Epidermal Growth Factor Receptor Resistance Mutation Research Articles

Abstract 964: Updated results of a prospective observational multicenter study on resistance mechanisms to osimertinib using circulating tumor DNA analyses in EGFR-mutated non-small cell lung cancer patients (ELUCIDATOR study)

Abstract Background: Although osimertinib (Osi) is the key standard therapy for patients (pts) with advanced non-small cell lung cancer (NSCLC) harboring sensitizing epidermal growth factor receptor (EGFR) mutations, most of cases experience the progressive disease (PD) after initial response to the treatment. Therefore, understanding the Osi resistance mechanisms and identifying the poor responders are essential to develop better treatment strategies. Thus, we conducted a prospective observational multicenter study and analyzed serially collected circulating tumor DNA (ctDNA). Here we report the results from updated analyses focusing on the association between genetic information from ctDNA and clinical outcome. Methods: Patient eligibility criteria were as follows: a definitive diagnosis of non-squamous NSCLC confirmed through biopsy or cytology, EGFR exon 19 deletion (19del) or L858R mutation, Osi planned as first-line treatment, and pts who could provide blood specimens. Paired plasma DNA samples were collected before starting Osi as baseline (BL) and at PD and analyzed to investigate Osi resistance by next-generation sequencing (NGS). Correlations between genetic information obtained from ctDNA and clinical outcome such as progression free survival (PFS) and overall survival (OS) were evaluated. The median follow-up time was 33.1 months. Results: One hundred eighty-eight pts were enrolled between May 2019 and January 2021, and 125 (66%) were female, 96 (51%) were EGFR 19del mutation, and 109 (58%) were never smoker. Among those, 178 pts were included in the analyses, and 111 experienced PD. In consistent with previous reports, median PFS (mPFS) and median OS (mOS) were 19.1 months and 36.0 months, respectively. The efficacy of Osi differed between 19del and L858R in both PFS and OS, with 19del being longer outcome [mPFS, 23.3 months vs. 17.2 months, HR: 1.56 (95%CI: 1.08-2.26), mOS, 40.3 months vs. 30.4 months, HR: 1.54 (95%CI: 0.98-2.45)]. Univariate analysis revealed that the presence of TP53 mutations and MET amplification (amp) at BL was a predictive factor of shorter PFS and OS. Multivariate analysis also revealed that the TP53 mutations at BL was a predictive factor of shorter PFS and OS. Plasma samples from 85 pts were available for ctDNA analysis. New adaptive mutations or amp at PD were detected in genes such as PIK3CA in 3 cases, MET amp in 4, TP53 in 4, BRIMP3 in 2, APC in 1, and BRAF in 2. Secondary EGFR C797S resistance mutation was detected in 1 case. Conclusions: TP53 mutation at BL could be predictive of poor response to Osi. MET amp and PIK3CA mutations were detected as the most common resistance mechanisms, and secondary EGFR resistance mutations were observed less frequently than previous reports. Citation Format: Mitsuo Osuga, Akihiro Tamiya, Daijiro Harada, Yasuyuki Mizumori, Shun-ichi Isa, Yoshihiko Taniguchi, Keiichi Nakamura, Tsutomu Shinohara, Hidetoshi Yanai, Katsumi Nakatomi, Masahide Oki, Masahide Mori, Tomohito Kuwako, Koji Yamazaki, Atsuhisa Tamura, Masahiko Ando, Yasuhiro Koh. Updated results of a prospective observational multicenter study on resistance mechanisms to osimertinib using circulating tumor DNA analyses in EGFR-mutated non-small cell lung cancer patients (ELUCIDATOR study) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 964.

A phase 1 study to assess BDTX-1535, an oral EGFR inhibitor, in patients with glioblastoma or non–small-cell lung cancer.

TPS9156 Background: The epidermal growth factor receptor (EGFR) is a potent oncogene commonly altered in many cancers, including glioblastoma (GBM) and non-small cell lung cancer (NSCLC). EGFR tyrosine kinase activity driven by common EGFR mutations can be inhibited by small molecules, however, resistance to available agents may be driven by mutations in the EGFR active kinase site or other regions. BDTX-1535 is an orally available, highly potent, selective, irreversible inhibitor of EGFR mutations, including extracellular variants and amplifications commonly expressed in GBM and inhibits the common and uncommon EGFR mutations found in NSCLC, including the C797S mutation acquired following 3rd generation EGFR inhibitor therapy. Preclinical data demonstrated the ability of BDTX-1535 to cross the blood-brain barrier and produce sustained inhibition of EGFR signaling. Preclinical studies suggest that BDTX-1535 has the potential to be clinically active in suppressing tumor growth in patients with GBM and NSCLC with or without CNS metastases, including a potential survival benefit. Methods: BDTX-1535-101 (NCT05256290) is Phase 1, open-label, multicenter study to assess the safety, tolerability, PK, CNS activity, and preliminary antitumor activity of BDTX-1535 in recurrent GBM (rGBM) or locally advanced or metastatic NSCLC with or without CNS disease. The Monotherapy Dose Escalation portion will evaluate BDTX-1535 in patients with either rGBM expressing EGFR alterations or locally advanced/metastatic NSCLC harboring sensitizing EGFR mutations with or without CNS disease. Patients with rGBM must have previously received available standard therapy of surgical resection followed by chemoradiotherapy and/or temozolomide (TMZ). Eligible NSCLC patients must have EGFR mutated NSCLC that has progressed following standard of care EGFR inhibitor therapy. Once a provisional recommended Phase 2 dose (RP2D) has been established, BDTX-1535 monotherapy will be explored in the following Dose Expansion cohorts to further evaluate safety, PK, and preliminary assessment of efficacy: 1) rGBM with confirmed EGFR alterations, 2) NSCLC with uncommon EGFR mutations following EGFR inhibitor therapy; 3) NSCLC with acquired EGFR resistance mutation following a 3rd generation EGFR inhibitor in 1L setting. NSCLC patients may enroll with or without CNS metastases and must not be known to express excluded resistance mutations such as EGFR T790M or MET. BDTX-1535 will also be studied in combination with TMZ to assess safety, tolerability, and a recommended combination dose for the treatment of patients with rGBM harboring EGFR mutations or variants. Enrollment was initiated in 2022 and dose escalation is ongoing. Dose Expansion cohorts are expected to open in 2023. For additional information, please contact BDTX_1535_101_Study@bdtx.com . Clinical trial information: NCT05256290 .

Abstract CT127: A phase 1 study to assess BDTX-1535, an oral EGFR inhibitor, in patients with glioblastoma or non-small cell lung cancer

Abstract Background: The epidermal growth factor receptor (EGFR) is a potent oncogene commonly altered in many cancers, including glioblastoma (GBM) and non-small cell lung cancer (NSCLC). EGFR tyrosine kinase activity driven by common EGFR mutations can be inhibited by small molecules, however, resistance to available agents may be driven by mutations in the EGFR active kinase site or other regions. BDTX-1535 is an orally available, highly potent, selective, irreversible inhibitor of EGFR mutations, including extracellular variants and amplifications commonly expressed in GBM and inhibits the uncommon EGFR mutations found in NSCLC, including the C797S mutation acquired following 3rd generation EGFR inhibitor therapy. Preclinical data demonstrated the ability of BDTX-1535 to cross the blood-brain barrier and produce sustained inhibition of EGFR signaling. Preclinical studies suggest that BDTX-1535 has potential to be clinically active in suppressing tumor growth in patients with GBM and NSCLC with or without CNS metastases, including a potential survival benefit. Methods: BDTX-1535-101 (NCT05256290) is Phase 1, open-label, multicenter study to assess the safety, tolerability, PK, CNS penetrance, and preliminary antitumor activity of BDTX-1535 in recurrent GBM (rGBM) or locally advanced or metastatic NSCLC with or without CNS disease. The Monotherapy Dose Escalation portion will evaluate BDTX-1535 in patients with either rGBM expressing EGFR alterations or locally advanced/metastatic NSCLC harboring sensitizing EGFR mutations with or without CNS disease. Patients with rGBM must have previously received available standard therapy of surgical resection followed by chemoradiotherapy and/or temozolomide (TMZ). Eligible NSCLC patients must have EGFR mutated NSCLC that has progressed following standard of care EGFR inhibitor therapy. Once a provisional recommended Phase 2 dose (RP2D) has been established, BDTX-1535 monotherapy will be explored in the following Dose Expansion cohorts to further evaluate safety, PK, and preliminary assessment of efficacy: 1) rGBM with confirmed EGFR alterations, 2) NSCLC with uncommon EGFR mutations following EGFR inhibitor therapy; 3) NSCLC with acquired EGFR resistance mutation following a 3rd generation EGFR inhibitor in 1L setting. NSCLC patients may enroll with or without CNS metastases and must not be known to express excluded resistance mutations such as EGFR T790M or MET. BDTX-1535 will also be studied in combination with TMZ to assess safety, tolerability, and a recommended combination dose for the treatment of patients with rGBM harboring EGFR mutations or variants. Enrollment was initiated in 2022 and dose escalation is ongoing. Dose Expansion cohorts are expected to open in 2023. For additional information, please contact BDTX_1535_101_Study@bdtx.com Citation Format: Melissa Johnson, Jason Henry, Alex Spira, James Battiste, Iyad Alnahhas, Manmeet Ahluwalia, Minal Barve, Jeffrey Edenfield, DoHyun Nam, Sudharshan Eathiraj, Julio Hajdenberg, Sergey Yurasov, Helena Yu, Patrick Wen. A phase 1 study to assess BDTX-1535, an oral EGFR inhibitor, in patients with glioblastoma or non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT127.

398TiP MARIPOSA-2: Randomized phase III study of amivantamab + lazertinib + chemotherapy vs chemotherapy alone in EGFR-mutant NSCLC after osimertinib failure

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) effectively treat patients with non-small cell lung cancer (NSCLC) with EGFR-sensitizing mutations; however, almost all will eventually relapse due to new mutations on or after osimertinib the 3rd generation TKI. The current standard of care is platinum doublet chemotherapy after progression on osimertinib, but with limited benefit. Amivantamab, an EGFR and MET bispecific humanized antibody, has shown clinical activity against tumors with primary activating EGFR mutations, EGFR resistance mutations, or MET pathway activation.

Pilot Study of Dacomitinib for Patients With Metastatic EGFR-Mutant Lung Cancers With Disease Progression After Initial Treatment With Osimertinib

In this phase II study, patients who had progressed on first-line osimertinib were treated with dacomitinib 45 mg orally daily until disease progression or intolerability. The primary end point was objective response rate. We enrolled 12 patients. Two partial responses were documented (17% objective response rate; 95% CI, 5 to 45). The median progression-free survival was 1.8 months (95% CI, 1.6 to not reached). One patient with an original sensitizing EGFR G719A mutation and one patient without molecular testing available had partial responses, whereas 0 of the 3 patients with second-site acquired EGFR resistance mutations (two C797S and one G724S) met the response criteria. The patient with EGFR G719A has an ongoing response at 17 months, which exceeds prior time on osimertinib (11 months). In the first trial evaluating a second-generation EGFR TKI after first-line third-generation osimertinib, we found that dacomitinib after disease progression on osimertinib has limited benefit.

A Real-World, Observational, Prospective Study to Assess the Molecular Epidemiology of Epidermal Growth Factor Receptor (EGFR) Mutations upon Progression on or after First-Line Therapy with a First- or Second-Generation EGFR Tyrosine Kinase Inhibitor in EGFR Mutation-Positive Locally Advanced or Metastatic Non-Small Cell Lung Cancer: The 'LUNGFUL' Study.

Simple SummaryNon-small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancer cases, with few patients carrying driver mutations in the gene encoding for epidermal growth factor receptor (EGFR). Advances in translational research have established EGFR tyrosine kinase inhibitors (TKIs) as the standard first-line therapy for NSCLC patients with activating EGFR mutations. The aim of our observational study was to assess the frequency of T790M acquired resistance and predictors of its presence, in patients with EGFR-mutated locally advanced or metastatic NSCLC who have progressed in the first-line EGFR-TKI treatment setting with first- or second-generation TKIs and have undergone molecular testing in tissue and/or plasma biopsy. The study highlights the challenges of performing tissue re-biopsy in routine care settings, which can lead to patients considered non-eligible for certain therapies from which they can benefit, and merits further actions from the healthcare community, in order to establish re-biopsy as a standard procedure.Background: Real-world data on the molecular epidemiology of EGFR resistance mutations at or after progression with first- or second-generation EGFR-TKIs in patients with advanced NSCLC are lacking. Methods: This ongoing observational study was carried out by 23 hospital-based physicians in Greece. The decision to perform cobas® EGFR Mutation Test v2 in tissue and/or plasma at disease progression was made before enrollment. For patients with negative/inconclusive T790M plasma-based results, tissue re-biopsy could be performed. Results: Ninety-six (96) eligible patients were consecutively enrolled (median age: 67.8 years) between July-2017 and September-2019. Of the patients, 98% were tested upon progression using plasma and 2% using tissue/cytology biopsy. The T790M mutation was detected in 16.0% of liquid biopsies. Tissue re-biopsy was performed in 22.8% of patients with a T790M-negative plasma result. In total, the T790M positivity rate was 21.9%, not differing between patients on first- or second-generation EGFR-TKI. Higher (≥2) ECOG performance status and longer (≥10 months) time to disease progression following EGFR-TKI treatment initiation were associated with T790M positivity. Conclusions: Results from plasma/tissue-cytology samples in a real-world setting, yielded a T790M positivity rate lower than previous reports. Fewer than one in four patients with negative plasma-based testing underwent tissue re-biopsy, indicating the challenges in routine care settings.

Routine Molecular Screening of Patients with Advanced Non-SmallCell Lung Cancer in Circulating Cell-Free DNA at Diagnosis and During Progression Using OncoBEAMTM EGFR V2 and NGS Technologies

Background and ObjectivesThe use of ultra-sensitive diagnostic tests to detect clinically actionable somatic alterations within the gene encoding the epidermal growth factor receptor (EGFR) within circulating cell-free DNA is an important first step in determining the eligibility of patients with non-small cell lung cancer to receive tyrosine kinase inhibitors.MethodsWe present the clinical validation (accuracy, sensitivity, and specificity) of a highly sensitive OncoBEAMTM EGFR V2 test, which we compare to a custom next-generation sequencing assay, for the treatment of patients with non-small cell lung cancer with EGFR tyrosine kinase inhibitor therapies. The OncoBEAMTM digital-polymerase chain reaction method detects 36 different EGFR alterations in circulating cell-free DNA, whereas the next-generation sequencing assay covers major solid tumor oncodrivers. Of the 540 samples analyzed with the OncoBEAMTM EGFR V2 test, 42.4% of patients had undergone molecular testing at diagnosis (N = 229/540) and 57.7% of patients during disease progression (N = 311/540).ResultsThe sensitivity and specificity were measured for this BEAMing assay. The number of mutant beads and mutant allelic fraction were measured for each EGFR alteration and the level of detection was established at 0.1% for a median of 2861 genome equivalent (GE) in each reaction using HD780 horizon control DNA, as well as by an internal quality reference standard. Approximately 10%, 27%, and 63% of the 540 samples contained < 1500 GE, a range of 1500–3000 GE, and > 3000 GE, which corresponded to a maximal assay sensitivity of 2.0%, 0.5–0.1%, and 0.1–0.05% mutant allelic fraction, respectively. In a routine hospital setting, 11.4% of non-small cell lung cancer tumors were positive at diagnosis for EGFR alterations, while 43.7% samples harbored EGFR mutations at progression, among which 40.3% expressed EGFR resistance mutations after first-line tyrosine kinase inhibitor treatment with first- and second-generation drugs.ConclusionsThe OncoBEAMTM EGFR V2 is a sensitive, robust, and accurate assay that delivers reproducible results. Next-generation sequencing and BEAMing technologies act complementarily in the routine molecular screening. We show that using a next-generation sequencing assay, despite its lower sensitivity, enables the identification of rare EGFR alterations or resistance mechanisms (mutation, deletion, insertion, and copy number variation) to orient first- and second-line treatments.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40291-021-00515-9.

A Highly Sensitive Next-Generation Sequencing-Based Genotyping Platform for EGFR Mutations in Plasma from Non-Small Cell Lung Cancer Patients.

Simple SummaryIn this study, Sel-CapTM, a next-generation sequencing (NGS)-based genotyping platform, showed high sensitivity for detection of epidermal growth factor receptor (EGFR) gene mutations in plasma samples collected from 185 patients with non-small cell lung cancer (NSCLC). In the early-stage NSCLC, Sel-Cap liquid biopsy was able to detect more than half the EGFR mutations, which were detected in tumor tissue (sensitivity: 50% and 78% for Ex19del and L858R respectively, with tumor results as the references), while the conventional NGS could not detect any. Sel-Cap liquid biopsy was particularly sensitive for resistant mutation T790M (sensitivity: 88%). In addition, we conducted a retrospective study to monitor T790M using Sel-Cap in 34 patients who progressed on first-line tyrosine kinase inhibitors (EGFR-TKIs). The study suggested that the first appearance of T790M in plasma, ranging from at treatment baseline to over three years post-EGFR-TKI initiation, may be useful for prediction of disease progression (around 5 months in advance).Sel-CapTM, a digital enrichment next-generation sequencing (NGS)-based cancer panel, was assessed for detection of epidermal growth factor receptor (EGFR) gene mutations in plasma for non-small cell lung cancer (NSCLC), and for application in monitoring EGFR resistance mutation T790M in plasma following first-line EGFR-tyrosine kinase inhibitor (EGFR-TKI) treatment. Using Sel-Cap, we genotyped plasma samples collected from 185 patients for mutations Ex19del, L858R, and T790M, and compared results to those of PNAclampTM tumor biopsy (reference method, a peptide nucleic acid-mediated polymerase chain reaction clamping) and two other NGS liquid biopsies. Over two-thirds of activating mutations (Ex19del and L858R), previously confirmed by PNAclamp, were detected by Sel-Cap, which is 4–5 times more sensitive than NGS liquid biopsy. Sel-Cap showed particularly high sensitivity for T790M (88%) and for early-stage plasma samples. The relationship between initial T790M detection in plasma and progression-free survival (PFS) following first-line EGFR-TKIs was evaluated in 34 patients. Patients with T790M detected at treatment initiation (±3 months) had significantly shorter PFS than patients where T790M was first detected >3 months post treatment initiation (median PFS: 5.9 vs. 26.5 months; p < 0.0001). However, time from T790M detection to disease progression was not significantly different between the two groups (median around 5 months). In conclusion, Sel-Cap is a highly sensitive platform for EGFR mutations in plasma, and the timing of the first appearance of T790M in plasma, determined via highly sensitive liquid biopsies, may be useful for prediction of disease progression of NSCLC, around 5 months in advance.

384P BLU-945, a highly potent and selective 4th generation EGFR TKI for the treatment of EGFR T790M/C797S resistant NSCLC

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have changed the treatment landscape for EGFR-mutant (L858R and ex19del) driven non-small-cell lung cancer (NSCLC). T790M is the most prevalent acquired EGFR resistance mutation to treatment with 1st and 2nd generation TKIs. 3rd generation TKIs, such as osimertinib, were developed to treat patients with tumors driven by this mutation. The C797S mutation is the most significant on-target resistance mechanism to osimertinib, leading to EGFR T790M/C797S double-resistant mutants. There are currently no approved targeted therapies for NSCLC patients with T790M/C797S mutations. BLU-945 is designed to target and selectively inhibit the EGFR T790M/C797S and T790M resistant mutations. BLU-945 activity on EGFR mutants and EGFR wild-type (WT) was tested in biochemical assays and cellular phosphorylation specific EGFR AlphaLisa assays. The in vivo anti-tumor activity of BLU-945 was evaluated in an NCI-H1975 cell line-derived tumor xenograft (CDX) model, as well as in osimertinib-resistant CDX- and patient-derived xenograft (PDX) models of NSCLC. BLU-945 inhibits EGFRex19del/T790M/C797S, EGFRL858R/T790M/C797S, EGFRex19del/T790M, and EGFRL858R/T790M mutants with sub-nanomolar IC50 values in an enzyme assay with a >1000-fold window over EGFRWT enzyme activity. BLU-945 achieves potent EGFR pathway inhibition in NCI-H1975 EGFRL858R/T790M, Ba/F3 EGFRL858R/T790M/C797S, and Ba/F3 EGFRex19del/T790M/C797S cell lines and a large window relative to EGFRWT inhibition. Oral administration of BLU-945 to tumor-bearing mice demonstrated potent EGFR pathway inhibition and anti-tumor activity at well-tolerated doses in the subcutaneous NCI-H1975 CDX model, and osimertinib-resistant CDX and PDX models, as well as in the intracranial luc-H1975 model of NSCLC. BLU-945 is a potent, selective, and orally available EGFR inhibitor that shows robust anti-tumor activity in osimertinib-resistant EGFR xenograft models. Because of its pharmacological activity and selectivity for mutant EGFR, BLU-945 has the potential to demonstrate activity in osimertinib-resistant EGFR-mutant NSCLC.

Lungful: An observational prospective study to assess the molecular epidemiology of EGFR mutations upon progression on or after first line EGFR-TKI therapy, in real-life clinical settings in Greece.

e21641 Background: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are the gold standard 1st line strategy for non-small-cell lung cancer (NSCLC) patients with activating EGFR mutations (EGFRm), associated with improved survival outcomes and quality of life compared to chemotherapy. Despite the high response rate with first- and second- generation TKIs, most patients develop resistance to treatment and progress. The acquisition of T790M mutation in exon 20 is considered the most common resistance mechanism. This study aims to investigate the molecular epidemiology of EGFR resistance mutations, focusing on T790M in EGFRm NSCLC patients treated with TKIs. Methods: The study included patients with locally advanced/metastatic EGFRm NSCLC who have progressed on or after 1st line treatment with first- or second- generation TKI. Samples either from plasma-based liquid biopsy and/or tissue re-biopsy were analysed using the Cobas EGFR Mutation Test v2. All patients signed informed consent and were enrolled between July 2017 and September 2019. Statistical analyses were performed using SAS software, Version 9.4. Results: Ninety-six eligible patients were enrolled. At the time of progression, T790M mutation was detected in 16.7%of the patients using plasma-based liquid biopsies. Among patients with negative T790M result, in plasma, tissue re-biopsy was performed in 22,7% with evaluable/valid results in 72.2% of them. T790M mutation was identified in 38.5% of re-biopsy samples. According to Cobas EGFR Mutation test results (combined plasma and tissue), T790M mutation was identified in 21.9% of the patients. Of T790M-positive patients 42.9% had previously received first and 57.1% second generation EGFR-TKI. Conclusions: Results from this study in real world clinical setting in Greece, show that EGFR-T790M acquired resistance positivity rate in plasma is lower compared to previous reports. Moreover, these data underline the challenges of implementing precision medicine using tissue re-biopsy in advanced/metastatic NSCLC. Clinical trial information: D133FR00126. [Table: see text]

Evaluation of the Idylla system to detect the EGFRT790M mutation using extracted DNA

IntroductionDuring the last few years, detection of epidermal growth-factor–receptor (EGFR)-activating mutations has become a routine part of clinical practice because of their importance in choosing the optimal treatment strategy for non-small-cell lung cancers (NSCLCs). The emergence of third-generation EGFR–tyrosine-kinase inhibitors required the implementation of sensitive methods to detect the subclonal EGFRT790M mutation. Clinical implications make it essential to rapidly search for the T790M mutation, which is a real challenge for laboratories. The aim of this study was to compare performances of next-generation sequencing (NGS), one of the most frequently used molecular biology methods, and Idylla EGFR-Mutation Assay (henceforth Idylla), a fully automated real-time polymerase chain reaction (PCR) that is increasingly used in pathology laboratories, to detect the EGFRT790M mutation using DNA. MethodsThis retrospective study used 47 DNA samples extracted from NSCLC biopsies that previous NGS identified as: 29 harboring EGFR and T790M resistance mutations, 11 EGFR-activating mutation without T790 M and 7 wild-type EGFR. EGFRT790M limit-of-detection (LOD) experiments used a commercial DNA known to harbor that mutation. ResultsIdylla detected primary EGFR-activating mutations and the T790 M mutation in 97.5 % and 65.5 % of the cases, respectively. The results of this retrospective analysis and LOD experiments showed that the Idylla should only be used to detect EGFR mutations in samples with > 25 ng of DNA and > 10 % tumor cells. ConclusionsIdylla was able to rapidly detect EGFR-activating mutations but detecting subclone mutations, like T790M, with < 25 ng of good-quality DNA or < 10 % tumor cells (variant allele frequency below the assay’s validated LOD) was not always reliable.

Multicenter Evaluation of the Fully Automated PCR-Based Idylla EGFR Mutation Assay on Formalin-Fixed, Paraffin-Embedded Tissue of Human Lung Cancer

Before initiating treatment of advanced non-small-cell lung cancer with tyrosine kinase inhibitors (eg, erlotinib, gefitinib, osimertinib, and afatinib), which inhibit the catalytic activity of epidermal growth factor receptor (EGFR), clinical guidelines require determining the EGFR mutational status for activating (EGFR exons 18, 19, 20, or 21) and resistance (EGFR exon 20) mutations. The EGFR resistance mutation T790M should be monitored at cancer progression. The Idylla EGFR Mutation Assay, performed on the Idylla molecular diagnostics platform, is a fully automated (<2.5 hours turnaround time) sample-to-result molecular test to qualitatively detect 51 EGFR oncogene point mutations, deletions, or insertions. In a 15-center evaluation, Idylla results on 449 archived formalin-fixed, paraffin-embedded tissue sections, originating from non-small-cell lung cancer biopsies and resection specimens, were compared with data obtained earlier with routine reference methods, including next-generation sequencing, Sanger sequencing, pyrosequencing, mass spectrometry, and PCR-based assays. When results were discordant, a third method of analysis was performed, when possible, to confirm test results. After confirmation testing and excluding invalids/errors and discordant results by design, a concordance of 97.6% was obtained between Idylla and routine test results. Even with <10 mm2 of tissue area, a valid Idylla result was obtained in 98.9% of the cases. The Idylla EGFR Mutation Assay enables sensitive detection of most relevant EGFR mutations in concordance with current guidelines, with minimal molecular expertise or infrastructure.

Co-occurrence of EGFR sensitising and resistance mutations at diagnosis in NSCLC.

De novo epidermal growth factor receptor (EGFR) resistance mutations in tyrosine kinase inhibitor-naïve patients are rare when assessed by standard genotyping methods. Patients with EGFR mutations were identified using PCR-based fragment length analysis, mass spectrometry-based genotyping (Sequenom), and Sanger sequencing. From 2008 to 2015, we observed de novo EGFR resistance mutations in 12.8 patients who received an EGFR TKI with an overall response rate of 25%, median PFS 24months, and median OS 34months. Five patients (63%) received erlotinib in the first-line setting with a 60% disease control rate (DCR) and a median duration of response of 6months (range 4-45months). Three (37%) received cytotoxic chemotherapy in the first-line setting with 67% DCR and a median duration of response of 11months (range 10-12months). In patients with de novo EGFR T790M mutations, 50% (2/4) had stable disease with one patient having an ongoing response to erlotinib of over 96months. In patients with de novo EGFR S768I mutations who received erlotinib, 50% (2/4) have ongoing partial responses at 30 and 6months. This is the largest Irish review of de novo synchronous EGFR mutations. The incidence of co-occurring EGFR mutations in our cohort of non-small cell lung carcinoma (NSCLCA) is 1% on routine assays. Erlotinib appears to have activity in this cohort in both in the first- and second-line setting. De novo S768I and T790M represent distinct clinical entities. For de novo T790M mutations cytotoxic chemotherapy may still be considered first line. For de novo S768I mutations, erlotinib appears to be a reasonable therapeutic option.

Monitoring of circulating tumor DNA in non-small cell lung cancer patients treated with EGFR-inhibitors.

11535 Background: Epidermal growth factor receptor (EGFR) inhibitors such as erlotinib and gefitinib are routinely used in the treatment of non-small cell lung cancer (NSCLC). Monitoring of EGFR mutations in circulating tumor DNA (ctDNA) derived from plasma has been proposed as an alternative for repeated tumor biopsies. Our aim was to investigate the dynamics of ctDNA in a cohort of NSCLC patients and explore the roles of EGFR driver and resistance mutations in predicting disease progression and progression free survival (PFS). Methods: NSCLC patients treated with either erlotinib or gefitinib as first-line anti-EGFR therapy were included. Clinical data was collected retrospectively from medical records. Plasma samples collected as part of routine care were analyzed. First DNA was isolated from plasma using the QIAsymphony SP (Qiagen). Then EGFR driver (L858R and exon 19 deletions) and resistance (T790M) mutations were quantified using the X100 Droplet Digital PCR and analyzed using QuantaSoft software (Bio-Rad). The dynamics of ctDNA mutations over time and the relationship between copy numbers and progression free survival were explored. Results: 68 NSCLC patients and 249 plasma samples (1-13 per patient) were included in the analysis. In 33 patients, the T790M mutation was detected. The median (range) T790M concentration in these samples was of 7.3 (5.1 - 3688.7) copies/mL. In 30 patients, the L858R or exon 19 deletion driver mutations were found in median concentrations of 11.7 (5.1 – 12393.3) and 27.9 (5.9 – 2896.7) copies/mL, respectively. Using local polynomial regression, the copies/mL of EGFR driver mutations increased several weeks prior to progression on standard response evaluation. In Kaplan-Meier analysis, patients with a detectable T790M mutation during the first 8 weeks of treatment had a shorter PFS (7.6 versus 14.4 months, p &lt; 0.01, log-rank test). Conclusions: Early detection of the T790M mutation in plasma ctDNA is related to poor PFS. Furthermore, an increase in the copies/mL of the EGFR driver mutation over time may predict clinical progression.

Response to tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) patients with de novo epidermal growth factor receptor (EGFR) T790M and S768I resistance mutations.

e20557 Background: Patients with synchronous de novo EGFR sensitising and resistance mutations are rare. Little is known about the response of these patients to EGFR TKIs, especially in a Caucasian population. Methods: We identified NSCLC patients found to have EGFR mutations using PCR-based fragment length analysis, mass spectrometry-based genotyping (Sequenom), and Sanger sequencing using a large multi-institutional database. Baseline clinical characteristics, response rate, progression free survival (PFS) and overall survival (OS) were calculated. Results: From 2008-2015, we observed de novo synchronous EGFR sensitising and resistance mutations in 12 patients representing an overall incidence of 3.6% of EGFR mutants and 0.4% of all NSCLC patients tested. Seven patients were treated using EGFR TKI therapy with erlotinib. In all cases, T790M (n = 4,50%) or S768I (n = 4, 50%) occurred concurrently with another sensitising EGFR mutation, either L858R (n = 4, 34%) or exon 19 deletion (n = 8, 66%). Objective responses were seen in two patients (29%). Three further patients had stable disease lasting 6, 23 and 54 months respectively. The median progression-free survival was 24 months and the median overall survival was 34 months. All patients with baseline EGFR S768I mutations (n = 3) had an objective response or stable disease on erlotinib while two of four patients with T790M demonstrated de novo resistance. Conclusions: This is the largest Irish review of synchronous de novo EGFR mutations. The incidence of co-occurring EGFR mutations was 0.4% and erlotinib demonstrated activity in this cohort of patients. Ongoing trials will determine whether next-generation EGFR TKIs such as osimertinib are preferable as first-line therapy in these patients.

Abstract 3491: Absolute quantification of EGFR activation and resistance mutations as well as copy number in circulating nucleic acids by droplet digital PCR.

Abstract Background: Non-small cell lung cancer (NSCLC) patients who harbor activating mutations in epidermal growth factor receptor (EGFR) such as L858R and the Exon 19 deletions (E746-A750) are treated with either erlotinib or gefitinib. However, a large percentage of patients acquire the gatekeeper resistance mutation, T790M. Limited access to post-treatment tumor tissue restricts monitoring options, and even when biopsy specimens are available, sampling bias may confound diagnosis of the resistance mechanism. Circulating, cell-free DNA (CNA) provides a solution to circumvent tissue access limitations. Low overall recovery of CNA requires assays be designed with increased sensitivity and specificity not currently available. We have developed and optimized a series of droplet digital PCR assays that can quantify both EGFR activating and resistance mutations as well as copy number. Methods: The ddPCR assays were developed on the BioRAD QX100 system. Primers and probes were designed to optimize quantification of EGFR L858R and T790M mutations, Exon 19 deletions and EGFR copy number. Genomic DNA from H1975 (L5858R and T790M mutations) and H1650 (Exon 19 deletion) cell lines were diluted into wild type gDNA to determine the assays’ limits of detection (LOD). Patient samples were analyzed as well. Finally, ddPCR data were compared with qPCR data using the Qiagen EGFR Rotor-Gene (RGQ) kit. Results: The ddPCR assays were designed to quantify EGFR activating and resistance mutations in relative to the wild type template. The T790M and L858R assays displayed linear dynamic ranges from ∼14000 copies to LODs of 9 and 6 copies, respectively. In addition, ddPCR assays were successfully multiplexed to an LOD of 0.2%, or 15 copies based upon a 95% confidence interval in 20ng gDNA. In healthy donor spiked plasma samples, the LOD was determined to be 1.2%, or 8 copies in 2.2ng purified CNA. By comparison, in plasma, the RGQ assays displayed an average LOD of 80 copies with a dynamic range of ∼8000 copies to 76 copies. Data on Exon 19 deletion and EGFR copy number ddPCR assays will be presented. Spiked normal plasma samples analyzed for the ratio of T790M to activating mutations such as L858R and Exon 19 deletion will be presented. Conclusion: Droplet digital PCR provides sensitive quantification of low abundance EGFR resistance and activating mutations in CNA isolated from plasma. These assays provide a useful method for selecting treatment, monitoring disease progression, and providing early detection of treatment failure as well as gene amplification associated with EGFR acquired resistance. Citation Format: Jessica Kristof, Eric Bruening, Stephane Wong, Sabita Sankar. Absolute quantification of EGFR activation and resistance mutations as well as copy number in circulating nucleic acids by droplet digital PCR. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3491. doi:10.1158/1538-7445.AM2013-3491

First Case of A859T Epidermal Growth Factor Receptor Mutation Responding to Erlotinib

A 59-year-old smoker consulted for a pulmonary nodule. Computed tomography (CT) scan lung biopsy diagnosed a non-small cell lung cancer (NSCLC) of adenocarcinoma subtype. Positron emission tomography demonstrated a left adrenal metastasis. First-line chemotherapy with a combination of pemetrexed and carboplatin was performed. After four cycles, a partial response was observed according to RECIST 1.1 criteria. During the same period, epidermal growth factor receptor (EGFR) mutations were characterized according to a sensitive method based on high-performance liquid chromatography and sequencing (Figures 1A, B). The rare mutation A859T was observed. Erlotinib was introduced after a wash-out period of 4 weeks. Tolerance was excellent. Eight weeks later, CT scan demonstrated a partial response (criteria RECIST 1.1 = − 41%). Maximal standard uptakes of 18F-fluorodeoxyglucose (FDG) showed a reduction for primary and secondary tumors, 3.9 to 1.0 and 2.9 to 2.3, respectively (Figure 2). The radiographic response was confirmed with a CT scan 3 months later. To date, the duration of response is 3 months.FIGURE 2Computed tomography and positron emission tomography scans and fused images before and 8 weeks after erlotinib.View Large Image Figure ViewerDownload (PPT) Activating EGFR mutations are predictive responses for the tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib. The most frequent deletions and mutations, located in exon 19 and 21, are observed from 10% in Caucasian populations1Rosell R Moran T Queralt C et al.Screening for epidermal growth factor receptor mutations in lung cancer.N Engl J Med. 2009; 361: 958-967Crossref PubMed Scopus (1901) Google Scholar up to 40 to 60% in female, nonsmoking Asian populations.2Mok TS Wu YL Thongprasert S et al.Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma.N Engl J Med. 2009; 361: 947-957Crossref PubMed Scopus (6514) Google Scholar Alongside the most frequent activating EGFR mutations, many other less frequent mutations have been described.3Avizienyte E Ward RA Garner AP Comparison of the EGFR resistance mutation profiles generated by EGFR-targeted tyrosine kinase inhibitors and the impact of drug combinations.Biochem J. 2008; 415: 197-206Crossref PubMed Scopus (73) Google Scholar In general, little is known about the sensitivity or the lack of sensitivity to TKIs in humans. A859T EGFR mutation has already been reported twice and has been associated with the absence of sensitivity to gefitinib.4Han SW Kim TY Hwang PG et al.Predictive and prognostic impact of epidermal growth factor receptor mutation in non-small-cell lung cancer patients treated with gefitinib.J Clin Oncol. 2005; 23: 2493-2501Crossref PubMed Scopus (715) Google Scholar, 5Cappuzzo F Bemis L Varella-Garcia M HER2 mutation and response to trastuzumab therapy in non-small-cell lung cancer.N Engl J Med. 2006; 354: 2619-2621Crossref PubMed Scopus (183) Google Scholar Functional analysis of A859T EGFR mutation exposed to erlotinib based on a cellular assay concluded that this variant could be a silent polymorphism.6de Gunst MM Gallegos-Ruiz MI Giaccone G et al.Functional analysis of cancer-associated EGFR mutants using a cellular assay with YFP-tagged EGFR intracellular domain.Mol Cancer. 2007; 6: 56Crossref PubMed Scopus (19) Google Scholar To the contrary of all these published data, our case supports the evidence of a therapeutic effect of erlotinib in a NSCLC patient bearing A859T EGFR mutation. The proofs are a tumor size reduction and a decrease of the FDG maximal uptake after 8 weeks of treatment and confirmed 3 months later. Although the follow-up of our case is limited, our observation seems similar to patients with TKI-sensitive adenocarcinomas harboring a frequent EGFR mutation: significant shrinking tumor size after TKI introduction, long period of stable disease, and good tolerance. For all the reasons, we consider that A859T EGFR mutation could be an activating mutation-associated response to erlotinib. Because of the lack of a reliable tool to predict EGFR mutation sensitivity of current TKIs, we recommend that physicians test both/several TKIs for every NSCLC patient harboring a rare/new EGFR mutation.

Chemogenomic Profiling Provides Insights into the Limited Activity of Irreversible EGFR Inhibitors in Tumor Cells Expressing the T790M EGFR Resistance Mutation

Reversible epidermal growth factor receptor (EGFR) inhibitors are the first class of small molecules to improve progression-free survival of patients with EGFR-mutated lung cancers. Second-generation EGFR inhibitors introduced to overcome acquired resistance by the T790M resistance mutation of EGFR have thus far shown limited clinical activity in patients with T790M-mutant tumors. In this study, we systematically analyzed the determinants of the activity and selectivity of the second-generation EGFR inhibitors. A focused library of irreversible as well as structurally corresponding reversible EGFR-inhibitors was synthesized for chemogenomic profiling involving over 79 genetically defined NSCLC and 19 EGFR-dependent cell lines. Overall, our results show that the growth-inhibitory potency of all irreversible inhibitors against the EGFR(T790M) resistance mutation was limited by reduced target inhibition, linked to decreased binding velocity to the mutant kinase. Combined treatment of T790M-mutant tumor cells with BIBW-2992 and the phosphoinositide-3-kinase/mammalian target of rapamycin inhibitor PI-103 led to synergistic induction of apoptosis. Our findings offer a mechanistic explanation for the limited efficacy of irreversible EGFR inhibitors in EGFR(T790M) gatekeeper-mutant tumors, and they prompt combination treatment strategies involving inhibitors that target signaling downstream of the EGFR.

Comparison of the EGFR resistance mutation profiles generated by EGFR-targeted tyrosine kinase inhibitors and the impact of drug combinations

Recent clinical data indicates that the emergence of mutant drug-resistant kinase alleles may be particularly relevant for targeted kinase inhibitors. In order to explore how different classes of targeted therapies impact upon resistance mutations, we performed EGFR (epidermal-growth-factor receptor) resistance mutation screens with erlotinib, lapatinib and CI-1033. Distinct mutation spectra were generated with each inhibitor and were reflective of their respective mechanisms of action. Lapatinib yielded the widest variety of mutations, whereas mutational variability was lower in the erlotinib and CI-1033 screens. Lapatinib was uniquely sensitive to mutations of residues located deep within the selectivity pocket, whereas mutation of either Gly(796) or Cys(797) resulted in a dramatic loss of CI-1033 potency. The clinically observed T790M mutation was common to all inhibitors, but occurred with varying frequencies. Importantly, the presence of C797S with T790M in the same EGFR allele conferred complete resistance to erlotinib, lapatinib and CI-1033. The combination of erlotinib and CI-1033 effectively reduced the number of drug-resistant clones, suggesting a possible clinical strategy to overcome drug resistance. Interestingly, our results also indicate that co-expression of ErbB2 (v-erb-b2 erythroblastic leukaemia viral oncogene homologue 2) has an impact upon the EGFR resistance mutations obtained, suggesting that ErbB2 may play an active role in the acquisition of drug-resistant mutations.