EGFR Mutations In Plasma Research Articles

The increase in activating EGFR mutation in plasma is an early biomarker to monitor response to osimertinib: a case report

BackgroundSystemic treatment of advanced non-small cell lung cancer (NSCLC) has changed dramatically since the introduction of targeted therapies. The analysis of circulating tumor DNA (ctDNA) is a valuable approach to monitor the clonal evolution of tumors during treatment with EGFR-tyrosine kinase inhibitors (TKIs) and to detect resistance mutations.Case presentationA NSCLC patient with exon 19 deletion (ex19del) of EGFR was treated with osimertinib after multiple lines of treatment and obtained a partial response that lasted over 26 months. Blood was collected at each visit and ctDNA was extracted to monitor ex19del by digital droplet PCR. Within a few weeks from the beginning of osimertinib, ex19del disappeared from plasma but appeared again and steadily increased a few months later anticipating tumor progression. Interestingly, the change in ex19del was much more pronounced than other mutations, since T790M appeared 3 months after the increase of ex19del, and C797S was detectable a few weeks before clinical disease progression. Then the patient received cytotoxic chemotherapy, which was associated with a decrease in ex19del and disappearance of T790M and C797S; however, at disease progression, all EGFR mutations increased again in plasma together with MET amplification which was detected by NGS.ConclusionsThe measurement of ex19del changes in ctDNA is a simple and sensitive approach to monitor clinical outcome to osimertinib and, potentially, to other therapeutic interventions.

From the beginning to resistance: Study of plasma monitoring and resistance mechanisms in a cohort of patients treated with osimertinib for advanced T790M-positive NSCLC

IntroductionAnalysis of circulating tumor DNA (ctDNA) for the identification of T790M mutation in advanced EGFR-mutated NSCLC patients can replace tissue re-biopsy for resistance characterization and, being non-invasive, may be applied for disease monitoring. We analysed ctDNA during osimertinib treatment to correlate mutational levels with clinical outcome and to predict pattern of resistance. Materials and MethodsForty patients with advanced NSCLC receiving osimertinib for T790M + disease after previous EGFR-TKI were enrolled in a pilot study to collect plasma at baseline and every 12 weeks until progression. Molecular analysis of ctDNA was performed by ddPCR and Therascreen®. When feasible at progression, tissue re-biopsy and NGS analysis were performed. ResultsThirty-eight patients had baseline plasma samples suitable for molecular analysis. Patients with low levels of the EGFR activating mutation in ctDNA [< 2200 copies/mL or allele frequency (AF) < 6.1%] showed better progression-free survival (17.8 or 17.8 months vs. 4.3 or 2.7, p = 0.022 or p = 0.018, respectively) and overall survival (23.6 or 23.6 vs. 7.7 or 7.3, p = 0.016 or p = 0.013, respectively) than patients with high levels (≥ 2200 copies/mL or AF ≥ 6.1%). Patients with detectable EGFR mutations in plasma (shedders) presented worse outcome than negative subjects (non-shedders). Low levels of T790M, higher T790M/activating mutation ratio and complete clearance after 2 months were associated with a trend towards better outcome. Tissue re-biopsy at resistance showed 3 patients with EGFR C797S, 1 with MET amplification, 1 with MYC amplification, 1 with PTEN loss, 3 with SCLC transformation. ConclusionsThe mutational analysis performed on plasma plays a significant role in prognostic stratification, especially for the EGFR activating mutation, since patients with absence or low levels of mutations presented a better outcome to osimertinib. At progression, tissue re-biopsy remains a crucial issue for the identification of resistance mechanisms.

Detection of Plasma EGFR Mutations in NSCLC Patients with a Validated ddPCR Lung cfDNA Assay.

Purpose: The clinical utility of cell-free DNA (cfDNA) to assess EGFR mutations is increasing. However, there are limited studies determining their clinical validity and utility. The value of cfDNA assays in cancer management remains controversial.Methods: In this study, we first evaluated the analytical performance of the ddPCR Lung cfDNA Assay. We next analyzed the concordance of the results with tissue amplification refractory mutation system PCR (ARMS-PCR) and plasma next-generation sequencing (NGS) genotyping. Finally, we assessed its clinical utility by exploring the association of cfDNA EGFR mutations with metastatic sites and the efficacy of EGFR-TKIs treatment.Results: The ddPCR Lung cfDNA Assay demonstrated a limit of blank of 1 droplet per reaction, an analytical specificity of 100%, and detection limit of 0.05%, 0.05%, and 0.1% for E746_A750del, L858R, and T790M, respectively. With tissue ARMS-PCR as a standard for comparison, the clinical sensitivity and specificity of ddPCR were 62.5% (15/24) and 100% (82/82) for E746_A750del, and 75.0% (15/20) and 94.2% (81/86) for L858R, respectively. The ddPCR showed high concordance with NGS in determining cfDNA EGFR mutations. Patients with bone and/or brain metastasis showed a higher detection rate and mutant abundance of cfDNA EGFR mutations compared to those with other sites of metastasis. Moreover, EGFR-TKIs treatment was effective in patients with sensitive EGFR mutations in either plasma cfDNA or tumor tissue-derived DNA.Conclusions: We validated in this study that the ddPCR Lung cfDNA Assay is reliable for detection of EGFR mutations in lung cancers, in terms of analytical performance, clinical validity and utility.

Electric Field–Induced Release and Measurement Liquid Biopsy for Noninvasive Early Lung Cancer Assessment

Previously, we detected circulating tumor DNA that contained two EGFR mutations (p.L858R and exon19 del) in plasma of patients with late-stage non-small-cell lung carcinoma (NSCLC) using the electricfield-induced release and measurement (EFIRM) platform. Our aim was to determine whether EFIRM technology can detect these mutations in patients with early-stage NSCLC. Prospectively, 248 patients with radiographically determined pulmonary nodules were recruited. Plasma was collected before biopsy and histologic examination of the nodule. Inclusion criteria were histologic diagnosis of benign nodule (control) and stage I or II adenocarcinoma harboring either p.L858R or exon19 delEGFR mutations. Plasma samples were available from 44 patients: 23 with biopsy-proven benign pulmonary nodules and 21 with stage I or II adenocarcinoma (12 p.L858R and 9 exon19 delEGFR variants). Samples were analyzed for the EGFR mutations using the EFIRM platform. Assay sensitivity was 92% for p.L858R (11 of 12 samples positive) and 77% for exon19 del (7 of 9 samples positive). Specificity was 91% with two false-positive results in 23 patients with EGFR-positive nodules and 95% for the entire 44-patient series. Concordance was 100% with identical mutations discovered in plasma and nodule biopsy. The EFIRM platform is able to noninvasively detect two EGFR mutations in individuals with early-stage NSCLC.

P3.01-58 Relative Abundance Of EGFR Mutations in Plasma Predicts Tumor Response to EGFR-TKI in Advanced Lung Adenocacarcinoma

It is unclear whether relative abundance of EGFR mutations in plasma predicts tumor response treatment with EGFR–TKIs in advanced lung adenocarcinoma. The present prospective study enrolled patients with advanced lung adenocarcinoma in the first affiliated hospital of Wenzhou Medical University from 1 April 2016 to 1 January 2017. EGFR mutations detection in tumor tissues by ADx-ARMS and in plasma cfDNA by ADx-ARMS and ADx-SuperARMS were all tested. We enrolled patients with EGFR-mutant in tumor tissue and receiving EGFR-TKIs. Mutation-positive plasma by both methods carried high abundance of EGFR mutations. Plasma that was mutation positive by ADx-SuperARMS but the result by ADx-ARMS was negative harbored medium abundance. Mutation-negative plasma by both methods was recognized as low abundance group. The correlation between EGFR mutation abundance and clinical benefit from EGFR-TKIs treatment was analyzed using Kaplan-Meier curve, Chi-square test and Cox proportional hazards model. The study protocol was approved by the Institutional Review Board of the first affiliated hospital of Wenzhou Medical University(2016-017). 71 patients were enrolled, 42 harbored EGFR mutations in plasma detected by ADx-ARMS, while 53 were found positive result in plasma by ADx-SuperARMS. Median PFS was 10.0m and ORR was 64.8% in the patients with EGFR-mutant in tumor tissue. According to the results of the plasma detected by ADx-ARMS and ADx-SuperARMS, 42 were found high abundances of EGFR mutations, 11 were recognized as medium abundances group while the other 18 as low abundances. The ORRs were 69.0%，63.6% and 55.6% for patients with high, medium and low abundance of EGFR mutations, respectively. Pwith high abundances of EGFR mutations were seemed to acquire high ORRs, the difference was significant between high and low abundances group (P=0.006) but the differences were not significant when compared high abundances group with medium abundances group (P=0.732) and medium abundances group with low abundances group (P=0.114). Medium PFS for high, medium and low abundances groups were 11.0m, 8.5m and 9.0m, respectively(P<0.001). For the patients with 19-Del in tumor tissues, the ORRs were 70.4%，57.1% and 54.5% in high, medium and low abundance of EGFR mutations groups, respectively. Medium PFS of high abundance group was longer than medium and low abundance groups (12.0m vs 9.0m vs 9.0m). As for L858R, the ORRs were 66.7%，50.0% and 50.0%，respectively, Medium PFS were 9.6m, 5.5m and 9.5m. The relative EGFR mutation abundance in plasma could predict tumor response to EGFR-TKI treatment for advanced adenocarcinoma. The higher the EGFR mutation abundance is, the better efficacy of EGFR-TKI is.

P1.15-08 Implementation and Feasibility of the Assessment of EGFR Mutation in NSCLC Using Liquid Biopsy at an Argentinean University Hospital

Molecular characterization of lung cancer is standard of care. Almost 85% of NSCLC patients have advanced disease at diagnosis, being a challenge get enough tissue for diagnosis. That' s why using less invasive methods as liquid biopsy by determining cftDNA (circulating free tumor DNA) represents a promising tool. The detection of EGFR mutations in plasma by Therascreen (EGFR RGQ Mutation Kit) showed a sensitivity and specificity of 65.4% and 100 % respectively. It is also useful to detect TKI's resistance previous to clinical progression. The advantages of using PCR, even there are more sensitive methods available, are its simplicity and cost. We will prospectively recruit EGFR mutation-positive (n=40) NSCLC patients. Plasma samples are collected: baseline, after three months of treatment, every six months and at the time of progression. We report our first interim analysis. We evaluate the concordance of EGFRm in tissue and plasma with the implementation of the Therascreen test (Qiagen). We test other available plasma EGFRm detection kit (EGFR Plasma Mutation Analysis Kit, Entrogen) (n=10) Since February 2017, plasma EGFRm status could be determined in 17 out of 22 NSCLC patients with EGFRm in tissue. L858R was present in 9 (53%), del19 in 7 (42%), del19+T790M in 1 (5%). Baseline plasma analyzed with Therascreen and Entrogen kits showed similar sensitivity of 61.1% and 66% respectively. Only 1/11 pt had discordance between both kits (Therascreen negative , entrogen DEL19+T790M ). In 17 analyzed ptes, median age was 59 (51-76) years. Most of the population were women (88%), were never smokers (58%), had stage IV disease (58%). Frequent site of metastasis was bone (n=7). With a median follow up of 18 months (12-23), 7/15 (56%) patients who experienced clinical progression were detected also in plasma. Two patients presented T790M in plasma without evidence of disease progression until last follow-up (July 2018). We also observed two patients with mutations detected out of range to be considered as positive which became positive for these mutations during the follow up. This is the first work showing a cohort of EGFRm patients prospectively analyzed by successive liquid biopsies in a public institution in Argentina. We could successfully obtain cftDNA and analyze it by two commercial kits. The sensitivity of both kits was comparable to previous reports. The study is ongoing and more samples will be analized to shed light about the plasma dynamics of EGFRm related to clinical behavior.

P1.09-23 Effect of Plasma Input on the Clinical Sensitivity of a Real-Time PCR Assay for the Detection Of EGFR Mutation in Plasma ctDNA From NSCLC Patients

Plasma based EGFR mutation testing is a less invasive and feasible testing option for patients with advanced non-small cell lung cancer (NSCLC) when traditional tissue biopsy testing is challenging. A fast and sensitive real-time PCR method has been developed for the detection of EGFR mutations in plasma circulating tumor DNA (ctDNA). To define the effect of plasma input on the clinical sensitivity of the assay, clinical evaluation was conducted in the present study. A total of 203 patients with advanced NSCLC were recruited who provided plasma and matched tumor tissue samples, 109 of whom providing samples had adequate plasma for both 2 mL and 4 mL plasma testing, and the rest providing 2 mL plasma samples. ctDNA was isolated and tested for the presence of EGFR mutation by the AmoyDx Super-ARMS EGFR Mutation Detection Kit (Super-ARMS EGFR). Tumor tissue DNA was isolated and tested for EGFR mutation by AmoyDx ARMS EGFR Mutation Detection Kit (ARMS EGFR). Clinical sensitivity and specificity of ctDNA testing were analyzed by using tumor tissue testing as a reference. In pairs of tumor biopsy and plasma samples, the sensitivity, specificity and overall concordance of EGFR mutation status determined in 4 mL of plasma by Super-ARMS EGFR test and matched tissue samples were 82.0% [50/61], 100% [48/48], and 89.9% [98/109] (N=109), respectively. These rates were much higher than those of mutation status determined in 2 mL of plasma and matched tissue samples (65.55% [78/119], 97.62% [82/84] and 78.82% [160/203], N=203). Super-ARMS EGFR assay is a highly reliable and sensitive method for the detection of EGFR mutation in lung cancer plasma ctDNA samples. Plasma samples of 4 mL volume is more appropriate for ctDNA EGFR mutation assessment than determination in 2 mL volume of plasma.

Detection of EGFR mutations in plasma circulating tumour DNA as a selection criterion for first-line gefitinib treatment in patients with advanced lung adenocarcinoma (BENEFIT): a phase 2, single-arm, multicentre clinical trial

Detection of EGFR mutations in tumour tissue is the gold-standard approach to ascertain if a patient will benefit from treatment with an EGFR tyrosine kinase inhibitor. However, if tissue is scant, another strategy is to use circulating tumour DNA (ctDNA), but this method needs validation in clinical trials. We did a prospective clinical trial to assess ctDNA-based EGFR mutation detection as a selection criterion for patients with lung adenocarcinoma receiving gefitinib as first-line treatment. BENEFIT is a multicentre, single-arm, phase 2 clinical trial at 15 centres in China. Patients aged 18-75 years with stage IV metastatic lung adenocarcinoma and EGFR mutations detected in ctDNA were given oral gefitinib 250 mg once daily as first-line treatment. The primary endpoint was the proportion achieving an objective response. Secondary endpoints included median progression-free survival and safety. Next-generation sequencing (NGS) of a 168-gene panel was used for genetic analysis of baseline blood samples. The primary efficacy analysis was done by intention to treat in patients who had at least one post-baseline tumour assessment. The safety analysis was done in all patients who received at least one dose of study treatment. This trial is registered with ClinicalTrials.gov, number NCT02282267. Between Dec 25, 2014, and Jan 16, 2016, 426 patients were screened for the trial, of whom 188 with EGFR mutations in ctDNA were enrolled and received gefitinib. 183 patients had one or more post-baseline tumour assessment and were included in the primary efficacy analysis. Median follow-up was 14·5 months (IQR 12·2-16·5). At the time of data cutoff (Jan 31, 2017), 152 patients had progressive disease or had died. The proportion achieving an objective response was 72·1% (95% CI 65·0-78·5). Median progression-free survival was 9·5 months (95% CI 9·07-11·04). Of 167 patients with available blood samples, 147 (88%) showed clearance of EGFR mutations in ctDNA at week 8, and median progression-free survival was longer for these patients than for the 20 patients whose EGFR mutations persisted at week 8 (11·0 months [95% CI 9·43-12·85] vs 2·1 months [1·81-3·65]; hazard ratio [HR] 0·14, 95% CI 0·08-0·23; p<0·0001). From baseline NGS data in 179 patients, we identified three subgroups of patients: those with EGFR mutations only (n=58), those with mutations in EGFR and tumour-suppressor genes (n=97), and those with mutations in EGFR and oncogenes (n=24). Corresponding median progression-free survival in these subgroups was 13·2 months (95% CI 11·5-15·0), 9·3 months (7·6-11·0), and 4·7 months (1·9-9·3), respectively (EGFR mutations only vs mutations in EGFR and tumour-suppressor genes, HR 1·78, 95% CI 1·23-2·58; p=0·002; EGFR mutations only vs mutations in EGFR and oncogenes, 2·66, 1·58-4·49; p=0·0003). The most common grade 3 or 4 adverse events were hepatic function abnormalities (n=24). Serious adverse events were reported in 17 (9%) patients. No unexpected safety events for gefitinib were recorded. Detection of EGFR mutations in ctDNA is an effective method to identify patients who might benefit from first-line gefitinib treatment. Further analyses of dynamic alterations of EGFR mutations and accompanying gene aberrances could predict resistance to gefitinib. Guangdong Association of Clinical Trials, AstraZeneca, National Natural Sciences Foundation Key Programme, and National Key Research and Development Programme of China.

Abstract 937: Longitudinal circulating-tumor DNA profiling of EGFR-mutated non-small cell lung cancer patients treated with EGFR-tyrosine kinase inhibitors

Abstract Background: In EGFR-mutant non-small cell lung cancer (NSCLC), progression disease (PD) under 1st-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs) is driven by the EGFR T790M mutation in about 50% of cases. This mutation is targeted with osimertinib, a specific and potent inhibitor that also showed superior efficacy to that of 1st-generation EGFR-TKIs in the 1st-line treatment EGFR+ NSCLC. Strategy: We performed a longitudinal circulating-tumor DNA (ctDNA) analysis to evaluate 1) the dynamics of sensitizing or resistant mutations over time and 2) the genomic alterations associated with EGFR-TKI resistance, in NSCLC patients treated with either 1st-generation EGFR-TKIs (n=24) or with osimertinib (n=20). ctDNA sequencing was performed using InVisionSeq™, an amplicon-based NGS (36-gene panel) assay. Results: Sensitivity was 80% and 100% for the detection of EGFR-activating mutations and the EGFR T790M mutation, respectively, at PD in ctDNA vs tissue biopsies. The type of progression appears to influence the detection of EGFR mutations in plasma. Patients (pts) with exclusively brain or thoracic PD had significantly lower allelic fractions (AFs) of EGFR-activating mutations than pts with systemic PD (P=0.006). AFs of both EGFR-activating and EGFR T790M mutations were highest in patients with progressive metastases in liver or bone. High cell-free DNA load was associated with the presence of TP53 mutations regardless of the time under treatment (n=248 samples). The total number of mutations detected in ctDNA was higher in pts treated with osimertinib vs. 1st-generation EGFR-TKIs (P=0.004), suggesting a potential increase of tumor heterogeneity over time. We observed that the AFs of mutant ctDNA were correlated with response to treatment. Complete clearance of EGFR T790M (AF detection limit: 0.01%) was observed in pts who presented a partial response to osimertinib (n=17), of these, EGFR-activating mutations were found in 4/17 pts (AF:0.05-1.03%). PD was evidenced in 36/44 pts. Emergence of mechanisms of resistance, before the confirmation of clinical PD, was evidenced in 5 and 3 pts under erlotinib and osimertinib, respectively. PD to 1st-generation EGFR-TKIs was mainly driven by EGFR T790M (86%), followed by mutations in PIK3CA or PTEN. We detected the emergence of heterogeneous potential mechanisms of resistance to osimertinib in 3/9 cases, including mutations in EGFR, KRAS, BRAF as well as HER2 and MET amplification. We did not identify genomic drivers of resistance in 16/36 pts, despite evidence of clinical PD. These pts had either exclusive brain or thoracic progression. ctDNA WES will be performed in these samples. Conclusion: Our results suggest that ctDNA sequencing might be a complementary, noninvasive tool to monitor response to treatment and heterogeneous mechanisms of resistance in NSCLC pts treated with EGFR inhibitors. Citation Format: Sandra Ortiz-Cuaran, Aurélie Swalduz, Camille Léonce, Solène Marteau, Séverine Martinez, Gilles Clapisson, Virginie Avrillon, Luc Odier, Lionel Falchero, Pierre Fournel, Emma Green, Clive Morris, Maurice Pérol, Pierre Saintingy. Longitudinal circulating-tumor DNA profiling of EGFR-mutated non-small cell lung cancer patients treated with EGFR-tyrosine kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 937.

EFIRM liquid biopsy (eLB): Detection of ultra-short circulating tumor DNA (usctDNA) in plasma and saliva of non-small cell lung cancer (NSCLC) patients.

e24062Background: We previously demonstrated Electric Field-Induced Release and Measurement (EFIRM)-based liquid biopsy (eLB) can detect two frontline actionable EGFR mutations in plasma and saliva...

Early clearance of plasma EGFR mutations as a predictor of response to osimertinib in the AURA3 trial.

9027Background: In the Phase III AURA3 trial (NCT02151981), osimertinib, a third-generation EGFR-TKI, had significantly greater efficacy than platinum-pemetrexed in patients (pts) with advanced NSC...

Comparison of the SuperARMS and Droplet Digital PCR for Detecting EGFR Mutation in ctDNA From NSCLC Patients

BACKGROUND: Liquid biopsy is emerging as an important approach for tumor genotyping in non-small cell lung cancer, ddPCR and SuperARMS are both methods with high sensitivity and specificity for detecting EGFR mutation in plasma. We aimed to compare ddPCR and SuperARMS to detect plasma EGFR status in a cohort of advanced NSCLC patients. METHOD: A total of 79 tumor tissues and paired plasma samples were collected. The EGFR mutation status in tissue was tested by ADx-ARMS, matched plasma was detected by ddPCR and SuperARMS, respectively. RESULTS: The EGFR mutation rates were identified as 64.6% (tissue, ARMS), 55.7% (plasma, ddPCR), and 49.4% (plasma, Super ARMS), respectively. The sensitivity of ddPCR was similar with Super-ARMS in plasma EGFR detection (80.4% vs 76.5%), as well as the specificity (89.3% vs 100%). And the McNemar’s test showed there was no significant difference (P = .125). The concordance rate between SuperARMS and ddPCR was 91.1%. A significant interaction was observed between cfDNA EGFR mutation status and EGFR-TKIs treatment tested by both methods. CONCLUSION: Super-ARMS and ddPCR share the similar accuracy for EGFR mutation detection in plasma biopsy; both methods predicted well the efficacy of EGFR-TKIs by detecting plasma EGFR status.

P3.01-031 ctDNA Assessment of EGFR Mutation Status in Chinese Patients with Advanced Non–Small-Cell Lung Cancer in Read World Setting

EGFR mutation in plasma circulating free tumor-derived DNA (ctDNA) by ARMS methods has been widely used in clinical settings in China. However, the prevalence of EGFR mutations in ctDNA was still unknown in the real world. This large-scale study (NCT02623257) aimed to explore the prevalence of EGFR mutations and determine the correlation of EGFR mutation status with clinical characteristics.

Quantification of plasma EGFR mutations in patients with lung cancers: Comparison of the performance of ARMS-Plus and droplet digital PCR

ObjectivesEGFR mutation is a key factor to predict EGFR-TKI efficacy. However, a significant number of advanced patients do not have sufficient tumor specimens for molecular testing. Also, there is a lack of quantitative assay to analyze the mutant abundance. This study aims to evaluate the detection efficiency and clinical feasibility of a new platform, namely ARMS-Plus, for the detection and quantification of EGFR mutations in plasma. Materials and methodsThe detection limit of ARMS-Plus was assessed by detecting spiked mutant plasmids which were serially diluted with normal human genomic DNA. The cutoff values were defined by examining the mutant copy numbers presented in 134 healthy controls. Plasma samples from 65 lung cancer patients were collected to evaluate the clinical performance of ARMS-Plus. EGFR mutations were concurrently tested by droplet digital PCR (ddPCR) for the plasma samples and conventional amplification refractory mutation system-PCR (ARMS-PCR) for the matched tumor tissue specimens to serve as a standard for comparison. ResultsIn this study, the analytical sensitivity of ARMS-Plus was 0.015%. The cutoff values of EGFR 19Del, L858R, T790M mutations were defined as 2, 5, and 3 copies/mL, respectively. With tumor specimens as the standard, the sensitivity, specificity, and concordance rate of ARMS-Plus and ddPCR were 60.7%, 94.6%, and 80.0%; and 50.0%, 97.3%, and 76.9%, respectively. For quantification, the plasma 19Del and L858R mutant abundance detected by ARMS-Plus and ddPCR were consistent (Spearman R=0.7956 and 0.7710, P<0.0001). ConclusionARMS-Plus is a reliable, convenient and cost-effective method for the detection and quantification of plasma EGFR mutations.

Patients with NSCLC may display a low ratio of p.T790M vs. activating EGFR mutations in plasma at disease progression: implications for personalised treatment.

IntroductionNSCLC harboring activating mutations of EGFR is highly sensitive to first-line EGFR-tyrosine kinase inhibitors (TKIs), but drug resistance depending on the EGFR mutation p.T790M will occur in about 50-60% of patients. Detailed information on the amount of p.T790M plasmatic level associated with resistance to EGFR-TKIs and guidance to treatment with p.T790M-effective TKI depending on these levels, is lacking.MethodsThis study enrolled p.T790M-positive patients (n=49) affected by EGFR-mutated NSCLC at progression to first-line EGFR-TKIs and, in selected cases (n=5), after second-line treatment with osimertinib. Cell-free circulating tumor DNA (cftDNA) was extracted from plasma and the quantitative analysis of EGFR ex19del, p.L858R and p.T790M was performed by digital droplet PCR.ResultsThe mean amount of mutated alleles at progression to first-line EGFR-TKIs was 108,492 copies/ml for ex19del, 97,336 copies/ml for p.L858R, but only 8,754 copies/ml for p.T790M. There was no significant correlation between progression-free survival and the ratio of p.T790M over EGFR activating mutations. The analysis of cftDNA in 5 patients treated with osimertinib revealed a marked decrease of all EGFR mutant alleles.ConclusionsThe amount of p.T790M in plasma can be much lower than activating EGFR mutations. Despite this finding, osimertinib is effective in p.T790M-positive patients. These results indicate that clones driving resistance to EGFR-TKIs represent a minority among cells bearing activating EGFR-mutations. In addition, the identification of a threshold level of p.T790M is not a strict requirement for the selection of patients to be treated with osimertinib, since treatment showed a decrease in all EGFR mutated cells.

Dynamic circulating tumor DNA quantificaton for the individualization of non-small-cell lung cancer patients treatment.

BackgroundLiquid biopsy has evolved from being a promising line to becoming a validated approach for biomarker testing. However, its utility for individualization of therapy has been scarcely reported. In this study, we show how monitoring levels of EGFR mutation in plasma can be useful for the individualization of treatment.ResultsLongitudinal EGFR mutation levels in plasma always correlated with tumor response ascertained by RECIST criteria. Moreover, decreasing EGFR mutation levels were detected in all patients benefiting from locoregional radiotherapy, whereas the opposite occurred when a patient progressed soon after radiotherapy treatment. Similarly, increasing EGFR mutation levels anticipated disease progression after TKI dose reduction, discontinuation of treatment, or reduced bioavailability due to drug interactions. In addition, EGFR mutation levels were useful to monitor treatment outcome of new therapies and constituted a decisive factor when the clinical situation of the patient did not correlate with responses ascertained by radiologist. Finally, our results indicate that cancer associated body fluids (pleural, pericardial or cerebrospinal fluid) are certainly a suitable source for biomarker testing that can extend EGFR mutation detection to biofluids other than blood.Materials and MethodsA total of 180 serial plasma samples from 18 non-small-cell lung cancer patients who carried an activating EGFR mutation were investigated by digital PCR.ConclusionsMonitoring levels of EGFR mutation in plasma allows resolving doubts that frequently arise in daily clinical practice and constitutes a major step towards achieving personalized medicine.

Complete clearance of plasma EGFR mutations as a predictor of outcome on osimertinib in the AURA trial.

9018 Background: Osimertinib, an EGFR T790M-selective tyrosine kinase inhibitor (TKI), provides high and durable response rates in patients (pts) with T790M positive advanced NSCLC whose disease has progressed following EGFR-TKI therapy. We investigated whether changes in the levels of plasma EGFRmutations post-osimertinib treatment are associated with clinical outcome. Methods: We studied pts from the AURA Phase I study (NCT01802632) with acquired resistance to EGFR-TKIs, T790M positive baseline genotyping (in tumor or plasma), and plasma samples collected at baseline and 6 weeks (wks) post- osimertinib (20–240 mg daily) treatment. Plasma samples were analyzed for the presence of detectable EGFR mutations (Ex19del, L858R and T790M) using BEAMing digital PCR (Sysmex/Inostics). In pts with detectable plasma EGFR mutations at baseline, clinical outcomes (by investigator, per RECIST 1.1) were compared for pts with or without detectable plasma EGFRmutations at 6 wks. Results: Evaluable baseline and 6 wk plasma samples were collected from 160 pts with T790M positive genotyping, of whom 143 had detectable EGFR mutations at baseline (median allelic fraction for Ex19del: 7.09%; L858R: 3.81%; T790M: 2.12%). In this cohort, the overall median progression-free survival (mPFS) was 9.3 months (m; 95% CI 8.2, 9.7). Clearance of plasma EGFRmutations at 6 wks was seen in 92/143 (64%) pts. mPFS was longer in pts with plasma clearance (10.9 m; 95% CI 9.5, 15.2) compared with pts without (5.5 m; 95% CI 3.9, 6.7), as was objective response rate (ORR; 70%; 95% CI 59%, 79% vs. 35%; 95% CI 22%, 50%). Conclusions: Clearance of plasma EGFR mutations after 6 wks of osimertinib therapy appears to be associated with improved ORR and mPFS in pts with T790M positive NSCLC. Evidence or lack of such a “plasma response” measured at 6 wks could, potentially, be used to predict subsequent outcomes on therapy. Further research is needed to better understand whether continued detection of plasma EGFR mutations at 6 wks may indicate the presence of heterogeneous resistance mechanisms, which could, potentially, be targeted by combination therapies. Validation of these results in an independent cohort of pts is ongoing. Clinical trial information: NCT02228369.

Predictive impact of complete molecular response in plasma: A phase II, liquid biopsy study in EGFR mutated NSCLC patients treated with afatinib (WJOG 8114LTR).

9027 Background: Liquid biopsy has been approved as an alternative method to detect clinically relevant EGFR mutations in NSCLC. However, it is unknown whether the early assessment of molecular response in plasma could be a surrogate of clinical response and/or longer efficacy. Here, we conducted a prospective, multi-institutional study of liquid biopsy in EGFRmutated NSCLC patients (WJOG 8114LTR). Methods: Chemotherapy naïve, advanced NSCLC patients with EGFR-sensitizing mutation received afatinib monotherapy (40 mg/body) until progressive disease (PD) or unacceptable toxicity. Plasma DNA was obtained from patients at baseline, weeks 2, 4, 8, 12, 24, 48, and at PD. Three types of clinically relevant EGFRmutations (exon 19 deletion, exon 20 T790M and exon 21 L858R) will be analyzed using plasma DNA with multiplexed, pico-droplet digital PCR assay (RainDrop system, RainDance Technologies, Billerica, MA). Complete molecular response (CMR) was defined as mutant allele event/frequency of exon 19 deletion or exon 21 L858R below the cutoff for the positivity by digital PCR in plasma. This study was registered at UMIN (ID: 000015847). Results: Fifty-seven patients were registered in the study. Efficacy of afatinib was comparable to previous reports (overall response rate: 78.6%, and median progression-free survival (mPFS): 14.2 months). At baseline, 62.5% of patients were positive for EGFR mutation in plasma. Plasma positive patients had slightly shorter PFS than plasma negative patients, but not significant (p = 0.24, log-rank). Among those who were positive at baseline, CMR was achieved in 60.6% of patients at two weeks, and 87.5% at four weeks, respectively. Patients with CMR at two weeks had significantly longer PFS than those without one (13.6 versus 7.5 months, p = 0.0001). Patients with CMR at four weeks also demonstrated longer PFS than those without one (13.6 versus 5.1 months, p &lt; 0.0001). Among patients who achieved CMR by week four, time to achieve CMR did not affect PFS ( p= 0.59). Conclusions: This is the first report on the predictive impact of early CMR in plasma for longer therapeutic efficacy of afatinib in EGFR mutated NSCLC patients. Clinical trial information: 000015847.

MA08.09 Monitoring Plasma EGFR Mutations during First Line Treatment with EGFR TKIs in NSCLC Patients

Genotyping cell free circulating DNA (cfDNA) is a non-invasive method of detecting EGFR mutations (EGFRmu) in plasma and may provide an option to identify patients who progress while treated with EGFR TKIs. The aim of our study was to monitor plasma EGFRmu and identify dynamic case specific changes in plasma EGFRmu during routine treatment of advanced EGFRmu NSCLC patients. Plasma was collected from patients with advanced EGFRmu NSCLC treated with first- or second-generation EGFR TKIs. Plasma EGFRmu were dynamically monitored consecutively at every scheduled visit. Cobas EGFR Mutation Test v1 and v2 (Roche, USA) was used to detect 42 mutations at EGFR gene in exons 18 to 21. Liquid biopsy progression (LBP) was determined as reappearance of EGFRmu in plasma after negativization during treatment or increase of EGFRmu levels expressed by semi-quantitative index (SQI). Radiologic progression was determined in accordance with RECIST1.1 criteria. From May 2014, 23 patients were treated with EGFR TKIs for advanced EGFRmu NSCLC; 20/23 had detectable activating mutations in plasma before any treatment and were therefore included in our analysis. Dynamic changes of plasma EGFRmu during 1st line EGFR TKI treatment are shown in Figure 1. Eight patients (40%) experienced RECIST 1.1 progression while on treatment, whereas one patient was inevaluable. In 4/8 patients (50%) LBP appeared at the same time as radiologic progression, in 3/8 patients (37%) LBP appeared before radiologic progression (8w, 14w, 20w before, respectively) and in 1 patient (12%) radiologic progression appeared 6w before LBP. Among patients who did not experience radiologic progression yet, some dynamic changes in cfDNA were also observed, but alterations in the SQI values were much smaller. Monitoring EGFR mutations in plasma is a feasible and less invasive method in routine clinical practice and could be used as a predictive marker of progression on treatment with EGFR TKIs.

P1.02-008 2-Year Single Institution Experience with EGFR Plasma Testing in Advanced NSCLC

Lung adenocarcinoma patients in advanced stage of disease that harbor EGFR sensitizing mutations are eligible for treatment with tyrosine kinase inhibitors (TKI) due to a high likelihood of response. Most patients will ultimately develop resistance at disease progression. The T790M mutation is a dominant resistance mechanism to TKI. EGFR plasma testing enables non-invasive monitoring and detection of T790M. We followed patients with EGFR sensitizing mutations by measuring EGFR mutations in plasma during TKI treatment. We analyzed patients who were diagnosed lung adenocarcinoma stage IV, detected EGFR sensitizing mutations in tumor tissue samples and treated with TKI at University Clinic Golnik. We collected baseline plasma samples prior to TKI treatment and consecutive plasma samples at different time intervals after initiation of therapy. At the beginning, two separate tests, cobas® EGFR Mutation Test for tissue (CE-IVD) and plasma (under development) were used, and since October 2015 one test for tissue and plasma, cobas® EGFR Mutation Test v2 (Roche, Pleasanton, CA, USA) is used. Detected EGFR mutations in plasma samples were expressed as semi-quantitative index (SQI) which reflects a proportion of mutated versus wild-type copies of the EGFR gene. During 2-year period we collected 414 peripheral blood samples from 63 patients and performed 619 EGFR plasma tests. There are 25 patients with baseline and serial follow-up EGFR plasma tests, 16 patients with only serial follow-up EGFR tests since they started with TKI treatment before EGFR plasma testing was available, 5 patients are included in adjuvant setting, and 17 patients had no monitoring due to various reasons. Maximum number of EGFR plasma tests done per patient was 27 at 20 time-points. When introducing EGFR plasma testing, we prepared two aliquots of plasma out of 10 ml blood sample in EDTA-tubes and run test for both aliquots. Results of reproducibility study showed 95% concordance rate between both aliquots and thus we modified protocol to run the second aliquot only if the first one was negative. At disease progression, reappearance of EGFR sensitizing mutations with increasing SQI levels was detected. In 14 patients who progressed we detected T790M mutation, in 10 of them during monitoring TKI treatment. We also observed daily variation in EGFR mutation levels in the plasma. These data support the value of EGFR plasma testing to monitor the patient's response to TKI and detect T790M resistance mutation prior to clinical progression in a routine clinical setting.