Epidermal Growth Factor Receptor Mutant Lung Cancer Research Articles

Abstract A38: Resistance to Afatinib and Cetuximab Combination Therapy in EGFR-mutant Lung Adenocarcinomas

Abstract The epidermal growth factor receptor (EGFR) T790M mutation confers acquired resistance to tyrosine kinase inhibitors (TKIs) in approximately 50% of drug-resistant EGFR mutant lung adenocarcinomas. Experiments using genetically engineered mouse models of EGFR mutant lung cancer have revealed that T790M-mediated resistance can be overcome by a second-generation TKI, afatinib, in combination with the anti-EGFR antibody, cetuximab. This drug combination is currently in clinical trials in patients with TKI-resistant tumors and is showing a promising ∼40% response rate. Nevertheless, cases of afatinib+cetuximab resistance are beginning to emerge. To identify the molecular mechanisms that play a role in afatinib+cetuximab resistance and test new therapies to overcome resistance to these drugs, we have developed mouse models of afatinib+cetuximab resistance in mice with inducible expression of the EGFRL858R+T790M mutant in type II pneumocytes. Mice harboring lung tumors were treated five days a week with afatinib and twice a week with cetuximab for four weeks. After the first round of treatment, drug-treatments were interrupted for one month. This on/off drug treatment was repeated until lung tumors no longer responded to treatment, as evidenced by imaging as well as clinical symptoms. Resistance to afatinib+cetuximab treatment was also studied in xenografts. Eight-week-old nu/nu athymic nude mice were injected subcutaneously with human lung adenocarcinoma cells, expressing the erlotinib-resistant EGFRDel19+T790M mutant. Notably, both transgenic and xenografts models harboring EGFRT790M-induced tumors developed resistance to EGFR-dual targeting and preliminary molecular studies on the resistant tumors show the absence of additional mutations in EGFR and in the ERBB2 tyrosine kinase domain in resistant tumors. These findings suggest that an alternative pathway or mechanism is involved in mediating resistance to the afatinib and cetuximab combination. We are further exploring mechanisms of resistance by performing genome-wide and signaling pathway analyses of drug-resistant tumors from these models. Findings from mouse models will be validated in repeat biopsy specimens from patients who have developed resistance to the drug combination. An improved understanding of the molecular mechanisms responsible for acquired resistance to EGFR inhibition will provide new insight into the biology of this subset of lung cancers with immediate therapeutic implications for patients.

Ligand‐triggered resistance to molecular targeted drugs in lung cancer: Roles of hepatocyte growth factor and epidermal growth factor receptor ligands

Recent advances in molecular biology have led to the identification of new molecular targets, such as epidermal growth factor receptor ( EGFR ) mutations and echinoderm microtubule-associated protein-like 4 (EML4) - anaplastic lymphoma kinase (ALK) fusion gene, in lung cancer. Dramatic response has been achieved with EGFR inhibitors (gefitinib and erlotinib) and an ALK inhibitor (crizotinib) in lung cancer expressing corresponding targets. However, cancer cells acquire resistance to these drugs and cause recurrence. Known major mechanisms for resistance to molecular targeted drugs include gatekeeper mutations in the target gene and activation of bypass survival signal via receptors other than the target receptors. The latter mechanism can involve receptor gene amplification and ligand-triggered receptor activation as well. For example, hepatocyte growth factor (HGF), the ligand of a tyrosine kinase receptor Met, activates Met and the downstream PI3K/Akt pathway and triggers resistance to EGFR inhibitors in EGFR mutant lung cancer cells. Moreover, EGFR ligands activate EGFR and downstream pathways and trigger resistance to crizotinib in EML4-ALK lung cancer cells. These observations indicate that signals from oncogenic drivers (EGFR signaling in EGFR -mutant lung cancer and ALK signaling in EML4-ALK lung cancer) and ligand-triggered bypass signals (HGF-Met and EGFR ligands-EGFR, respectively) must be simultaneously blocked to avoid the resistance. This review focuses specifically on receptor activation by ligand stimulation and discusses novel therapeutic strategies that are under development for overcoming resistance to molecular targeted drugs in lung cancer.

Abstract 1907: Heat shock protein 90 inhibition overcomes hepatocyte growth factor-triggering resistance to EGFR tyrosine kinase inhibitors in EGFR mutant lung cancer by decreasing client protein expression and angiogenesis

Abstract The three major, clinically relevant mechanisms of acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR mutant lung cancer are a second mutation in the EGFR gene (T790M), Met amplification, and increased expression of hepatocyte growth factor (HGF). Heat shock protein90 (Hsp90) is a 90kDa molecular chaperone for proteins that include EGFR, Met and EML4-ALK. Hsp90 inhibitors have been reported to reverse resistance to EGFR-TKIs caused by EGFR T790M mutation and Met amplification. Here, we determined whether inhibition of Hsp90 could overcome HGF-triggered EGFR-TKI resistance in EGFR mutant lung cancer cells. EGFR-TKI erlotinib inhibited the growth of PC-9, HCC827, and Ma-1 cells, with deletions in exon19 of EGFR, but not HGF-gene transfected Ma-1 (Ma-1/HGF) cells and H1975 cells, which contain the EGFR L858R and T790M mutations, respectively. Erlotinib also did not inhibit the growth of PC-9 and Ma-1 cells in the presence of HGF. However, an Hsp90 inhibitor, 17-DMAG, induced apoptosis and markedly inhibited the growth of these cell lines, even in the presence of HGF. This inhibition by 17-DMAG was associated with decreased expression of EGFR and Met, thereby decreasing Akt phosphorylation in tumor cells. In an in vivo model of HGF-triggered erlotinib-resistance, using Ma-1/HGF cells, 17-DMAG markedly suppressed tumor growth by decreasing angiogenesis and increasing apoptosis. These results indicate that Hsp90 inhibitors may overcome HGF-triggered resistance to EGFR-TKIs and that the use of Hsp90 inhibitors may result in more successful treatment of EGFR mutant lung cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1907. doi:1538-7445.AM2012-1907

Abstract 1897: RAS signaling pathway gene mutations and acquired resistance to EGFR tyrosine-kinase inhibitors in EGFR mutant lung cancer

Abstract EGFR mutant lung cancers are highly sensitive to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), but in the metastatic setting, acquired drug resistance is inevitable. Second-site EGFR mutations, MET amplification, PIK3CA mutations, and histologic changes (transformation to small cell lung cancer, epithelial-mesenchymal transition (EMT)) have been reported as resistance mechanisms. Up to 40% of cases are still mechanistically unexplained. Surprisingly, despite the importance of the RAS signaling pathway downstream of EGFR, no RAS pathway signaling gene mutations have been reported to occur in acquired resistance. Here, we investigated further the significance of this pathway in preclinical models and tumor specimens. First, we screened 21 lung tumors from 11 mice with acquired resistance to erlotinib (Politi et al. Dis Model Mech. 2010; 3(1-2): 111-9) and found one tumor with a KrasG12V mutation. Next, we established 6 new cell-line based models of acquired erlotinib resistance in 5 different drug-sensitive EGFR mutant cell lines. While 5 lines developed known mechanisms of resistance (i.e. EGFR T790M, MET amplification, EMT), 1 (11-18 cells) developed de novo a NRASQ61K mutation. Single cell cloning suggested that both the EGFR mutation (L858R) and NRASQ61K mutations were in the same cell. Consistent with these findings, NRAS activity was higher in 11-18 resistant cells (11-18R) compared to parental cells. In growth inhibition assays, 11-18R were sensitive only to combined EGFR inhibition with siRNA knockdown of NRAS or with MEK inhibitors. Expression of NRASQ61K in drug-sensitive PC-9 cells conferred resistance to erlotinib. Finally, we screened 127 tumor samples from patients with acquired resistance patients to EGFR-TKIs. No NRAS or KRAS mutations were found, but one patient's tumor harbored in addition to EGFR L858R both EGFRT790M and BRAFV600E mutations. BRAFV600E expression also conferred resistance to EGFR inhibition, both in PC-9 (EGFR exon 19 deletion) and PC-9R cells (EGFR exon 19 deletion plus T790M). Collectively, these data shown that mutations in RAS pathway signaling genes may be infrequent but occur and could influence therapeutic outcomes in trials for patients with acquired resistance to EGFR TKIs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1897. doi:1538-7445.AM2012-1897

Abstract IA4: Defining the mechanisms of tumorigenesis by mutant EGFR using mouse models

Abstract Mutations in exons encoding the epidermal growth factor receptor (EGFR) are found in 10–15% of lung adenocarcinomas. The two most common mutations: a point mutation in exon 21 that leads to substitution of an arginine for a leucine at position 858 and small in-frame deletions in exon 19 are associated with sensitivity to the specific tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib. Approximately 70% of tumors harboring EGFR mutations respond to treatment with these drugs, however 30% of these tumors exhibit primary resistance to treatment with these agents. Moreover, responses are transient and, on average, within a year of initiating treatment patients who initially respond eventually develop TKI-inhibitor-resistant disease. Drug resistance, in most cases, is due to a secondary mutation in EGFR (EGFR T790M). Other less frequent mechanisms of resistance include MET amplification, PIK3CA mutations and transformation to small cell lung cancer. However, the mechanism of resistance is still unknown in approximately 30% of cases. More effective treatment of patients with EGFR mutant lung cancer requires a better understanding of the mechanisms of primary and acquired resistance to EGFR TKIs and the development of strategies to overcome this resistance. To study these problems in vivo, we developed tetracycline-inducible transgenic mouse models of EGFR mutant lung cancer. Expression of lung cancer associated EGFR mutants gives rise to lung adenocarcinomas with bronchioloalveolar carcinoma features that are dependent upon the continuous activity of mutant EGFR for survival. Thus, treatment of the mice with erlotinib leads to tumor regression and long-term treatment with the drug gives rise to drug-resistant tumors that harbor some of the alterations observed in human TKI-resistant tumors such as the T790M mutation. Current efforts to use these mouse models to: 1) study mechanisms of primary and acquired resistance to EGFR TKIs and 2) evaluate new therapies to overcome resistance to first generation TKIs will be discussed.

Hepatocyte Growth Factor Expression in EGFR Mutant Lung Cancer with Intrinsic and Acquired Resistance to Tyrosine Kinase Inhibitors in a Japanese Cohort

This study was performed to determine the incidence rates of resistance factors, i.e., high-level hepatocyte growth factor (HGF) expression, epidermal growth factor receptor (EGFR) T790M secondary mutation, and MET amplification, in tumors with intrinsic and acquired EGFR tyrosine kinase inhibitor (TKI) resistance in EGFR mutant lung cancer. Ninety-seven specimens from 93 EGFR mutant lung cancer patients (23 tumors with acquired resistance from 20 patients, 45 tumors with intrinsic resistance from 44 patients [nonresponders], 29 sensitive tumors from 29 patients) from 11 institutes in Japan were analyzed. HGF expression, EGFR T790M secondary mutation, and MET amplification were determined by immunohistochemistry, cycleave real-time polymerase chain reaction, and fluorescence in situ hybridization, respectively. High-level HGF expression, EGFR T790M secondary mutation, and MET amplification were detected in 61, 52, and 9% of tumors with acquired resistance, respectively. High-level HGF expression was detected in 29% of tumors with intrinsic resistance (nonresponders), whereas EGFR T790M secondary mutation and MET amplification were detected in 0 and 4%, respectively. HGF expression was significantly higher in tumors with acquired resistance than in sensitive tumors (p < 0.001, Student's t test). Fifty percent of tumors with acquired resistance showed simultaneous HGF expression with EGFR T790M secondary mutation and MET amplification. High-level HGF expression was detected more frequently than EGFR T790M secondary mutation or MET amplification in tumors with intrinsic and acquired EGFR-TKI resistance in EGFR mutant lung cancer in Japanese patients. These observations provide a rationale for targeting HGF in EGFR-TKI resistance in EGFR mutant lung cancer.

"Pulsatile" high-dose weekly erlotinib for CNS metastases from EGFR mutant non-small cell lung cancer

Erlotinib is effective for epidermal growth factor receptor (EGFR) mutant lung cancer, but CNS penetration at standard daily dosing is limited. We previously reported that intermittent "pulsatile" administration of high-dose (1500 mg) erlotinib once weekly was tolerable and achieved concentrations in cerebrospinal fluid exceeding the half maximal inhibitory concentration for EGFR mutant lung cancer cells in a patient with leptomeningeal metastases; we now expand this paradigm to a series of 9 patients. We retrospectively identified patients with EGFR mutant lung cancer treated with pulsatile erlotinib for CNS metastases (brain and/or leptomeningeal) that occurred despite conventional daily erlotinib or other EGFR tyrosine kinase inhibitors. Mutations in available lung and CNS tissue were correlated with efficacy. Erlotinib was administered as monotherapy at a median dose of 1500 mg weekly. Best CNS radiographic response was partial in 67% (6/9, including 2 with isolated leptomeningeal metastases), stable disease in 11% (1/9), and progressive disease in 22% (2/9). Median time to CNS progression was 2.7 months (range, 0.8-14.5 months) and median overall survival was 12 months (range, 2.5 months-not reached). Treatment was well tolerated. No acquired resistance mutations in EGFR were identified in the CNS metastases of 4 patients, including 1 harboring T790M outside the CNS. Pulsatile erlotinib can control CNS metastases from EGFR mutant lung cancer after failure of standard daily dosing. CNS disease may not harbor acquired resistance mutations that develop systemically. A prospective trial is planned.

Modified Restriction Enzyme-based Detection of EGFR Mutations in Lung Cancer

背景与目的表皮生长因子受体(epidermal growth factor receptor, EGFR)基因突变是肺癌靶向药物疗效的可靠预测指标，因此基因突变的检测具有非常重要的临床意义。本研究建立使用常规实验仪器、高灵敏度、简便的检测表皮生长因子受体突变的方法，以利于临床中快速的检测EGFR基因突变。方法采用改良的内切酶法富集法检测251例肺腺癌DNA标本中EGFR基因外显子19缺失突变和21(L858R)点突变，并与直接测序进行比较。利用混合突变/野生型EGFR基因的细胞系测定改良方法的灵敏度。结果在251例腺癌标本DNA中使用测序法检测出EGFR外显子19突变46例、外显子21突变26例。采用改良的突变体富集法检另外测出外显子19突变78例、外显子21突变57例，总突变率53.8%。灵敏度检测显示对于外显子19和21，新方法的检测灵敏度达0.5%。结论本方法具有简便、经济、灵敏度高等特点，便于临床快速筛查非小细胞肺癌病理组织中的EGFR基因突变。

E7080 Suppresses Hematogenous Multiple Organ Metastases of Lung Cancer Cells with Nonmutated Epidermal Growth Factor Receptor

While epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors improve the prognosis of patients with EGFR mutant lung cancer, the prognosis of patients with nonmutant EGFR lung cancer, especially those with metastases, is still extremely poor. We have assessed the therapeutic efficacy of E7080, an orally available inhibitor of multiple tyrosine kinases including VEGF receptor 2 (VEGFR-2) and VEGFR-3, in experimental multiple organ metastasis of lung cancer cell lines without EGFR mutations. E7080 markedly inhibited the in vitro proliferation of VEGF-stimulated microvascular endothelial cells. Intravenous inoculation into natural killer cell-depleted severe combined immunodeficient mice of the small cell lung cancer cell lines H1048 (producing low amounts of VEGF) and SBC-5 (producing intermediate amounts of VEGF) resulted in hematogenous metastases into multiple organs, including the liver, lungs, kidneys, and bones, whereas intravenous inoculation of PC14PE6, a non-small cell lung cancer cell line producing high amounts of VEGF, resulted in lung metastases followed by massive pleural effusion. Daily treatment with E7080 started after the establishment of micrometastases significantly reduced the number of large (>2 mm) metastatic nodules and the amount of pleural effusion, and prolonged mouse survival. Histologically, E7080 treatment reduced the numbers of endothelial and lymph endothelial cells and proliferating tumor cells and increased the number of apoptotic cells in metastatic nodules. These results suggest that E7080 has antiangiogenic and antilymphangiogenic activity and may be of potential therapeutic value in patients with nonmutant EGFR lung cancer and multiple organ metastases.

Correlation of 18F-FDG uptake and EGFR/KRAS mutations in surgically resected lung cancer.

10547 Background: The mutation status of both the epidermal growth factor receptor (EGFR) and KRAS genes have been reported to be important predictors of the efficacy of EGFR tyrosine kinase inhibitors such as gefitinib or erlotinib. 18F-fluoro-2-deoxy-glucose (18F-FDG) positron emission tomography (PET) is a functional imaging modality based on glucose metabolism, which has recently been used as a common staging tool for non-small cell lung cancer. We investigated the correlations between the standardized uptake value (SUV) of 18F-FDG and the mutation status of the EGFR and KRAS genes in lung cancer. Methods: We conducted a retrospective review of 189 patients with surgically resected lung cancers (167 adenocarcinomas and 22 non-adenocarcinomas) who underwent 18F-FDG PET/computed tomography scans preoperatively and who were examined for EGFR/KRAS mutations using surgically resected specimens. Correlations between the EGFR/KRAS mutation status and the clinicopathological factors including SUVmax were examined by the chi-square test and a logistic regression analysis. Results: EGFR and K-ras mutations were detected in 82 (44%) and 21 (11%) of the 189 lung cancers, respectively. The univariate analyses showed that EGFR mutations were more frequently observed in females (61%), non-smokers (56%), adenocarcinomas (49%), tumors ≤3 cm (51%), tumors without lymph node involvement (49%), lymphatic permeation (53%) and blood vessel invasion (53%), and with SUVmax <10 (51%). In the multivariate analyses, gender and SUVmax were the significant predictors of EGFR mutation. In the 75 female patients with a tumor with SUVmax <10, 49 (65%) patients had EGFR mutations. The univariate analyses showed that KRAS mutations were more frequently observed in males (17%), smokers (16%), and tumors with SUVmax ≥10 (21%). In the multivariate analyses, only gender was a significant predictor of KRAS mutations. Conclusions: The probability of EGFR mutation was inversely correlated with the SUVmax. In contrast, the probability of KRAS mutation was directly correlated with the SUVmax. EGFR mutated lung cancer may therefore be biologically less aggressive than KRAS mutated lung cancer.

Abstract 1730: Transient PI3K inhibition induces apoptosis and overcomes HGF-mediated resistance to EGFR-TKIs in EGFR mutant lung cancer

Abstract Purpose: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, show favorable response to EGFR mutant lung cancer. However, the responders acquire resistance almost without exception. We recently reported that hepatocyte growth factor (HGF) induces EGFR-TKI resistance by activating MET which restores downstream MAPK-ERK1/2 and PI3K-Akt signaling. The purpose of this study was to determine whether inhibition of PI3K, a downstream molecule of both EGFR and MET, could overcome HGF-mediated EGFR-TKI resistance in EGFR mutant lung cancer cells, PC-9 and HCC827. Experimental Design: We explored therapeutic effect of a class I PI3K inhibitor PI-103 on HGF-induced EGFR-TKI resistance in vitro and in vivo. Results: Unlike gefitinib or erlotinib, continuous exposure with PI-103 inhibited proliferation of PC-9 and HCC827 cells, even in the presence of HGF. On the other hand, in gefitinib-resistant xenograft model using PC-9 cells mixed with HGF high producing fibroblasts, PI-103 monotherapy did not inhibit tumor growth. However, PI-103 combined with gefitinib successfully regressed gefitinib-resistant tumor. In vitro experiments considering short half life of PI-103 reveal that transient exposure of PI-103 combined with gefitinib caused sustained inhibition of Akt phosphorylation, but not ERK1/2 phosphorylation, resulting in induction of tumor cell apoptosis even in the presence of HGF. Conclusions: These results indicate that transient blockade of PI3K-Akt pathway by PI-103 and gefitinib could overcome HGF-mediated resistance to EGFR-TKIs by inducing apoptosis, further suggesting usefulness of double blockade of PI3K-Akt signaling at upstream (EGFR) and downstream (PI3K) for more successful treatment of EGFR mutant lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1730. doi:10.1158/1538-7445.AM2011-1730

Transient PI3K Inhibition Induces Apoptosis and Overcomes HGF-Mediated Resistance to EGFR-TKIs inEGFRMutant Lung Cancer

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), such as gefitinib and erlotinib, show favorable response to EGFR mutant lung cancer. However, the responders acquire resistance almost without exception. We recently reported that hepatocyte growth factor (HGF) induces EGFR-TKI resistance by activating MET that restores downstream mitogen activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK)1/2 and phosphoinositide 3-kinase (PI3K)/Akt signaling. The purpose of this study was to determine whether inhibition of PI3K, a downstream molecule of both EGFR and MET, could overcome HGF-mediated EGFR-TKI resistance in EGFR mutant lung cancer cells PC-9 and HCC827. We explored therapeutic effect of a class I PI3K inhibitor PI-103 on HGF-induced EGFR-TKI resistance in vitro and in vivo. Unlike gefitinib or erlotinib, continuous exposure with PI-103 inhibited proliferation of PC-9 and HCC827 cells, even in the presence of HGF. On the other hand, in gefitinib-resistant xenograft model by using PC-9 cells mixed with HGF high producing fibroblasts, PI-103 monotherapy did not inhibit tumor growth. However, PI-103 combined with gefitinib successfully regressed gefitinib-resistant tumor. In vitro experiments by considering short half-life of PI-103 reveal that transient exposure of PI-103 combined with gefitinib caused sustained inhibition of Akt phosphorylation, but not ERK1/2 phosphorylation, resulting in induction of tumor cell apoptosis even in the presence of HGF. These results indicate that transient blockade of PI3K/Akt pathway by PI-103 and gefitinib could overcome HGF-mediated resistance to EGFR-TKIs by inducing apoptosis in EGFR mutant lung cancer.

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.

Screening for EGFR mutations in lung cancer, a report from India

Epidermal growth factor receptor (EGFR) is one of the targeted molecular markers in many cancers including the lung malignancy. Genetic modifications such as deletions, insertions and Single Nucleotide Polymorphisms in the tyrosine kinase (TK) domain of EGFR is a common feature observed in most lung cancers. Gefitinib and erlotinib are commonly available therapeutic drugs which act as specific inhibitors for the tyrosine kinase domain of EGFR and associated with EGFR mutations in exons 18–21. However the prevalence of mutation varies among ethnicity, grade, age and gender. This is the first report on the prevalence of EGFR mutation in non-small cell lung cancer patients using DNA obtained from samples such as biopsy/cytology/pleural fluid and Fine Needle Aspiration (FNA), across India. We have screened for 29 somatic mutations which span exons 18, 19, 20 and 21 of EGFR gene using Scorpion probe based ARMS-PCR technique. DNA from 220 NSCLC tissue samples were analyzed for EGFR mutations and mutations were detected in 51.8% of the study population. Among the mutant positive cases, the deletions in exon 19 (52%) and a missense mutation L858R in exon 21 (26%) were most predominant. There was a significant increase in overall mutations (p=0.01) as a function of age, mutation in exons 19 and 21 together (p=0.003), mutations in exons 18, 19 and 21 (p=0.04) and mutations in exons 18 and 19 (p=0.03) in females. Mutations did not seem to significantly correlate metastases or disease progression. Mutations in exons [19] and 21 together were significant in non-smokers compared to smokers (p=0.01) using Mann–Whitney tests. The study suggests high prevalence of EGFR positivity in NSCLC in Indian sub-population and provides opportunities for targeted therapies for this group.

Predictive biomarkers in the management of EGFR mutant lung cancer

Activating mutations in the form of deletions in exon 19 (del 19) or the missense mutation L858R in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) predict outcome to use of EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. Pooled data from several phase II studies show that gefitinib and erlotinib induce responses in over 70% of NSCLC patients harboring EGFR mutations, with progression-free survival (PFS) ranging from 9 to 13 months. Two studies in Caucasian and Asian patients have confirmed that these subgroups of patients attain PFS up to 14 months. These landmark outcomes have been accompanied by new challenges, primarily the additional role of chemotherapy and the management of tumors with the secondary T790M mutation that confers resistance to EGFR TKIs. Mechanisms of resistance to reversible EGFR TKIs should be further clarified and could be related to modifications in DNA repair.

Abstract 4688: A relationship between environmental tobacco smoke (ETS) and epidermal growth factor receptor (EGFR) mutations in never smoker patients with non-small cell lung cancer

Abstract Purpose The etiology of EGFR-mutant lung cancer remains unknown. The high incidence of EGFR mutations are observed in life-time never smokers. We investigated whether the risk factors of lung cancer in never smoker may be associated with a mutant-EGFR dependent carcinogenesis. Especially, we focused on the effect of ETS on EGFR-mutant lung cancer. Patients and Methods We enrolled 179 consecutive never smokers who were newly diagnosed with NSCLC. The history of ETS exposure was obtained with a standardized questionnaire that included exposure period, place, and duration. The nucleotide sequences of exons 18-21 on EGFR gene were determined using nested PCR amplification. Those patients with an exon 19 deletion and exon 21 point mutations were selected as a subgroup with activating mutations for further analysis. Results The median total smoker-year of patients with ETS exposure was higher than that of those without (25 years vs. 15 years; P = 0.002). The incidence of EGFR mutations was significantly lower in patients with ETS exposure than in those without (38.5% vs. 61.4%; P = 0.008). The proportion of exon 19 mutation was lower in patients with ETS exposure (59.6% vs. 81.5%; P = 0.050). In a logistic regression model that adjusted for gender and histology, an adjusted odds ratio (AOR) for the risk of EGFR mutations with exposure to ETS was 0.40 (95% confidential interval [CI], 0.20-0.81; P = 0.011). In quartile groups based on total smoker-year, the AORs for the lowest- to highest-quartile groups were 0.59 (95% CI, 0.23-1.49), 0.50 (0.17-1.50), 0.48 (0.20-1.18), and 0.22 (0.08-0.62) (Ptrend = 0.028). The period and place of ETS exposure had no different effect on the incidence of EGFR mutations. Patients with ETS exposure showed a lower response rate to EGFR tyrosine kinase inhibitors than did patients without ETS exposure (24.6% vs. 44.8%; P = 0.053). Conclusions Exposure to ETS is negatively associated with the incidence of EGFR mutations in lung cancer in never smokers. There was an inverse trend between the cumulative dose of ETS exposure and the frequency of EGFR mutations. A mutant-EGFR-dependent carcinogenesis may be different from a tobacco-smoke-associated carcinogenesis. 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 4688.

EGFR mutations and the terminal respiratory unit

Considerable knowledge has accumulated about mutations of the epidermal growth factor receptor (EGFR)-tyrosine kinase domain since these were first identified in 2004. Patients with nonsmall cell lung cancer with this mutation show dramatic clinical responses to treatment with EGFR-tyrosine kinase inhibitors, whose effectiveness has been established recently in large clinical trials. Most of the mechanisms responsible for resistance to treatment, which most responders experience eventually, have been elucidated, and methods to overcome resistance have been developed. In addition to the clinical benefit, understanding EGFR mutations sheds new light on the molecular and pathological aspects of this adenocarcinoma subset, which include frequent development in nonsmokers or females, and particular clusters within the molecular classification in lung cancer. In contrast to the involvement of EGFR mutations in the early stage of lung adenocarcinoma development, EGFR amplification is superimposed on the progression to invasive cancer. In this review, I summarize the clinicopathological characteristics of EGFR mutations in lung cancer. I also provide an overview of the current understanding of the lung adenocarcinoma subset harboring EGFR mutations with special reference to the molecular classification of lung cancer and the novel concept of the "terminal respiratory unit."

Lung Cancer in Never Smokers: Molecular Profiles and Therapeutic Implications

The majority of lung cancers are caused by long term exposure to the several classes of carcinogens present in tobacco smoke. Although a significant fraction of lung cancers in never smokers may also be attributable to tobacco, many such cancers arise in the absence of detectable tobacco exposure, and may follow a very different cellular and molecular pathway of malignant transformation. Recent studies summarized here suggest that lung cancers arising in never smokers have a distinct natural history, profile of oncogenic mutations, and response to targeted therapy. The majority of molecular analyses of lung cancer have focused on genetic profiling of pathways responsible for metabolism of primary tobacco carcinogens. Limited research has been conducted evaluating familial aggregation and genetic linkage of lung cancer, particularly among never smokers in whom such associations might be expected to be strongest. Data emerging over the past several years show that lung cancers in never smokers are much more likely to carry activating mutations of the epidermal growth factor receptor (EGFR), a key oncogenic factor and direct therapeutic target of several newer anticancer drugs. EGFR mutant lung cancers may represent a distinct class of lung cancers, enriched in the never-smoking population, and less clearly linked to direct tobacco carcinogenesis. These insights followed initial testing and demonstration of efficacy of EGFR-targeted drugs. Focused analysis of molecular carcinogenesis in lung cancers in never smokers is needed, and may provide additional biologic insight with therapeutic implications for lung cancers in both ever smokers and never smokers.

Induction of BIM Is Essential for Apoptosis Triggered by EGFR Kinase Inhibitors in Mutant EGFR-Dependent Lung Adenocarcinomas

BackgroundMutations in the epidermal growth factor receptor (EGFR) gene are associated with increased sensitivity of lung cancers to kinase inhibitors like erlotinib. Mechanisms of cell death that occur after kinase inhibition in these oncogene-dependent tumors have not been well delineated. We sought to improve understanding of this process in order to provide insight into mechanisms of sensitivity and/or resistance to tyrosine kinase inhibitors and to uncover new targets for therapy.Methods and FindingsUsing a panel of human lung cancer cell lines that harbor EGFR mutations and a variety of biochemical, molecular, and cellular techniques, we show that EGFR kinase inhibition in drug-sensitive cells provokes apoptosis via the intrinsic pathway of caspase activation. The process requires induction of the proapoptotic BH3-only BCL2 family member BIM (i.e., BCL2-like 11, or BCL2L11); erlotinib dramatically induces BIM levels in sensitive but not in resistant cell lines, and knockdown of BIM expression by RNA interference virtually eliminates drug-induced cell killing in vitro. BIM status is regulated at both transcriptional and posttranscriptional levels and is influenced by the extracellular signal-regulated kinase (ERK) signaling cascade downstream of EGFR. Consistent with these findings, lung tumors and xenografts from mice bearing mutant EGFR-dependent lung adenocarcinomas display increased concentrations of Bim after erlotinib treatment. Moreover, an inhibitor of antiapoptotic proteins, ABT-737, enhances erlotinib-induced cell death in vitro.ConclusionsIn drug-sensitive EGFR mutant lung cancer cells, induction of BIM is essential for apoptosis triggered by EGFR kinase inhibitors. This finding implies that the intrinsic pathway of caspase activation may influence sensitivity and/or resistance of EGFR mutant lung tumor cells to EGFR kinase inhibition. Manipulation of the intrinsic pathway could be a therapeutic strategy to enhance further the clinical outcomes of patients with EGFR mutant lung tumors.

Possible correlation between SOCS1 methylation and EGFR exon 19 mutation but not exon 21 mutation in Chinese NSCLC patients

18113 Background: Suppressor of cytokine signaling-1 (SOCS1) and -3 (SOCS3) interact with the epidermal growth factor receptor (EGFR) to bring about the ubiquination and degradation of the receptor. Silencing of SOCS1 and SOCS3 by methylation was postulated to regulate the IL-6R/JAK-mediated STAT3 activation. Its relationship to EGFR and EGFR mutations in lung cancer remains unclear. Here, we examined the relationship between EGFR mutations and methylation of SOCS1 and SOCS3 in Chinese NSCLC patients. Methods: EGFR mutations were assessed in DNAs from microdissected tumor cells. PCR products were purified and sequenced using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystem) and the ABI 3100 Genetic Analyzer. For methylation analyses of SOCS1 and SOCS3, DNAs were extracted from paraffin sections of tumor tissues, bisulfite treated and analyzed by methylation- specific PCR (MSP). Results: Data is available from 9 lung cancer cell lines and 20 tumor samples. Five EGFR-wild type cell lines had methylation for SOCS1. We found EGFR exon 19 mutation in 5 (25%) tumors, exon 21 mutation (L858R) in 2 (10%) tumors, and 1 (5%) tumor had double mutations. Four of 5 (80%) tumors with exon 19 mutation and none (0%) of the tumors with exon 21 mutation had SOCS1 methylation, comparing to only 2 of 12 (17%) EGFR-wild type tumors had SOCS1 methylation. SOCS3 methylation was not detected in any of the primary tumors or cell lines. Conclusions: Our preliminary findings suggest that SOCS1 methylation might be positively correlated with EGFR exon 19 mutation but exclusive from exon 21 mutation. A following study with larger sample size is currently under way. SOCS1 methylation may explain the better tumor response associated with EGFR exon 19 mutation. No significant financial relationships to disclose.