Secondary EGFR T790M Mutation Research Articles

Repeat biopsy versus initial biopsy in terms of complication risk factors and clinical outcomes for patients with non-small cell lung cancer: a comparative study of 113 CT-guided needle biopsy of lung lesions.

The safety and feasibility of repeat biopsy after systemic treatment for non-small cell lung cancer have received extensive attention in recent years. The purpose of this research was to compare complication rates between initial biopsy and rebiopsy in non-small cell lung cancer patients with progressive disease and to assess complication risk factors and clinical results after rebiopsy. The study included 113 patients initially diagnosed with non-small cell lung cancer who underwent lung biopsy at initial biopsy and rebiopsy after progression while on epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) and/or chemotherapy from January 2018 to December 2021. We compared the incidence of complications between the initial biopsy and rebiopsy and analyzed the predictors factors that influenced complications in patients who underwent rebiopsy. The successful rate of rebiopsy was 88.5% (100/113). With the exception of two cases where lung adenocarcinoma changed into small cell lung cancer with gefitinib treatment, 98 individuals retained their initial pathological type. The secondary EGFR T790M mutation accounts for 55.6% of acquired resistance. The total number of patients with complications in initial biopsy was 25 (22.1%) and 37 (32.7%) in the rebiopsy. The incidence of pulmonary hemorrhage increased from 7.1% at the initial biopsy to 10.6% at rebiopsy, while the incidence of pneumothorax increased from 14.2% to 20.4%. Compared with the initial biopsy, the incidence of overall complications, parenchymal hemorrhage, and pneumothorax increased by 10.6%, 3.5%, and 6.2%, respectively. In all four evaluations (pneumorrhagia, pneumothorax, pleural reaction, and overall complication), there were no significant differences between the rebiopsy and initial biopsy (all p > 0.05). The multivariate logistic regression analysis suggested that male sex (odds ratio [OR] = 5.064, p = 0.001), tumor size ≤ 2cm (OR = 3.367, p = 0.013), EGFR-TKIs with chemotherapy (OR = 3.633, p =0.023), and transfissural approach (OR = 7.583, p = 0.026) were independent risk factors for overall complication after rebiopsy. Compared with the initial biopsy, the complication rates displayed a slight, but not significant, elevation in rebiopsy. Male sex, tumor size ≤ 2cm, transfissural approach, and EGFR-TKIs combined with chemotherapy were independent risk factors for rebiopsy complications.

Increased expression of SSEA-4 on TKI-resistant non–small cell lung cancer with EGFR-T790M mutation

Non-small cell lung cancer (NSCLC), a major life-threatening disease accounting for 85% of all lung cancer cases, has been treated with tyrosine kinase inhibitors (TKIs), but often resulted in drug resistance, and approximately 60% of TKI-resistant cases are due to acquired secondary (epithelial growth factor receptor) EGFR-T790M mutation. To identify alternative targets for TKI-resistant NSCLC with EGFR-T790M mutation, we found that the three globo-series glycosphingolipids are increasingly expressed on this type of NSCLC cell lines, and among them, the increase of stage-specific embryonic antigen-4 (SSEA-4) expression is the most significant. Compared to TKI-sensitive cell lines, SSEA-4 and the key enzyme β3GalT5 responsible for the synthesis of SSEA3 are more expressed in TKI-resistant NSCLC cell lines with EGFR-T790M mutation, and the expression levels strongly correlate with poor survival in patients with EGFR mutation. In addition, we demonstrated that a SSEA-4 targeted monoclonal antibody, especially the homogeneous glycoform with well-defined Fc glycan designed to improve effective functions, is highly effective against this subpopulation of NSCLC in cell-based and animal studies. These findings provide a direction for the prediction of tumor recurrence and treatment of TKI-resistant NSCLC with EGFR-T790M mutation.

Comparison of afatinib and erlotinib combined with bevacizumab in untreated stage IIIB/IV epidermal growth factor receptor-mutated lung adenocarcinoma patients: a multicenter clinical analysis study.

Background:Although bevacizumab in combination with afatinib or erlotinib is an effective and safe first-line therapy for advanced epidermal growth factor receptor (EGFR)-mutated non-small-cell lung cancer (NSCLC), there are very few clinical data comparing afatinib and erlotinib combined with bevacizumab. We performed a retrospective multicenter analysis for the comparison of two combination therapies.Methods:Between May 2015 and October 2020, data of 135 stage IIIB/IV EGFR-mutated NSCLC patients receiving first-line afatinib or erlotinib combined with bevacizumab combination therapy in Linkou, Keelung, Chiayi, and Kaohsiung Chang Gung Memorial Hospitals were retrieved and retrospectively analyzed.Results:In all, 67 patients received afatinib plus bevacizumab, and 68 patients received erlotinib plus bevacizumab. Afatinib combined with bevacizumab had an objective response rate (ORR) of 82.1% and a disease control rate (DCR) of 97.0%, and the ORR and DCR were 83.8 and 95.6%, respectively, in the erlotinib combined with bevacizumab group (p = 0.798 and p = 1.000). The median progression-free survival was 20.7 and 20.3 months for the afatinib plus bevacizumab group and the erlotinib plus bevacizumab group, respectively [hazard ratio (HR) = 1.02; 95% confidence interval (CI), 0.891–1.953; p = 0.167). The overall survival was 41.9 and 51.0 months for the afatinib plus bevacizumab group and erlotinib plus bevacizumab group, respectively (HR = 1.42; 95% CI, 0.829–2.436; p = 0.201). The secondary EGFR-T790M mutation rates after disease progression were 44% in the afatinib plus bevacizumab group and 58.8% in the erlotinib plus bevacizumab group (p = 0.165). Skin toxicity was the most frequent treatment-related adverse event (AE) in both treatment groups. Diarrhea, an AE, occurred significantly more frequently in the afatinib plus bevacizumab group than in the erlotinib plus bevacizumab group (p < 0.05).Conclusion:Afatinib combined with bevacizumab was equally as effective as erlotinib combined with bevacizumab for untreated advanced EGFR-mutated NSCLC. Prospective clinical studies that explore bevacizumab combined with afatinib or erlotinib for advanced EGFR-mutated NSCLC are warranted.

A Real-World Analysis of Patients with Untreated Metastatic Epidermal Growth Factor Receptor (EGFR)-Mutated Lung Adenocarcinoma Receiving First-Line Erlotinib and Bevacizumab Combination Therapy.

IntroductionThe clinical features of patients with metastatic epidermal growth factor receptor (EGFR)-mutated lung adenocarcinoma receiving first-line therapy based on erlotinib combined with bevacizumab are unclear. Here, we sought to analyze the clinical features of this patient group.MethodsData were analyzed for the period from January 2015 to August 2019 for 49 patients with metastatic EGFR-mutated lung adenocarcinoma receiving first-line erlotinib-and-bevacizumab combination therapy from the Linkou and Kaohsiung Chang Gung Memorial Hospitals.ResultsThe combination of erlotinib and bevacizumab showed an 83.7% objective response rate and a 97.9% disease control rate. The median progression-free survival (PFS) and overall survival (OS) were 22.0 [95% CI (19.7–22.33)] and 47.6 [95% CI (38.87–56.37)] months, respectively, for all patients. The secondary EGFR-T790M mutation rate in the patients with acquired resistance to the combination was 72.4%. No predictive factor associated with the appearance of secondary EGFR-T790M mutations was found. The most frequent adverse event (AE) caused by the combination therapy was dermatitis (100%), and most of the AEs were manageable and grades 1 and 2.ConclusionErlotinib combined with bevacizumab is an effective and safe therapy for untreated metastatic EGFR-mutated lung adenocarcinoma. The combination does not alter secondary EGFR-T790M mutations in patients with acquired resistance and is feasible in real-world clinical practice.Graphic Supplementary InformationThe online version contains supplementary material available at 10.1007/s40487-021-00152-6.

Remodeling Tumor-Associated Macrophages and Neovascularization Overcomes EGFRT790M -Associated Drug Resistance by PD-L1 Nanobody-Mediated Codelivery.

Precision medicine has made a significant breakthrough in the past decade. The most representative success is the molecular targeting therapy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) in non-small-cell lung cancer (NSCLC) with oncogenic drivers, approved by the US Food and Drug Administration (FDA) as first-line therapeutics for substituting chemotherapy. However, the rapidly developed TKI resistance invariably leads to unsustainable treatment. For example, gefitinib is the first choice for advanced NSCLC with EGFR mutation, but most patients would soon develop secondary EGFRT790M mutation and acquire gefitinib resistance. TKI resistance is a severe emergency issue to be solved in NSCLC, but there are a few investigations of nanomedicine reported to address this pressing problem. To overcome EGFRT790M -associated drug resistance, a novel delivery and therapeutic strategy is developed. A PD-L1 nanobody is identified, and first used as a targeting ligand for liposomal codelivery. It is found that simvastatin/gefitinib combination nanomedicine can remodel the tumor microenvironment (e.g., neovascularization regulation, M2-macrophage repolarization, and innate immunity), and display the effectiveness of reversing the gefitinib resistance and enhancing the EGFRT790M -mutated NSCLC treatment outcomes. The novel simvastatin-based nanomedicine provides a clinically translatable strategy for tackling the major problem in NSCLC treatment and demonstrates the promise of an old drug for new application.

P49 Lung Adenocarcinoma with Double Heterozygote EGFR mutation and Combined Resistance: ALK Translocation and EGFR T790M

About 14% of patients with metastatic lung adenocarcinomas in our population have tumors harboring EGFR mutations or deletions. Herein we report the case of a patient with lung adenocarcinoma with a double deletion of EGFR who had a partial response for 6 months treated with afatinib and, upon progression, an EGFR T790M mutation and an ALK translocation were detected. Not Applicable Case Report: A 32-year-old male, non-smoker, presents with abdominal pain, cough and fatigue. Abdominal ultrasound showed a large left adrenal lesion and the chest X-Rays, a consolidation in the right inferior lobe. PET-CT with 18FDG scan was performed evidencing a 7.2 cm tumor in the right lower lobe with high affinity for FG, as well as mediastinal and retroperitoneal enlarged lymph nodes, right adrenal mass, left acetabular and posterior costal lesion. A diagnostic transbronchial tumor-biopsy showed an undifferentiated carcinoma TTF1 + (T3N3M1c). EGFR status, assessed by PCR and sequencing, showed an exon 19 deletion (delE746_A750) with no ALK (clon D5F3) nor ROS1 (clon D4D6) translocations by immunohistochemistry (IHC). Next generation sequencing (NGS) with the Oncomine TM Focus Assay panel (Ion 520 Chip) revealed a double deletion in EGFR (p.delE746_A750 and p.delL747_T751insQ) in separate reads. The patient started treatment with afatinib and achieved a partial response at 3 months. After 6 months he experienced progressive lung abnormalities (metastasis???) and brain and cerebellar metastasis consistent with disease progression. Afatinib was stopped and both CNS lesions were resected. Standard ALK, ROS1 and EGFR determinations were repeated on brain tissue. An ALK translocation was identified by IHC and confirmed by FISH as well as the EGFR exon 19 deletion p.delE746_A750, without T790M mutation. The pre-treatment lung biopsy was reviewed and was negative for ALK translocation. EGFR testing in circulating tumor DNA was positive for the T790M secondary resistance mutation. Patient had rapid retroperitoneal progression with ascites, bleeding and abdominal pain requiring surgical intervention. Crizotinib was started without response and the patient died. Here we report a case of a patient achieving a partial response with afatinib in a tumor with two EGFR deletions and the co-occurrence of secondary EGFR T790M mutation and ALK translocation as mechanism of resistance. In addition, the lack of detection of the EGFR deletion p.delL747_T751insQ, suggests a complex subclonal evolution secondary to tumor heterogeneity.

Targeting EGFRL858R/T790M and EGFRL858R/T790M/C797S resistance mutations in NSCLC: Current developments in medicinal chemistry.

Both the first-generation reversible epidermal growth factor receptor (EGFR) inhibitors gefitinib and erlotinib and the second-generation covalent epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) afatinib have significantly improved the survival of non-small-cell lung cancer (NSCLC) patients with activating EGFR mutations. However, a secondary EGFRT790M mutation leads to the clinically acquired resistance to the first- and second-generation EGFR-TKIs drugs. A number of the third-generation wild-type sparing EGFR inhibitors, for example, WZ4002, CO1686, AZD9291, HM61713, EGF816, ASP8173, and PF0674775, have been developed, among which AZD9291 has been approved by US FDA for the treatment of NSCLC patients with EGFRT790M . More recently, a tertiary EGFRC797S mutation was reported as the dominant resistance mechanism to the third-generation irreversible inhibitors. It is highly desirable to develop the fourth-generation EGFR inhibitors. This review summarizes the mechanisms of acquired resistance and the latest medicinal chemistry advances on the third- and fourth-generation EGFR inhibitors, with special attention being paid to the allosteric and reversible inhibitors combating the tertiary EGFRC797S mutation.

Identification of Clinically Approved Drugs Indacaterol and Canagliflozin for Repurposing to Treat Epidermal Growth Factor Tyrosine Kinase Inhibitor-Resistant Lung Cancer.

In advanced lung cancer, epidermal growth factor tyrosine kinase inhibitors (EGFR TKIs) have extraordinary clinical efficacy. However, their usefulness is severely compromised by drug resistance mediated by various mechanisms, the most important of which is the secondary EGFR T790M mutation. The mutation blocks the binding of EGFR TKIs to the receptor kinase, thereby abolishing the therapeutic efficacy. In this study, we used our free and open-source protein-ligand docking software idock to screen worldwide approved small-molecule drugs against EGFR T790M. The computationally selected drug candidates were evaluated in vitro in resistant non-small cell lung cancer (NSCLC) cell lines. The specificity of the drugs toward the mutant EGFR was demonstrated by cell-free kinase inhibition assay. The inhibition of EGFR kinase activity and its downstream signaling pathways in NSCLC cells was shown by immunoblot analysis. The positive hints were revealed to be indacaterol, canagliflozin, and cis-flupenthixol, all of which were shown to induce apoptosis in NSCLC cells harboring the EGFR T790M mutation. Moreover, the combination of indacaterol with gefitinib was also found to produce synergistic anticancer effect in NSCLC cells bearing EGFR T790M. The observed synergistic effect was likely contributed by the enhanced inhibition of EGFR and its downstream signaling molecules.

Novel resistance mechanisms to first-generation EGFR tyrosine kinase inhibitors: A perspective study in NSCLC patients using targeted next generation sequencing.

e20576 Background:Patients with non-small-cell lung cancer (NSCLC) harboring sensitive epithelial growth factor receptor (EGFR) mutations invariably develop acquired resistance to EGFR tyrosine kinase inhibitors (TKIs). Identification of actionable mutations conferring drug-resistance can be helpful for guiding the subsequent treatment decision. Currently, the known mechanisms of acquired resistance includes: the secondary gatekeeper EGFR-T790M mutation, activation of members of downstream signaling pathways such as PI3K/AKT/mTOR pathway, activation of bypass signaling such as MET, and changes in tumor histology. However, the mechanisms in the remaining patients are still unknown. Methods:In this prospective study, thirty-one advanced NSCLC patients initially carrying sensitive EGFR mutations and subsequently developing acquired resistance to the first-generation EGFR-TKIs were enrolled. Pre-treatment tumor samples as well as re-biopsies of tumor and plasma when the patients were diagnosed with EGFR-TKI resistance were acquired, followed by mutation profiling using targeted next generation sequencing (NGS) on 416 cancer-related genes. Results: In total, 55% of patients were identified to carry acquired secondary EGFR-T790M mutation. Three patients (~10%) harbor EGFR-T854A mutation, which has been reported as another TKI resistant mutation. 26% and 19% of cases accumulated TP53 and RB1 mutations, respectively. In T790M/T854A-negative cases, 30% of patients acquired MET amplification. Other potential acquired resistance mechanisms includes single nucleotide variants (SNVs) in genes such as SMAD4, DNMT3A, GNAS, ATM, KRAS, PIK3CA and TET2, and copy number variations (CNVs) in genes such as CDK4, MDM2, MYC, RICTOR and ERBB2. Conclusions:The study depicted the genetic landscapes comprehensively in matched pre- and post-EGFR-TKIs samples of NSCLC population resistant to first generation TKI treatments. Our analysis demonstrates new perspectives for further study of resistance and putting forward corresponding relevant tactics against the challenge of disease progression. Clinical trial information: NCT02804217.

Mutational profiling of non-small-cell lung cancer patients resistant to first-generation EGFR tyrosine kinase inhibitors using next generation sequencing.

Patients with advanced non-small-cell lung cancer (NSCLC) harboring sensitive epithelial growth factor receptor (EGFR) mutations invariably develop acquired resistance to EGFR tyrosine kinase inhibitors (TKIs). Identification of actionable genetic alterations conferring drug-resistance can be helpful for guiding the subsequent treatment decision. One of the major resistant mechanisms is secondary EGFR-T790M mutation. Other mechanisms, such as HER2 and MET amplifications, and PIK3CA mutations, were also reported. However, the mechanisms in the remaining patients are still unknown. In this study, we performed mutational profiling in a cohort of 83 NSCLC patients with TKI-sensitizing EGFR mutations at diagnosis and acquired resistance to three different first-generation EGFR TKIs using targeted next generation sequencing (NGS) of 416 cancer-related genes. In total, we identified 322 genetic alterations with a median of 3 mutations per patient. 61% of patients still exhibit TKI-sensitizing EGFR mutations, and 36% of patients acquired EGFR-T790M. Besides other known resistance mechanisms, we identified TET2 mutations in 12% of patients. Interestingly, we also observed SOX2 amplification in EGFR-T790M negative patients, which are restricted to Icotinib treatment resistance, a drug widely used in Chinese NSCLC patients. Our study uncovered mutational profiles of NSCLC patients with first-generation EGFR TKIs resistance with potential therapeutic implications.

Abstract 1890: IKBKE is a substrate of EGFR and a therapeutic target in NSCLCs with activating mutations of EGFR

Abstract EGFR is one of key driver pathways of non-small cell lung cancer (NSCLC). EGFR inhibitors have shown significant response in patients with EGFR mutation. However, patients develop resistance in short period of time, which results from secondary EGFRT790M mutation and other mechanisms. Here, we demonstrated that activating mutations of EGFR significantly activated IKBKE, an IκB kinase family member previously shown to activate Akt and NF-κB pathways. Furthermore, we showed that EGFR directly interacts with and phosphorylates IKBKE on tyrosine 153 and tyrosine 179 residues. IKBKE-Y153F/179-F, a mutant that could not be phosphorylated by EGFR, largely reduced its kinase activity and failed to activate Akt and NF-κB as well as inhibited EGFR signaling. Furthermore, phospho-IKBKE-Y153 correlated with EGFR activation in NSCLC patients. Notably, depletion of IKBKE by either small molecule inhibitor amlexanox or shRNA selectively inhibited cell viability in NSCLC cells with EGFR mutations. Moreover, we observed that inhibition of IKBKE led to feedback activation of the MAPK pathway. Combination of amlexanox with MEK inhibitor AZD6244 significantly inhibits cell survival and tumor growth in NSCLC cells driven by activating EGFR mutations including EGFRT790M. Thus, our findings not only identify IKBKE as a direct downstream target of EGFR but also provide a potential therapeutic strategy for EGFR-TKI resistant NSCLC driven by secondary EGFR mutation. Citation Format: Sridevi Challa, Jian-Ping Guo, Cheng-xiong Xu, Yajuan Li, Donghwa Kim, Douglas Cress, Eric Haura, Domenico Coppola, Jin Cheng. IKBKE is a substrate of EGFR and a therapeutic target in NSCLCs with activating mutations of EGFR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1890.

Abstract IA31: Mechanisms of resistance to EGFR inhibitors in lung cancer

Abstract Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitors (TKIs), including geftinib, erlotinib and afatinib are the standard of care for patients with advanced EGFR mutant non-small cell lung cancer. Treatment with an EGFR TKI leads to a response rate of ~60% and a median progression free survival ranging from 9 to 11 months. Acquired drug resistance inevitably develops in most of not all EGFR mutant lung cancer patients treated with EGFR inhibitors. The most common mechanism of acquired resistance is the development of a secondary EGFR T790M mutation. Other mechanism of resistance include activation of bypass signaling pathways (such as MET or HER2) and histological transformation. Over the last few years a series of new structurally distinct EGFR inhibitors have been identified and entered the clinic that are capable of inhibiting EGFR T790M. This class of mutant selective EGFR TKIs are effective both against the activating EGFR mutation and the drug resistant T790M but spare inhibition of wild type (WT) EGFR. This has enormous potential therapeutic implications as the side effects of all approved EGFR inhibitors is derived from inhibition of WT EGFR. Several of these agents, including AZD9291 and rociletinib (CO-1686) are in advanced stage clinical development. Both agents have been granted FDA’s breakthrough designation. In EGFR inhibitor resistant patients who have developed resistance to current clinical EGFR inhibitors through T790M, the response rates to either AZD9291 or rociletinib is over 60% with median progression free survivals of ~ 10 months. Current research efforts and clinical studies are focusing on a.) understanding mechanisms of drug resistance to this new class of EGFR inhibitors, b.) the development of rationale combination therapies and c.) use of these agents as initial treatment (instead of currently approved EGFR inhibitors) for patients with advanced EGFR mutant NSCLC. Citation Format: Pasi Jänne. Mechanisms of resistance to EGFR inhibitors in lung cancer. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA31.

NF-κB drives acquired resistance to a novel mutant-selective EGFR inhibitor.

The clinical efficacy of EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) harbouring activating EGFR mutations is limited by the emergence of acquired resistance, mostly ascribed to the secondary EGFR-T790M mutation. Selective EGFR-T790M inhibitors have been proposed as a new, extremely relevant therapeutic approach. Here, we demonstrate that the novel irreversible EGFR-TKI CNX-2006, a structural analog of CO-1686, currently tested in a phase-1/2 trial, is active against in vitro and in vivo NSCLC models expressing mutant EGFR, with minimal effect on the wild-type receptor. By integration of genetic and functional analyses in isogenic cell pairs we provide evidence of the crucial role played by NF-κB1 in driving CNX-2006 acquired resistance and show that NF-κB activation may replace the oncogenic EGFR signaling in NSCLC when effective and persistent inhibition of the target is achieved in the presence of the T790M mutation. In this context, we demonstrate that the sole, either genetic or pharmacologic, inhibition of NF-κB is sufficient to reduce the viability of cells that adapted to EGFR-TKIs. Overall, our findings support the rational inhibition of members of the NF-κB pathway as a promising therapeutic option for patients who progress after treatment with novel mutant-selective EGFR-TKIs.

Non-invasive and quantitative analysis for EGFR T790M mutation in the patients with advanced non-small cell lung cancer received EGFR-TKI therapy.

e19101 Background: Approximately 50% of advanced non-small cell lung cancer (A-NSCLC) patients with EGFR sensitive mutation who develop acquired resistance to EGFR-TKIs reportedly have a secondary EGFR T790M mutation. Establishing a dynamical, quantitative and noninvasive detection system of EGFR T790M mutation in process of disease therapy for NSCLC is critical to personalized targeted therapy. Methods: 135 A-NSCLC patients with EGFR mutation who received EGFR-TKIs and presented acquired resistance (PFS≥6 months) were included into this study. All patients provided the plasma samples for molecular analysis when disease progressed. 109 patients of them had matched TKI-naive plasma. T790M mutation was measured qualitatively and quantitatively by ARMS and Digital PCR (DggPCR), respectively. Association of T790M mutation with clinical charateristics were evaluated. Results: DgPCR was more sensitive than ARMs to detect T790M mutation in plasma [pre-treatment 29.4% (32/109) VS 5.5% (6/109); post-treatment: 43.0% (58/135) VS 25.2% (34/135)]. 32 patients with pre-treatment T790M mutation predicted shorter PFS and OS compared with 77 T790 M negative patients (PFS, F 12.7 VS 9.2 months, P=0.004, GOS, F 27.0 VS 18.8 months, P=0.002). Patients with or without post-treatment T790M mutation have no significantly different PFS and OS. However, quantified the ratio of copy number of mutant T790M to wild-type by DgPCR, patients were divided into high-frequency groups (≥5%), low-frequency group (0%-5%) and wild-group (0%) according to the number of positive signals observed from DgPCR results. 12 patients in high-frequency group showed shorter PFS and OS compared with wild group and low-frequency group (PFS 9.5 VS 11.9 months, P=0.033, G9.5 VS 13.6 months, P=0.028, GOS, F 18.5 VS 21.2 months, P=0.044, 18.5 VS 28.8 months, P=0.001). Conclusions: Non-invisive and quantitative detection of T790m mutation by digital PCR is feasible in clinical practice. High contents of T790M when disease progression after EGFR-TKIs therapy predicted poor prognosis.

Abstract A149: Quantitative detection of the EGFR activating and resistance mutations in plasma DNA of lung cancer patients.

Abstract EGFR-TKIs are effective therapeutic agents for EGFR-mutation-positive lung cancer patients, but most patients develop resistance to EGFR-TKIs. A secondary EGFR T790M mutation is reported to correlate with acquired resistance and accounts for about half of the resistance cases. Circulating tumor DNA (ctDNA) is a promising target for monitoring T790M mutations in non-invasive cancer diagnostics and evaluations, although detection of rare mutation alleles is technically demanding. BEAMing (beads, emulsion, amplification, magnetics) is currently the most sensitive technique which can quantitate the mutated allele. With the technique, we can detect one mutated allele in 10,000 normal alleles. Activating mutations were identified from plasma DNA in 72% (31/43). The T790M mutation was identified in 43% (10/23) of the patients with progressive disease during EGFR-TKI treatment. The T790M mutation appeared in 13–94% of alleles with activating mutations. Judging from this incidence, BEAMing could detect T790M in the majority of eligible cases. The major advantage over other techniques is the ability to calculate the fraction of T790M-positive alleles in cancer cells, irrespective of normal cell DNA contamination. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A149.

Abstract 4738: Cellular resistance screening with a novel selective c-Met inhibitor reveals a spectrum of missense mutations that partially overlap with activating mutations found in cancer patients

Abstract Emergence of resistance is a major concern when treating cancer patients with targeted kinase inhibitors, as exemplified by the appearance of point mutations in Bcr-Abl in CML patients treated with imatinib or a secondary EGFR-T790M mutation in lung cancer patients treated with gefitinib or erlotinib. In vitro approaches to identify resistance mutations in Bcr-Abl have yielded mutation spectra that faithfully recapitulated clinical observations. In order to predict resistance mutations in the receptor tyrosine kinase c-Met that could emerge during inhibitor treatment in patients, we performed a resistance screen in BaF3 cells transformed with Tpr-Met using the novel selective c-Met inhibitor NVP-BVU972. The observed spectrum of mutations in resistant cells was dominated by substitutions of the residue Y1230 in the activation loop of the c-Met kinase domain, but also included other missense mutations. Intriguingly, some of the c-Met resistance mutations observed in this cellular screen were identical with previously described activating mutations in cancer patients that interfere with inhibitory interactions in the inactive conformation. Co-crystallization of the c-Met kinase domain in complex with NVP-BVU972 revealed a key role for Y1230 in binding of NVP-BVU972. This binding mode has also been reported for multiple other selective c-Met inhibitors, some of which have entered clinical trials, suggesting a broader relevance of the resistance profile obtained with NVP-BVU972. A second resistance screen in the same format with the c-Met inhibitor AMG 458 yielded a distinct spectrum of mutations that was rich in F1200 alterations. This is consistent with a different predicted binding mode. Again, the mutation profile observed with AMG 458 could be predictive for several other inhibitors that bind c-Met in a similar way. Collectively, our findings suggest that amino acid substitutions in the c-Met kinase domain of cancer patients need to be carefully monitored prior to and during treatment with c-Met inhibitors, as resistance may pre-exist or emerge. Compounds binding in the same manner as NVP-BVU972 might be particularly susceptible to the development of resistance through mutations in Y1230, a complication that could potentially be overcome by c-Met inhibitors with alternative binding modes. 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 4738. doi:10.1158/1538-7445.AM2011-4738

Abstract LB-399: Chronic inhibition of mutant EGFR in NSCLC leads to EGFR TKI resistance by TGF-β1 mediated epithelial to mesenchymal transition

Abstract In NSCLC, activating EGFR mutations underlie responsiveness of NSCLCs to reversible EGFR tyrosine kinase inhibitors (TKIs), including gefitinib and erlotinib. Despite initial responses, acquired resistance invariably develops, mediated by the emergence of the secondary T790M mutation and by focal amplification of MET, in approximately 50% and 30% of patients, respectively. The resistance mechanisms for the remaining 20% of cases remain elusive. EGFR TKI-sensitive HCC827 cells were exposed to graded concentrations of erlotinib for 6 months. Approximately 70% of the isolated clones were resistant to erlotinib and harbored MET amplification, and were sensitive to dual EGFR/MET inhibition. The remaining 30% of clones were resistant to EGFR/MET dual-inhibition and demonstrated less than 1.4-fold increase in MET copy number and no secondary T790M mutation. Interestingly, 38 plex Luminex growth factor assays revealed that the 30% of clones for which the mechanism of resistance to erlotinib is unknown secrete high levels of TGF-1. In NSCLC, TGF-β1 has been identified as a potent inducer of epithelial to mesenchymal transition (EMT). Subsequent gene expression profiles of HCC827 erlotinib-resistant clones without MET amplification and secondary EGFR T790M mutation exhibit significant enrichment of genes unique to EMT and clustered separately from gene expression profiles of clones with MET amplification. In agreement with genomic analyses, Western blots confirmed the EMT phenotype in the subset of clones. Additionally, phosphoprotein arrays of the clones demonstrated markedly decreased expression of EGFR, ERBB3, and MET receptors, while maintaining high level of PI3-K MAPK, and SRC activities. Importantly, clones from HCC827 cells with stable EGFR knockdown (HCC827shEGFR) was established and all clones displayed EMT markers, suggesting induction of the EMT phenotype is not due to off-target effects of erlotinib, but rather is primarily due to the sustained loss of EGFR signaling in HCC827 cells. Multiplex Luminex growth factor assays identified TGF-β1 as the most abundant growth factor secreted from HCC827shEGFR clones. Overexpression of mutant EGFR in HCC827shEGFR clones re-sensitized the clones to EGFR TKIs and partially reversed EMT phenotype. To test the roles of TGF-β1in the emergence of EGFR TKI resistance, HCC827 cells were cultured in the media supplemented with TGF-1 for 30 days. MTS assays revealed that HCC827 cells became insensitive to EGFR TKIs after chronic exposure to TGF-β1 for 30 days. However, subsequent removal of TGF-β1 for 30 days re-sensitizes the cells to EGFR TKIs. Consequently, it is hypothesized that sustained suppression of EGFR in HCC827 cells promotes TGF-β1 secretion and subsequent induction of EMT processes, which modulate signaling and apoptosis pathways contributing to the development of resistance to erlotinib. 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 LB-399. doi:10.1158/1538-7445.AM2011-LB-399

Abstract 1782: Detection of T790M gefitinib resistance mutation in EGFR using the BEAMing method

Abstract Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), such as gefitinib or erlotinib, are effective therapies for non-small cell lung cancer (NSCLC) patients with primary EGFR mutations. However, most patients who initially respond to treatment with these drugs will develop resistance to EGFR TKIs. The secondary EGFR T790M mutation is reported to correlate with acquired resistance. We examined the T790M mutation in primary tumors with BEAMing (beads, emulsion, amplification, magnetic), which is the most sensitive technique to detect minor mutated alleles. Currently, we can detect point mutation at the level of 1/10,000 using an improved version of the BEAMing. We detected T790M mutant alleles in NSCLCs in 19 out of 270 NSCLCs with primary EGFR mutations. The detection of small fraction of T790M mutant alleles may be useful for predicting resistance of NSCLCs to TKIs. 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 1782.

Non-invasive detection of EGFR T790M in gefitinib/erlotinib resistant NSCLC

8075 Background: Fifty percent of NSCLC patients (pts) that develop a partial (PR) or complete (CR) response to gefitinib (G) or erlotinib (E) will develop a secondary EGFR T790M mutation as a mechanism of drug resistance. As most pts do not undergo repeated tumor biopsies, we evaluated the ability to detect EGFR T790M non-invasively from plasma DNA. Methods: NSCLC pts (n= 55) previously treated with G (n= 15) or E (n=36) or untreated (n=4) were included. Best response to prior treatment: CR/PR (n=28); SD (n=13); PD (n=10). EGFR mutant/wild type/unknown: 27/16/12. DxS real time PCR and WAVE technologies were used to screen for EGFR mutations in 72 plasma samples (median no. of samples per pt = 1; range = 1–5). Two methods of whole genome amplification (WGA) were explored to increase the yield of plasma DNA. Results: EGFR activating mutations were identified from plasma DNA in 32 (58%) pts. Concordance with primary EGFR tumor mutation was 70%. In 9 pts with a tumor EGFR mutation, we were unable to detect the mutation in plasma DNA. We detected EGFR T790M in 15/28 (53%) clinically resistant pts with prior CR/PR; 3/13 (23%) resistant pts with prior SD; and 0/10 (0%) resistant pts with prior PD. EGFR T790M was not detected in untreated pts. WGA was successful in 51/55 (93%) pts. There was no correlation between plasma DNA concentration and ability to detect EGFR activating or T790M mutation. Conclusions: We detected EGFR T790M from plasma DNA in 15 (53%) G or E resistant pts with prior CR/PR. This correlates closely with established estimates of EGFR T790M frequency (50%) from tumor biopsies in this pt population. Detection of EGFR T790M using non-invasive methods may aid in directing the course of subsequent therapy for G or E resistant pts. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration AstraZeneca, Boehringer Ingelheim, Roche Pfizer Genzyme