L858R point mutation. Among the exon 19 deletions, E746-A750 occurs in 61% of cases. In this study we compared novel IHC antibodies for E764-A750 and L858R to direct DNA sequencing in specimens from Japanese patients with NSCLC treated with gefitinib. Methods: IHC using the E746-A750 and L858R mutation specific antibodies (Cell Signaling Technologies, Danvers, MA) was performed on resected NSCLC Japanese patients who were subsequently treated with gefitinib at relapse. Extracted DNA was sequenced for mutational analysis of EGFR exons 18 to 21. Results: DNA sequencing of all 70 patients showed exon 19 deletions in 18 patients (25.7%), 11 had a deletion at E746-A750, exon 20 mutations in 18 patients, exon 21 mutations in 12 (17.1%), and exon 18 mutations in one patient. The mutation specific antibodies detected 9/11 of the E746-A750 mutations (2 false negatives, 0 false positive, sensitivity 81.8%, and specificity 100%) and 9/12 of the L858R point mutations (2 false positives, 3 false negatives, sensitivity 75%, and specificity 96.6%). Objective response rates (ORR) were associated significantly with exon 19 deletions (44.4%, p = 0.006) and with exon 21 mutations (36.4%, p = 0.009). Conclusions: The exon 19 and 21 mutation specific antibodies for IHC staining have high sensitivity and specificity for the pre-defined mutations. Such techniques would be very cost-effective for mutation screening in NSCLC. False positives could result from low-sensitivity sequencing, whereas false negative could result from small sample size of TMA relative to tumor size sequenced. For clinical decisions, negative IHC staining may require further EGFR mutation analyses. No significant financial relationships to disclose.

Cetuximab for the treatment of advanced bronchioloalveolar carcinoma (BAC): An Eastern Cooperative Oncology Group phase II study (ECOG 1504).

7522 Background: Inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase have demonstrated modest anticancer activity in pts with advanced BAC, a disease that is refractory to standard chemotherapy regimens. We conducted a phase II study to evaluate cetuximab, an antibody against EGFR, for the treatment of advanced BAC. Methods: Pts with advanced stage pure BAC or adenocarcinoma with BAC features, presence of measurable disease, 0 or 1 prior chemotherapy regimen, and ECOG performance status (PS) of 0-2 were eligible. Prior EGFR inhibitor therapy was not permitted. Cetuximab was given as a weekly i.v. infusion at 250 mg/m2 from week 2 onwards after an initial loading dose of 400 mg/m2 in week 1 until disease progression. The primary endpoint was determination of response rate. EGFR and KRAS mutations were evaluated by pyrosequencing. Results: 72 pts were enrolled and 68 met all eligibility criteria. Pt characteristics were: median age- 71 yrs, female– 57%; ECOG PS 0 or 1– 88%; no prior chemotherapy- 68%; and never smokers- 18%. Central pathology review confirmed the diagnosis in 45 out of 47 available specimens (mucinous- 35%; non-mucinous- 20%; Adeno with BAC features- 16%, pure BAC -29%). Approximately 50% of pts received more than 2 cycles of therapy (> 8 weeks). Skin rash was the most common toxicity (any grade 71%; grade 3- 15%) and hypomagnesemia was noted in 26%. The confirmed response rate was 6% and SD was observed in 37% of pts. The median survival was 13 m and the 1- and 2-year survival rates were 52% and 37% respectively. The median PFS was 3.2 m with a 6- months PFS rate of 22%. Tumor tissues were available from 55 pts for evaluation of EGFR and KRAS mutation status. Analysis of EGFR exon 20 polymorphisms demonstrate an allele frequency of Q787Q G = 0.55, A = 0.45; T790M, C = 1.0. Complete EGFR and KRAS mutation analysis will be presented at the meeting. Conclusions: The efficacy of cetuximab is comparable with that of EGFR TK inhibitors in the treatment of advanced BAC despite low response rates by RECIST. Supported in part by PHS Grants CA23318, CA66636, CA21115, CA21076 and from the NCI, NIH and the Department of Health and Human Services. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Bristol-Myers Squibb, ImClone Systems, Lilly ImClone Systems

Combined mutation pattern analyses of epidermal growth factor receptor and K-ras genes for the differential diagnosis of primary versus metastatic lung adenocarcinomas.

e21010 Background: The differential diagnosis of multiple primary lung cancers (MPLCs) from pulmonary metastases (PMs) is sometimes difficult based solely on the Martini and Melamed's criteria. We investigated whether combined mutation pattern analyses of the epidermal growth factor receptor (EGFR) and K-ras genes were useful for differentiating between MPLCs and PMs. Methods: We conducted a retrospective review of 84 tumors (81 adenocarcinomas and three atypical adenomatous hyperplasias) in 36 patients with multifocal lung adenocarcinomas. Genomic DNA was extracted from paraffin-embedded tissue. The peptide nucleic acid (PNA)-locked nucleic acid polymerase chain reaction clamp method was used for EGFR mutation analysis. The PNA-mediated PCR clamping method was used for K-ras mutation analysis. We determined clonality status of secondary tumors based on the mutation patterns of EGFR and K-ras by comparing them with primary tumors. Correlations between the mutation status and the proportion of bronchioloalveolar carcinoma (BAC) component in a tumor were examined. Results: EGFR and K-ras mutations were detected in 38 (45%) and 24 (29%) of 84 tumors, respectively. Thirty-four (71%) of secondary tumors were diagnosed as a different clonality (MPLC). Seven (15%) of secondary tumors were classified into the same clonality (PM). Clonality status could not be determined for the seven (15%) remaining secondary tumors. A clonality assessment based on the EGFR and K-ras mutation status was possible in 41 (84%) of 48 secondary tumors, and it was consistent with the Martini and Melamed's criteria in 29 (71%) of 41 secondary tumors. The proportion of BAC component in a tumor did not correlate with the EGFR/K-ras mutation status. Conclusions: The combined mutation pattern analyses of EGFR and K-ras enabled a definite distinction between PMLCs and PMs in a large portion of multifocal lung adenocarcinomas. Mutations of EGFR and K-ras in multifocal lung adenocarcinomas frequently occurred randomly, which support the concept of “field cancerization.” No significant financial relationships to disclose.

Abstract LB-77: A randomized double-blind placebo-controlled phase II trial of sorafenib and erlotinib or erlotinib alone in previously treated advanced non-small cell lung cancer: Correlative analyses

Abstract Background: Sorafenib is an oral multikinase inhibitor of angiogenesis and cellular proliferation which has shown preliminary activity in non-small-cell lung cancer (NSCLC). We conducted a multicenter phase II trial of erlotinib +/− sorafenib in 2nd/3rd line NSCLC. Methods: Patients with previously treated stage IIIB/IV NSCLC were eligible (1-2 prior regimens). Additional eligibility criteria included: ECOG performance status 0-2, all NSCLC histologies, measurable disease, adequate organ function, and the absence of major cardiovascular disease. Patients with treated brain metastases and/or on stable anticoagulation were eligible. Patients were stratified according to squamous/non-squamous histology, prior bevacizumab use, and line of therapy and were randomized 2:1 to receive erlotinib 150mg orally once daily and sorafenib 400mg orally twice daily, or erlotinib and placebo. Patients were also consented for biomarker testing for epidermal growth factor receptor (EGFR) mutations, EGFR amplification, and K-ras mutations. The primary endpoint was to assess the efficacy of each arm in terms of objective response rate (ORR) and progression-free survival (PFS). The trial was designed to enroll 168 patients to demonstrate a 40% improvement on an historical 2.2 month PFS (> 80%, 1-sided 0.1). Results: 168 patients were enrolled from February 2008 to February 2009. Baseline characteristics were well-matched between cohorts (median age, 65 years; 3rd line therapy, 39%; prior bevacizumab, 36%; squamous histology, 30%). The ORRs for the sorafenib/erlotinib and erlotinib/placebo cohorts were 8.1% v. 10.9%, respectively (p=.55). However, the disease control rates (ORR + proportion of patients with stable disease) were 54.1% (sorafenib/erlotinib) v. 38.2% (erlotinib/placebo) (p=.06). The median PFS was 3.25 months (sorafenib/erlotinib) v. 1.87 months (erlotinib/placebo) (HR 0.86 [95% CI 0.60, 1.22], p=.20). 72 (44%) patients had material assessable for EGFR mutation analysis. Among the 67 patients who were found to have EGFR wild-type (WT) tumors, the median PFS was 3.25 months (sorafenib/erlotinib) v. 1.71 months (erlotinib/placebo) (HR 0.55 [95% CI 0.32, 0.96], p=.02). Conclusions: Additional preplanned subset analyses based on histology, EGFR amplification, K-ras mutation, prior bevacizumab treatment, and line of therapy will be presented, as well as updated overall response, PFS, survival, and toxicity data. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-77.

EGFR fluorescence in situ hybridization-positive lung adenocarcinoma: incidence of coexisting KRAS and BRAF mutations

Despite growing evidence that epidermal growth factor receptor (EGFR) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation analysis is the most reliable predictor of the lung carcinoma response to EGFR-targeted therapies, there is still discussion about the role of EGFR fluorescence in situ hybridization (FISH). Studies focusing on EGFR FISH as a predictor of response to EGFR-targeted therapies mostly focused on the relationship between EGFR FISH and EGFR mutations. The incidence of KRAS and V-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations in EGFR-amplified or EGFR FISH-positive lung adenocarcinomas remains unknown. The aim of this study was to prospectively characterize the incidence of KRAS and BRAF mutations in EGFR FISH-positive surgically treated lung adenocarcinomas. Of 386 primary lung adenocarcinomas, 77 (20%) were EGFR FISH positive by University of Colorado criteria. The incidence of KRAS mutations in EGFR FISH-positive lung adenocarcinomas was 23% and was not significantly different from the incidence of KRAS mutations in EGFR FISH-negative subsets of adenocarcinoma (32%). A higher mean ratio between EGFR and chromosome 7 enumeration probe (EGFR/CEP7) was observed in EGFR-mutated tumors when compared to cases with KRAS mutation (13 versus 4.5, respectively). Our results showed significant number of EGFR FISH positive/amplified lung adenocarcinomas harboring KRAS mutation. It appears that an increase in EGFR/CEP7 ratio to cutoff point of 4.5 may distinguish between coexisting EGFR (FISH ratio of >5) or KRAS (FISH ratio of 2 to 5) mutations. Observations presented here indicate that the patient selection for EGFR-targeted therapies should include EGFR and KRAS mutational analysis, probably complemented by EGFR FISH studies.

Pulmonary Pleomorphic Carcinoma: A Clinicopathological Study Including EGFR Mutation Analysis

Pulmonary pleomorphic carcinoma is a rare epithelial tumor and has an aggressive clinical course. As few studies of pulmonary pleomorphic carcinoma have been described, the clinicopathological characteristics of the disease remain unclear. Especially, the information on the epidermal growth factor receptor (EGFR) mutation status of pulmonary pleomorphic carcinoma is sparse. We retrospectively examined 17 patients with pulmonary pleomorphic carcinoma. EGFR mutation and Ki-67 labeling index were investigated in these patients. The median age of the patients was 72 years (range, 47-84 years). Thirteen patients were men and four were women. EGFR mutation was observed in 3 (18%) of 17 patients. The median value of Ki-67 labeling index was 62% (range, 20-87%). Positron emission tomography with 18-fluorodeoxy-glucose was performed in 16 patients, and the standardized uptake value tended to be high (median 19.3). The survival of patients without surgery demonstrated a significantly poor prognosis compared with those with surgery (P = 0.0096). Palliative chemotherapy was almost poor response in advanced pulmonary pleomorphic carcinoma. The response to gefitinib in a patient with EGFR mutation was small and transient. EGFR mutation was recognized in approximately 20% of patients with pulmonary pleomorphic carcinoma. It is necessary to investigate whether the use of a molecular targeting drug improves outcome for pulmonary pleomorphic carcinoma.

Efficacy and safety of erlotinib as monotherapy for advanced non-small cell lung cancer

To explore the efficacy and safety of erlotinib monotherapy for advanced non-small cell lung cancer (NSCLC). Totally 50 patients with advanced NSCLC received oral erlotinib 150 mg/d treatment, and tumor specimen in 19 patients were collected for epidermal growth factor receptor (EGFR) gene mutation tests. Median survival (MS) was calculated using the Kaplan-Meier method. The most common adverse events (AEs) were skin rash (96%) and diarrhea (32%). The overall survival (OS) of all patients was 21.8 months 95% confidential interval (CI): 17.1-26.4 months and the median progression-free survival (PFS) of all patients was 7.0 months (95% CI: 3.9-10.1 months). EGFR mutation analysis showed gene mutation in 8 cases and wild type in 11 cases. The objective response rate in patients with or without EGFR gene mutations were 62.5% and 9.1%, respectively (chi(2)=6.631, P=0.036). PFS in patients with or without EGFR gene mutations were 16.330 (95% CI: 2.803-29.857 months) and 5.570 months (95% CI: 2.441-8.699 months), respectively (chi(2)=8.799, P=0.003). Erlotinib monotherapy is safe and effective for some Chinese NSCLC patients after failure of prior chemotherapy.

Detection of epidermal growth factor receptor mutations in plasma by mutant‐enriched PCR assay for prediction of the response to gefitinib in patients with non‐small‐cell lung cancer

The high frequency of epidermal growth factor receptor (EGFR) mutations in tyrosine kinase inhibitor-responsive non-small-cell lung cancer (NSCLC) cases is now well established, highlighting the predictive value of activating EGFR mutations in guiding the clinical use of EGFR-targeted therapies. However, specimen source and methods for EGFR mutation analysis are limited by tissue availability and technical feasibility in clinical application. Therefore, the current study is designed to establish a blood-based approach for the assessment of EGFR mutations in NSCLC patients, in particular the advanced stage, and to test its clinical application. Plasma samples were obtained from the enrolled 134 NSCLC patients. The detection rate of the EGFR exon19 deletions and exon21 L858R was 49.3% (66/134) by the blood-based, mutant-enriched polymerase chain reaction. In the paired tumor and plasma samples, the detected mutant types of each pair respectively by direct sequencing and mutant-enriched polymerase chain reaction were concordant in 17 of 18 (94.4%). In the patients treated with gefitinib as a second-line therapy, those with plasma EGFR mutation have a prolonged median progression-free survival compared with those with EGFR wild type (7.609 vs. 2.877 months, p = 0.002). On comparing the efficacy of gefitinib with that of docetaxel, it was found that the median progression-free survival was significantly longer for patients treated with gefitinib than those with docetaxel in those harboring plasma EGFR mutation (7.609 vs. 3.192 months, p = 0.006). These results suggest that the blood-based EGFR mutations test has the ability to provide a reliable guidance for clinical decision making for the treatment of the advanced NSCLC patients.

A comparison of EGFR and KRAS status in primary lung carcinoma and matched metastases

Epidermal growth factor receptor (EGFR) and v-Ki-ras 2 (KRAS; viral Kirsten rat sacoma 2 oncogene homolog) oncogenes are predictors of response to EGFR-targeted therapy in lung carcinomas. Morphologic heterogeneity of lung carcinomas is reflected at the molecular level and may confound interpretation of immunohistochemistry, fluorescence in situ hybridization, and mutational assays, which are all used for analysis of KRAS and EGFR genes. Furthermore, molecular characteristics may differ between the primary tumor and corresponding metastases. The aim of this study was to determine if the KRAS and/or EGFR status of primary and metastatic lung carcinoma differs. Three hundred thirty-six cases of primary lung carcinomas were tested for EGFR and KRAS, and 85 cases had a metastasis (25%). Of the 40 cases (47%) with sufficient material for EGFR and KRAS mutational analysis, there were 11 (27.5%) primary tumors and 4 (10%) metastases identified with a KRAS mutation. Of the cases with EGFR fluorescence in situ hybridization results, there were 3 (8%) primary tumors and 8 (24%) metastases that were fluorescence in situ hybridization positive. Overall, there were 9 cases (22.5%) with discordant KRAS status and 11 cases (32.5%) with discordant EGFR fluorescence in situ hybridization status. Our results suggest that the EGFR and KRAS status of primary lung carcinomas may not predict the status in the corresponding metastases. This observation may have important implications for molecular testing for targeted therapies.

Frequency of and variables associated with the EGFR mutation and its subtypes

Mutation in the epidermal growth factor receptor (EGFR) is frequently seen in non-small cell lung cancers (NSCLCs), especially in Asian females with adenocarcinoma. The frequency of mutation and the factors associated requires to be elucidated by analyzing a large number of consecutive clinical samples. We summarized the result of the EGFR mutation analysis for 1,176 patients performed at the time of diagnosis or relapse. The PNA-LNA PCR clamp, a highly sensitive detection method for the EGFR mutation, was employed. For fresh cases a portion of samples isolated to establish the diagnosis of lung cancer was used. For cases with a relapsed disease archival tissue were tested. The variables associated with the EGFR mutation after removing the confound factors were investigated by the logistic analysis using the samples collected in our university (n = 308) where detailed information on patients were available. The frequency of the EGFR mutation and its subtypes were investigated using all samples (n = 1,176). The EGFR mutation was significantly associated with adenocarcinoma (p = 0.006) and light-smoking (p < 0.0001), but not gender. The deletions in exon 19 were more frequently associated with male gender while exon 21 deletions were with female gender (p = 0.0011). The overall frequency of the EGFR mutation was 31%. Our result suggests that the female predominance in the EGFR mutation rate is a reflection of a higher frequency of adenocarcinoma in females. The gender difference in the mutation subtypes may provide a clue for the mechanism of the occurrence of the EGFR mutation.

Epidermal Growth Factor Receptor Gene in Primary Tumor and Metastatic Sites from Non-small Cell Lung Cancer

The majority of patients with non-small cell lung cancer (NSCLC) develop distant metastases. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors are capable of reducing brain and adrenal metastases. However, the EGFR status may be discordant between primary NSCLC and the corresponding metastases. Using fluorescence in situ hybridization (FISH) analysis, the EGFR gene status was evaluated in a series of 38 cerebral or adrenal metastases collected from two institutions and in the corresponding primary tumors. Also, EGFR mutational analysis was performed using direct sequencing on the cerebral metastases. EGFR FISH was positive in 28% of the primary tumors and in 45% of the metastases (p < 0.05). Among the seven cases FISH-positive at the metastatic site but negative in the primary tumor, six were brain metastases, and one was an adrenal metastasis; all were polysomic for chromosome 7, none were amplified. No EGFR mutations have been found in the cerebral metastases. Because the molecular asset of EGFR may change during the metastatic progression of NSCLC to brain (but not to adrenal), the selection of patients with brain metastasis for specific targeted therapies by EGFR FISH analysis should be performed on metastatic lesions rather than on their corresponding primary tumors.

Phase II study of dasatinib in non-small cell lung cancer (NSCLC)

e19015 Background: Lung cancer is the leading cause of cancer-related deaths. Better systemic therapies are needed. One potential therapeutic target is c-Src which is expressed and activated in NSCLC patient tumors where it can mediate invasion, angiogenesis, and proliferation. Additionally, epidermal growth factor receptor (EGFR) and c-Src cooperate to promote NSCLC survival. We are conducting a phase II study of dasatinib, a tyrosine kinase inhibitor of c-Src, Abl, c-Kit, PDGFR, Btk, and EphA2. The primary objective is to determine the rate of progression free survival at 12 weeks in patients with metastatic NSCLC treated with dasatinib as front line therapy. Methods: Patients with metastatic NSCLC were treated with dasatinib (100 mg BID), with PET/CT scans every 6 weeks. KRAS and EGFR mutations, EGFR copy number, and pSrc expression were measured in pre-treatment biopsies. Blood was collected pretreatment and on day 21 to measure drug exposure (PK), pharmacodynamics (PD), and serum cytokine levels. Results: Twenty five patients have enrolled on study. Of the 16 patients evaluable for response: 1 had a partial response (PR) with no evidence of recurrence for at least 18 months (male smoker with adenocarcinoma and KRAS mutation); 6 patients had stable disease (SD) which includes 3 patients with prolonged stable disease for 4, 6, and 18 months; 9 had progressive disease (PD). Only 4 patients had a significant change in SUV (&gt;25%): decreased in 2 with PR and SD and increased in 2 with PD and SD. There is currently no clear association between EGFR and KRAS mutational analysis and response, although the sample number is small. Only 2 patients have activating EGFR mutations: one with SD and one with PD. PK, PD, and cytokine data will be presented. The most common grade 3/4 toxicity is dyspnea/pleural effusion that has led to a reduced dasatinib starting dose in subsequent patients. Conclusions: Dasatinib as a single agent has activity in a subset of patients with NSCLC. Planned correlative studies may lead to the discovery of biomarkers that predict response. Toxicities observed were consistent with prior dasatinib phase I studies in solid tumor patients. Supported by NCI/CTEP (NIH contract N01-CM-62202), The Commonwealth Foundation for Cancer Research and Bristol-Myers Squibb. [Table: see text]

Toward Noninvasive Genomic Screening of Lung Cancer Patients

Toward Noninvasive Genomic Screening of Lung Cancer Patients

Epidermal Growth Factor Receptor Mutations in Plasma DNA Samples Predict Tumor Response in Chinese Patients With Stages IIIB to IV Non–Small-Cell Lung Cancer

Mutations in the epidermal growth factor receptor (EGFR) kinase domain can predict tumor response to tyrosine kinase inhibitors (TKIs) in non-small-cell lung cancer (NSCLC). However, obtaining tumor tissues for mutation analysis is challenging. We hypothesized that plasma-based EGFR mutation analysis is feasible and has value in predicting tumor response in patients with NSCLC. Plasma DNA samples and matched tumors from 230 patients with stages IIIB to IV NSCLC were analyzed for EGFR mutations in exons 19 and 21 by using denaturing high-performance liquid chromatography. We compared the mutations in the plasma samples and the matched tumors and determined an association between EGFR mutation status and the patients' clinical outcomes prospectively. In 230 patients, we detected 81 EGFR mutations in 79 (34.3%) of the patients' plasma samples. We detected the same mutations in 63 (79.7%) of the matched tumors. Sixteen plasma (7.0%) and fourteen tumor (6.1%) samples showed unique mutations. The mutation frequencies were significantly higher in never-smokers and in patients with adenocarcinomas (P = .012 and P = .009, respectively). In the 102 patients who failed platinum-based treatment and who were treated with gefitinib, 22 (59.5%) of the 37 with EGFR mutations in the plasma samples, whereas only 15 (23.1%) of the 65 without EGFR mutations, achieved an objective response (P = .002). Patients with EGFR mutations had a significantly longer progression-free survival time than those without mutations (P = .044) in plasma. EGFR mutations can be reliably detected in plasma DNA of patients with stages IIIB to IV NSCLC and can be used as a biomarker to predict tumor response to TKIs.

Comparison of epidermal growth factor receptor mutations between primary and corresponding metastatic tumors in tyrosine kinase inhibitor-naive non-small-cell lung cancer

BackgroundMutations of the epidermal growth factor receptor (EGFR) gene in non-small-cell lung cancer (NSCLC) patients predict the patients who will respond to EGFR tyrosine kinase inhibitor (TKI) treatment. A recent study has suggested that 33% of NSCLC showed primary tumor/metastasis discordance of EGFR expression by immunohistochemistry analysis. We intended to find out whether the EGFR mutations of primary lung cancers are concordant to that of corresponding metastatic tumors. Materials and methodsWe analyzed EGFR exons 18–21 from paired primary and metastatic tumors in 67 lung cancer patients who had not received TKI before tissues were sampled. ResultsUsing the direct sequencing method, 9 of 18 (50%) patients with EGFR mutation-positive primary lung tumors had lost the mutations in metastases. For 26 patients who were EGFR mutation positive in the metastatic tumors, 17 (65%) were negative in the primary tumors. We analyzed these paired tissues with discrepant EGFR mutations by the Scorpion Amplified Refractory Mutation System assay. Finally, the discordant rate reached 27% (18 of 67 cases). ConclusionEGFR mutations in primary lung tumors do not always reflect the same situation in metastases. Analysis of EGFR mutations in the primary lung tumor would be inadequate for planning the use of TKI for advanced NSCLC.

Single-Molecule Detection of Epidermal Growth Factor Receptor Mutations in Plasma by Microfluidics Digital PCR in Non–Small Cell Lung Cancer Patients

We aim to develop a digital PCR-based method for the quantitative detection of the two common epidermal growth factor receptor (EGFR) mutations (in-frame deletion at exon 19 and L858R at exon 21) in the plasma and tumor tissues of patients suffering from non-small cell lung cancers. These two mutations account for >85% of clinically important EGFR mutations associated with responsiveness to tyrosine kinase inhibitors. DNA samples were analyzed using a microfluidics system that simultaneously performed 9,180 PCRs at nanoliter scale. A single-mutant DNA molecule in a clinical specimen could be detected and the quantities of mutant and wild-type sequences were precisely determined. Exon 19 deletion and L858R mutation were detectable in 6 (17%) and 9 (26%) of 35 pretreatment plasma samples, respectively. When compared with the sequencing results of the tumor samples, the sensitivity and specificity of plasma EGFR mutation analysis were 92% and 100%, respectively. The plasma concentration of the mutant sequences correlated well with the clinical response. Decreased concentration was observed in all patients with partial or complete clinical remission, whereas persistence of mutation was observed in a patient with cancer progression. In one patient, tyrosine kinase inhibitor was stopped after an initial response and the tumor-associated EGFR mutation reemerged 4 weeks after stopping treatment. The sensitive detection and accurate quantification of low abundance EGFR mutations in tumor tissues and plasma by microfluidics digital PCR would be useful for predicting treatment response, monitoring disease progression and early detection of treatment failure associated with acquired drug resistance.