Epidermal Growth Factor Receptor Mutations In Plasma Research Articles

Clinical validation of a highly sensitive assay to detect EGFR mutations in plasma cell-free DNA from patients with advanced lung adenocarcinoma.

BackgroundCirculating tumor DNA (ctDNA) is a promising biomarker for noninvasive epidermal growth factor receptor (EGFR) mutations detection in lung cancer patients, but the existing methods have limitations in sensitivity or in availability. In this study, we evaluated the performance of a novel assay called ADx-SuperARMS in detecting EGFR mutations in plasma cell-free DNA from patients with advanced lung adenocarcinoma.MethodsA total of 109 patients with metastatic advanced adenocarcinoma were recruited who provided both blood samples and matched tumor tissue samples. EGFR mutation status in plasma samples were tested with ADx-SuperARMS EGFR assay and tumor tissue samples were tested with ADx-ARMS EGFR assay. The clinical sensitivity, specificity, positive prediction value (PPV), and negative prediction value (NPV) of ADx-SuperARMS EGFR assay were calculated by using EGFR mutation status in tumor tissue as standard reference. A receiver operating characteristic (ROC) analysis was implemented and an area under the curve (AUC) was calculated to evaluate sensitivity and specificity of exon 19 deletion (E19Del) and L858R mutation detection. The objective response rate (ORR) were calculated according to the EGFR mutation status determined by ADx-superARMS as well.Results0.2% analytical sensitivity and 100% specificity of the ADx-SuperARMS EGFR assays for EGFR E19Del, L858R, and T790M mutants were confirmed by using a series of diluted cell line DNA. In the clinical study, EGFR mutations were detected in 45.9% (50/109) of the plasma samples and in 56.9% (62/109) of the matched tumor tissue samples. The sensitivity, specificity, PPV and NPV of the ADx-SuperARMS EGFR assay for plasma EGFR mutation detection were 82.0% (50/61), 100% (48/48), 100% (50/50), and 81.4% (48/59), respectively. In ROC analysis, ADx-SuperARMS achieved sensitivity and specificity of 88% and 99% in E19Dels as well as sensitivity and specificity of 89% and 100% in L858R, respectively. Among the 35 patients who were plasma EGFR mutation positive and treated with first generation of EGFR-tyrosine kinase inhibitors (TKIs), 23 (65.7%) achieved partial response, 11 (31.4%) sustained disease, and 1 (2.9%) progressive disease. The ORR and disease control rate (DCR) were 65.7% and 97.1%, respectively.ConclusionsADx-SuperARMS EGFR assay is likely to be a highly sensitive and specific method to noninvasively detect plasma EGFR mutations of patients with advanced lung adenocarcinoma. The EGFR mutations detected by ADx-SuperARMS EGFR assay could predict the efficacy of the treatment with first generation of EGFR-TKIs. Hence, EGFR blood testing with ADx-SuperARMS could address the unmet clinical needs.

Dynamics of EGFR mutations in plasma recapitulates the clinical response to EGFR-TKIs in NSCLC patients.

ObjectivesGenomic profiling using plasma cell-free DNA (cfDNA) represents a non-invasive alternative to tumor re-biopsy, which is challenging in clinical practice. The feasibility of dynamically monitoring epidermal growth factor receptor (EGFR) mutation status using serial plasma samples from non-small cell lung cancer (NSCLC) patients treated by tyrosine kinase inhibitors (TKIs) and its application in tracking clinical response and detection of resistance were investigated.Patients and methodsForty-five NSCLC patients with EGFR mutation-positive pre-TKI plasma and at least two post-TKI plasma collections were recruited to this study. EGFR mutations including L858R, exon 19 deletion (19-del) and T790M were analyzed using droplet digital PCR (ddPCR) in longitudinally collected plasma samples.ResultsWe observed a significant reduction in plasma EGFR mutation abundance during the first two-month of TKI treatment. Acquiring of secondary T790M gatekeeper mutation or completed “loss” of EGFR mutations represented two major categories of resistance profiles. Moreover, we demonstrated that levels of plasma EGFR mutations highly correlated with changes of tumor diameter as determined by radiographic imaging, or development of new lesions. In a subset of patients, we further showed that reappearance of EGFR mutations could be detected in plasma up to 5 months ahead of progressive disease (PD), suggesting an early detection of drug resistance.ConclusionsOur findings suggest that genomic analysis using plasma cfDNA may offer an effective approach to monitor clinical response and emergence of resistance.

Detection of EGFR mutation in plasma using multiplex allele-specific PCR (MAS-PCR) and surface enhanced Raman spectroscopy

In this study, surface enhanced Raman spectroscopy (SERS) in combination with multiplexed polymerase chain reaction (PCR) was utilized to detect mutations of exons 19 and 21 of the epidermal growth factor receptor (EGFR) gene. Through the use of multiplexed PCR, the two mutation types were amplified in a single reaction. SERS was used on the PCR products to detect mutations. DNA mixtures with increasing mutation percentages showed good linear relationship between mutation rates and peak height. Then, this PCR-SERS method was used on the plasma of 48 patients with non-small cell lung cancer (NSCLC) to detect EGFR mutations. Analysis of variance (ANOVA) and receiver operating characteristic (ROC) analysis revealed that the peak height ratios were significant for identifying different mutation types. The specificity, sensitivity and accuracy obtained were all 100%. The proposed method was then validated through comparison with high resolution melting (HRM) and showed high concordance with HRM (Pearson correlation is 0.92). Finally, logistic regression was performed on EGFR mutation status and the clinical features of the 48 patients. Our study indicates that PCR-SERS is an effective, noninvasive, and economical method for the detection and monitoring of EGFR mutations in the plasma of patients with NSCLC.

Association between clinical characteristics and the diagnostic accuracy of circulating single-molecule amplification and resequencing technology on detection epidermal growth factor receptor mutation status in plasma of lung adenocarcinoma.

Lung cancer is the leading cause of cancer-related mortality in the world. Circulating single-molecule amplification and resequencing technology (cSMART) can successfully detect epidermal growth factor receptor (EGFR) mutation in non-small cell lung cancer (NSCLC). However, few studies have investigated the association between clinical characteristics and the diagnostic accuracy of cSMART technique in lung adenocarcinoma. We enrolled 95 patients, which included paraffin embedded tumor tissues and matched plasma samples. Retrospectively analyzed the correlation between clinical characteristics and sensitivity, specificity, accuracy, positive predictive value (PPV) and negative predictive value (NPV) of cSMART. Of the 95 lung adenocarcinoma cancer patients, 49 (51.5%) and 40 (42.1%) harbored EGFR mutations respectively in tissue and plasma. In younger than 60years group, sensitivity, specificity and consistency for cSMART were 81.0%, 100%, and 90.9% (P<.001). In metastasis group, sensitivity, specificity, and consistency for cSMART were 92.9%, 77.8%, and 87.0% (P=.001). By univariate analysis, younger than 60years (OR=5.938; 95% confidence interval: 1.835-19.210; P=.001); metastasis group (OR=4.482; 95% confidence interval: 1.432-14.024; P=.007) were significantly correlated with a higher accuracy. By multivariate analysis, younger than 60years (P=.003) and metastasis (P=.004) were confirmed as independent factors for diagnostic accuracy of EGFR mutation in plasma through cSMART. cSMART is feasible for detection EGFR mutation in plasma when tissue is unavailable. Age and metastasis might be considered as independent factors in diagnostic accuracy of cSMART in lung adenocarcinoma.

Association of mutant EGFR L858R and exon 19 concentration in circulating cell-free DNA using droplet digital PCR with response to EGFR-TKIs in NSCLC.

The present study aimed to determine the diagnostic concordance of plasma epidermal growth factor receptor (EGFR) mutation using droplet digital polymerase chain reaction (ddPCR) with tumor tissue samples and the predictive clinical significance of plasma EGFR mutation concentration. Plasma DNA samples from patients with non-small cell lung cancer (NSCLC) were analyzed for EGFR exon 21 codon 858 (L858R) mutation, deletion of exon 19 (ex19del) and exon 20 codon 790 (T790M) mutation using ddPCR. Firstly, the mutations in the plasma samples were compared with the matched tumor samples to determine the concordance. Secondly, image examination follow-ups were analyzed to assess the association between plasma EGFR mutation concentration and patients' response to EGFR-tyrosine kinase inhibitors (TKIs). A total of 51 patients with NSCLC were enrolled, including 48 newly diagnosed patients. Compared with tumor tissue samples, the sensitivity and specificity of ddPCR were 76.19% (16/21) and 96.55% (28/29) for mutant L858R, and 88.89% (8/9) and 100% (41/41) for ex19del, respectively. No patient exhibited the T790M mutation in the tumor tissue or plasma samples. Furthermore, 5 patients with the L858R mutation and 4 patients with ex19del in plasma and tumor tissue samples had been followed up with image examination for ≥3 months following EGFR-TKI treatment. The baseline mutant EGFR concentrations were positively correlated with a reduction in tumor burden (Spearman's r=0.7000, P=0.0358). When analyzed separately, ex19del concentrations (Spearman's r=1.0000, P<0.0001) were also positively correlated with the reduction, while mutant L858R concentrations were not (Spearman's r=0.7000, P=0.1881). In the present study, detection of plasma EGFR mutations using ddPCR exhibited sufficient concordance with tumor tissue sample results. Baseline plasma mutant EGFR and ex19del concentrations were significantly and positively correlated with response to EGFR-TKIs.

Assessment of EGFR mutations in plasma from advanced NSCLC patients by modified cell-free DNA extraction method and blocker PCR assays.

e23115 Background: Epidermal growth factor receptor (EGFR) mutation testing in plasma cell-free DNA (cfDNA) for lung cancer patients is an emerging clinical tool.However,the sensitivity of plasma testing,compared with tissue testing, is not high enough. We aimed to investigate the likelihood of improving the plasma testing sensitivity by increase the cell-free DNA(cfDNA )yields using a modified extraction method and blocker PCR. Methods: Blood samples were collected from 106 patients without EGFR-TKI treatment and 127 patients with acquired EGFR-TKI resistance.CfDNA was extracted from plasma by the modified extraction method and mutations were identified by blocker PCR.The yields of circulating tumor DNA (ctDNA) and cfDNA from 106 patients were quantified by standard curve.Next generation sequencing (NGS)was employed to verify the reliability of blocker PCR. Meanwhile,paired tumor tissues were examined by amplification refractory mutation system (ARMS). Results: The novel extraction method can extract small fragment less than 100bp.The yield of total cfDNA of 106 plasma was mostly between4 000and 200,000 copies per milliliter.With regard to exon 19 deletions(E19-DELs) , the ctDNA proportion of 85% samples (17/20)was from 0.1% to 5% . As for L858R mutations, the fraction of ctDNA of 84.6%(11/13)cases was calculated with the range of 0.01% to 0.1%.For 106 patients,the overall concordance rate,sensitivity and specificity between plasma and tumor tissue was 99.06%[k = 0.97; 95% confidence interval (CI), 0.87-0.99; P &lt; 0.0001],94.87%(95% CI, 83.11%-98.58%) and 100 % (95% CI,94.58%-100%).Among the 127patients,40.16%(51/127)T790M was detected in the plasma, 78.44%(40/51) of which coexists with an EGFR activating mutation. Conclusions: The modified extraction method can improve ctDNA yields and thus improve the sensitivity of liquid biopsy.The blocker PCR offers the possibility of detecting mutations in cfDNA with high concordance with tumor tissues.

Detection of activating and acquired resistant mutation in plasma from EGFR-mutated NSCLC patients by peptide nucleic acid (PNA) clamping-assisted fluorescence melting curve analysis.

This study was designed to prospectively examine whether peptide nucleic acid clamping-assisted fluorescence melting curve analysis (PANAMutyper™) is feasible for the detection of activating and acquired resistant epidermal growth factor receptor (EGFR) mutation in plasma. Patients with non-small cell lung cancer harboring activating EGFR mutations who were scheduled to undergo EGFR-tyrosine kinase inhibitors (EGFR-TKIs) were enrolled between September 2011 and March 2015. A total of 102 patients with EGFR-mutated lung cancer were enrolled, 53 had available plasma samples at disease progression, and 28 underwent serial plasma sampling during EGFR-TKI treatment. EGFR-TKI-sensitizing and T790M mutations were detected in the plasma of 68.6% (70/102) at baseline and 30.2% (16/53) at disease progression, respectively. The concordance rates for matched tissue and plasma samples were 80.4% and 90.2% for E19del and L858R mutations at baseline and 56.3% for T790M mutation at disease progression. The sustained presence of plasma EGFR mutations four weeks after EGFR-TKI predicted a poor objective response rate (30.0% vs. 87.5%, P = 0.025), as well as worse progression-free survival (hazard ratio [HR], 4.381) and overall survival (HR, 5.475). Longitudinal analysis could detect T790M mutations earlier than disease progression based on imaging study (median time from appearance of T790M in plasma samples to progression at imaging scan, 103 days). In conclusion, PANAMutyper™ is reliable for detecting activating and acquired resistant EGFR mutation in plasma, and predicts responses to EGFR-TKI via longitudinal monitoring of EGFR mutation during treatment.

Estimation of cell-free circulating EGFR mutation concentration predicts outcomes in NSCLC patients treated with EGFR-TKIs.

Detection of circulating tumor DNA using droplet digital polymerase chain reaction (ddPCR) is a highly-sensitive, minimally invasive alternative to serial biopsies for assessment and management of cancer. We used ddPCR to assess the utility of measuring plasma concentrations of common epidermal growth factor receptor (EGFR) mutations (L858R, exon 19 deletion, and T790M) in 57 non-small cell lung cancer (NSCLC) patients treated with EGFR tyrosine kinase inhibitors (EGFR-TKIs). High baseline plasma EGFR mutation (pEGFRmut) concentrations were associated with shorter progression-free survival (8.43 months) than low baseline pEGFRmut (16.23 months; p = 0.0019). By contrast, there were no differences in tumor shrinkage or overall survival between groups. During EGFR-TKI treatment, pEGFRmut levels decreased to zero in 89.58% of patients. Twenty-five of the 27 patients who progressed had basal pEGFRmut, and 18 also had circulating T790M. All 20 patients with dramatic progression (according to a categorization system for EGFR-TKIs failure) had basal pEGFRmut, and 13 had T790M mutation at progression. These results support the use of ddPCR for analysis of plasma EGFR mutations for prediction of PFS and to monitor clinical responses to EGFR-TKIs in NSCLC patients.

JCES01.04 Liquid Biopsy in Monitoring Dynamic Changes of Driver Genes in Advanced NSCLC

JCES01.04 Liquid Biopsy in Monitoring Dynamic Changes of Driver Genes in Advanced NSCLC

P3.02b-018 Detection of Epidermal Growth Factor Receptor Mutations in Circulating Cell-Free DNA versus Tumor Biopsy: Topic: EGFR Biomarkers

Epidermal growth factor receptor (EGFR) mutations are predictive marker of EGFR-tyrosine kinase inhibitor (TKI) therapy. We compared the sensitivity of EGFR mutation detection techniques between matched tumor tissue and peripheral blood sample in patients with lung adenocarcinoma. We collected the paired samples from plasma and paraffin-embedded tumor tissue in 202 patients before EGFR-TKIs. DNA extraction was performed using the QIAamp MinElute virus spin kit and EGFR mutation analysis was done by two detection methods. One is the PNAClampTM which is the PNA-based PCR clamping that selectively amplifies only the mutated target DNA sequence as minor portion in mixture with the major wild type DNA sequences. The other is the PANAMutyperTM EGFR kit, which use PNA clamping-assisted fluorescence melting curve analysis to perform mutation detection and genotyping. The degree of agreement was evaluated by Cohen's kappa value. The EGFR mutation rates by PANAMutyperTMR and PNAClampTM were 51.0% vs. 47.0% in tissue, and 22.2% vs. 11.4% in plasma sample, respectively. Overall concordance rates and the degree of agreement between tissue and plasma samples was better in PANAMutyperTMR (69.3%, k=0.393, p<0.001) than PNAClampTM (62.3%, k=0.211, p<0.001). Sensitivity of plasma EGFR mutations was higher (41.7% vs. 22.1%, p<0.001) and false negative rate was lower in PANAMutyperTMR test (58.3% vs. 77.9%, p<0.001). Response to EGFR-TKI was correlated with plasma EGFR mutation by PANAMutyperTMR (p=0.025) unlike PNAClampTM (p=0.829). The sensitivity and concordance rate of PANAMutyperTMR test were better than standard PNAClampTM test. So this technique can be useful to detect EGFR mutation in circulating cell-free DNA sample.

Comparison of plasma and tissue samples in epidermal growth factor receptor mutation by ARMS in advanced non-small cell lung cancer

ObjectiveThe aim of this study was to assess the effectiveness and accuracy of blood-based circulating-free tumor DNA on testing epidermal growth factor receptor (EGFR) gene mutations. MethodsIn total, 219 non-small cell lung cancer patients in stages III-IV were enrolled into this study. All patients had tissue samples and matched plasma DNA samples. EGFR gene mutations were detected by the Amplification Refractory Mutation System (ARMS). We compared the mutations in tumor tissue samples with matched plasma samples and determined the correlation between EGFR mutation status and clinical pathologic characteristics. ResultThe overall concordance rate of EGFR mutation status between the 219 matched plasma and tissue samples was 82% (179/219). The sensitivity and specificity for the ARMS EGFR mutation test in the plasma compared with tumor tissue were 60% (54/90) and 97% (125/129), respectively. The positive predictive value was 93% (54/58) and the negative predictive value was 78% (125/161). The median overall survival was longer for those with EGFR mutations than for those without EGFR mutations both in tissue samples (23.98 vs. 12.16months; P<0.001) and in plasma (19.96 vs. 13.63months; P=0.009). For the 68 patients treated with EGFR- tyrosine kinase inhibitors (TKIs), the median progression-free survival (PFS) was significantly prolonged in the EGFR mutant group compared to the non-mutation group in tumor tissue samples (12.26months vs. 2.40months, P<0.001). In plasma samples, the PFS of the mutant group was longer than that of the non-mutant group. However, there was no significant difference between the two groups (10.88months vs. 9.89months, P=0.411). ConclusionsThe detection of EGFR mutations in plasma using ARMS is relatively sensitive and highly specific. However, EGFR mutation status tested by ARMS in plasma cannot replace a tumor tissue biopsy. Positive EGFR mutation results detected in plasma are fairly reliable, but negative results are hampered by a high rate of false negatives.

EGFR mutation status in plasma and tumor tissues in non-small cell lung cancer serves as a predictor of response to EGFR-TKI treatment

ABSTRACTObjective: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) can effectively control non-small cell lung cancer (NSCLC). Therefore, EGFR mutations should be detected before lung cancer patients undergo EGFR-TKI therapy. This study assessed the feasibility and predictive value of EGFR mutations in peripheral blood samples.Methods: EGFR mutations in exons 19 and 21 were analyzed in tumor tissue and plasma DNA samples from 121 NSCLC patients using amplification refractory mutation system (ARMS) and the integrated technique of mutant enriched PCR (me-PCR) and denaturing high performance liquid chromatography (DHPLC), respectively.Results: EGFR mutations were detected in 36.4% of tumor tissues and 34.7% of the plasma at a concordance rate of 85.1% (103/121). The sensitivity and specificity of plasma EGFR mutations were 77.3% and 89.6%, respectively. The gender and tumor histology of patients served as independent predictors of EGFR mutations in both tumor tissues and plasma, while CEA level was an independent predictor of EGFR mutations in the plasma. Furthermore, EGFR-TKI treatment showed a significantly higher objective response rate (ORR), median progression-free survival (mPFS), and overall survival (mOS) in patients harboring EGFR mutation than those that did not exhibit EGFR mutation (ORR: 69.4% versus 13.0% in tissues, P < 0.001; 64.5 % vs. 28.6% in the plasma, P = 0.006. mPFS: 10.4 months versus 4.1 months in tissues, P<0.001; 10.5 months vs. 5.2 months in the plasma, P=0.001. mOS: 25.7 months versus 8.3 months in tissues, P=0.005; 25.7 months vs. 13.5 months in the plasma, P=0.038).Conclusions: EGFR mutations can be detected in the plasma using the integrated technique of me-PCR and DHPLC, which enables us to predict patient response to EGFR-TKI therapy. High serum CEA levels served as an independent predictor for plasma EGFR mutations.

Detection of epidermal growth factor receptor mutation in plasma as a biomarker in Chinese patients with early-stage non-small cell lung cancer.

PurposeThis preplanned exploratory analysis was conducted to reveal the true status of correlation between tissue and plasma detection for early-stage non-small cell lung cancer (NSCLC) epidermal growth factor receptor (EGFR) mutations, knowing that specific subgroups of NSCLC patients may be potential candidates for EGFR mutation analysis by using plasma samples.Materials and methodsTissue samples were surgically resected from 198 patients with stage I–IV NSCLC, where stage IA to IIIA accounted for 92.4%. EGFR mutations in all these tissues were positive. Paired plasma EGFR mutations were detected by real-time polymerase chain reaction; concentration of cell-free DNA (cfDNA) in plasma was measured by ultraviolet spectrophotometry.ResultsEGFR-activating mutation was detected in 34 plasma samples, and their mutation types were matched with that in tissue. The sensitivity of EGFR mutation for the 198 paired tissue and plasma samples was 17.2%. The sensitivity positively correlated with disease stage and negatively correlated with tumor differentiation. The sensitivity of stage IA, IB, IIA, IIB, and IIIA was 1.6%, 7.9%, 11.1%, 20%, and 33.3%, respectively; the sensitivity of high differentiation was 0% versus 36.8% for poor differentiation. There was no correlation between plasma cfDNA concentration and patient characteristics.ConclusionWe recommend using plasma cfDNA as a biomarker in stage IIIA or poorly differentiated tumors for gene diagnosis, especially in patients whose tissue samples cannot be obtained by surgery. Plasma samples can really reflect the patients’ EGFR mutation types and may contain comprehensive genotypic information that comes from different parts of the tumor than tissue specimens. The concentration of plasma cfDNA does not vary with patient characteristics.

Early Prediction of Response to Tyrosine Kinase Inhibitors by Quantification of EGFR Mutations in Plasma of NSCLC Patients

The potential to accurately quantify epidermal growth factor receptor (EGFR) mutations in plasma from non-small-cell lung cancer patients would enable more rapid and more frequent analyses to assess disease status; however, the utility of such analyses for clinical purposes has only recently started to explore. Plasma samples were obtained from 69 patients with EGFR-mutated tumors and 21 negative control cases. EGFR mutations in plasma were analyzed by a standardized allele-specific polymerase chain reaction (PCR) test and ultra-deep next-generation sequencing (NGS). A semiquantitative index (SQI) was derived from dilutions of known EGFR mutation copy numbers. Clinical responses were evaluated by Response Evaluation Criteria in Solid Tumors 1.1 criteria and expressed as percent tumor shrinkage. The sensitivity and specificity of the PCR test and NGS assay in plasma versus tissue were 72% versus 100% and 74% versus 100%, respectively. Quantitative indices by the PCR test and NGS were significantly correlated (p < 0.001). EGFR testing at baseline and serially at 4 to 60 days during tyrosine kinase inhibitor therapy revealed a progressive decrease in SQI, starting from day 4, in 95% of cases. The rate of SQI decrease correlated with percent tumor shrinkage at 2 months (p < 0.0001); at 14 days, it was more than 50% in 70% of patients (rapid responders). In two patients with slow response, an early increase in the circulating levels of the T790M mutation was observed. No early T790M mutations were seen in plasma samples of rapid responders. Quantification of EGFR mutations from plasma with a standardized PCR test is feasible. To our knowledge, this is the first study showing a strong correlation between the EGFR SQI in the first days of treatment and clinical response with relevant implications for patient management.

Plasma EGFR Mutation Detection Associated With Survival Outcomes in Advanced-Stage Lung Cancer

BackgroundNoninvasive detection of epidermal growth factor receptor (EGFR) mutation in plasma is feasible and could be adjunct for therapeutic monitoring especially when repeated biopsy of tumor tissue is challenging. The aims of this study were to establish the diagnostic performance of peptide nucleic acid-locked nucleic acid polymerase chain reaction followed by custom array for plasma EGFR mutation and to evaluate the association of detection with clinical characteristics and survival outcomes. Materials and MethodsPlasma genomic DNA from consecutive advanced lung cancer subjects was tested for EGFR mutations before anticancer treatment, and compared with mutation status in tumor tissue. Clinical characteristics were compared between patients who were EGFR-mutant and wild type; and within EGFR mutants, whether EGFR mutations could be detected in plasma. ResultsIn 74 lung cancer patients, the sensitivity, specificity, and positive and negative predictive values of plasma EGFR detection were 79.1%, 96.8%, 97.1%, and 76.9%, respectively. EGFR mutants with concomitant detection of plasma EGFR mutation showed worse survival compared with mutants with no concomitant plasma mutation detected in biopsy specimens. ConclusionPlasma EGFR mutation detected using this method demonstrated high diagnostic performance. In EGFR mutants, plasma EGFR mutation detection correlated not only EGFR mutation status in biopsy but was also associated with worse prognosis compared with EGFR mutant without plasma EGFR mutation detection.

Dynamic Plasma EGFR Mutation Status as a Predictor of EGFR-TKI Efficacy in Patients with EGFR-Mutant Lung Adenocarcinoma

Epidermal growth factor receptor (EGFR) mutation status in lung cancer can effectively predict EGFR-tyrosine kinase inhibitor (TKI) efficacy. We evaluated the role of dynamic plasma cell-free DNA EGFR mutation status in outcome prediction. Advanced lung adenocarcinoma patients were enrolled and prospectively observed for outcomes of EGFR-TKI treatment. Peptide nucleic acid-zip nucleic acid polymerase chain reaction clamp method was developed to assess EGFR mutations in matched tumor and serial plasma cell-free DNA specimens. A total of 72 patients were enrolled in this study, of which 62 patients (86.1%) had EGFR-mutant tumors (34 patients with exon 19 deletions, and 28 patients with L858R). Pretreatment plasma used for EGFR mutation testing showed a sensitivity of 59.7% and a specificity of 100%. Detection sensitivity was significantly higher in stage IV-M1b patients compared with stage IIIb and IV-M1a patients (78.0% versus 23.8%, p < 0.001). All patients who presented with EGFR-mutant tumors received first-line EGFR-TKI therapy. The objective response rate and disease control rate were 74.2% and 82.3%, respectively. Median progression-free survival and overall survival were 8.8 months (95% CI: 6.6-11.0) and 20.5 months (95% CI 15.1-26.0), respectively. Failure to clear plasma EGFR mutations after EGFR-TKI treatment was an independent predictor of lower disease control rate (odds ratio 5.26 [95% CI: 1.13-24.44]; p = 0.034), shorter progression-free survival (hazard ratio: 1.97 [95% CI: 1.33-2.91]; p = 0.001), and shorter overall survival (hazard ratio: 1.82 [95% CI: 1.04-3.18], p = 0.036). Changes in plasma EGFR mutation status can be successfully assessed using the peptide nucleic acid-zip nucleic acid polymerase chain reaction clamp method and can serve as an independent outcome predictor.

Can EGFR mutations in plasma or serum be predictive markers of non-small-cell lung cancer? A meta-analysis.

The detection of epidermal growth factor receptor (EGFR) mutations in plasma or serum has previously been reported to be feasible for non-small-cell lung cancer (NSCLC). However, not all results indicate a consistency between EGFR mutation status in the plasma or serum and that in tissues. A meta-analysis was performed to evaluate the overall accuracy of EGFR mutation detection in plasma or serum. Publications up to December 2014 were searched for using the PubMed, Embase and Web of Science databases. Sensitivity, specificity and other accuracy measures were pooled using the bivariate mixed-effects regression model. Twenty-six studies were included in this meta-analysis. The pooled specificity, sensitivity, positive and negative likelihood ratios, and diagnostic odds ratios were 0.97 (95% confidence interval (CI): 0.93-0.99), 0.65 (95% CI: 0.54-0.74), 24.9 (95% CI: 9.2-67.2), 0.36 (95% CI: 0.27-0.48), and 69 (95% CI: 24-202), respectively. The summary receiver operating characteristic curve was 0.89 (95% CI: 0.86-0.91). The detection of EGFR mutations in plasma or serum is a noninvasive method to confirm EGFR mutation status in patients with NSCLC. However, more work is necessary to identify which method can raise the sensitivity of EGFR mutation detection.

Highly Sensitive Droplet Digital PCR Method for Detection of EGFR-Activating Mutations in Plasma Cell–Free DNA from Patients with Advanced Non–Small Cell Lung Cancer

Epidermal growth factor receptor (EGFR) mutation testing in plasma cell-free DNA from lung cancer patients is an emerging clinical tool. However, compared with tissue testing, the sensitivity of plasma testing is not yet satisfactory because of the highly fragmented nature of plasma cell-free DNA, low fraction of tumor DNA, and limitations of available detection technologies. We therefore developed a highly sensitive and specific droplet digital PCR method for plasma EGFR mutation (exon19 deletions and L858R) testing. Plasma from 86 EGFR-tyrosine kinase inhibitor-naive lung cancer patients was tested and compared with EGFR mutation status of matched tumor tissues tested by amplification refractory mutation system. By using EGFR mutation-positive cell DNA, we optimized the droplet digital PCR assays to reach 0.04% sensitivity. The plasma testing sensitivity and specificity, compared with the matched tumor tissues tested by amplification refractory mutation system, were 81.82% (95% CI, 59.72%-94.81%) and 98.44% (95% CI, 91.60%-99.96%), respectively, for exon19 deletions, with 94.19% concordance rate (κ=0.840; 95% CI, 0.704-0.976; P<0.0001), whereas they were 80.00% (95% CI, 51.91%-95.67%) and 95.77% (95% CI, 88.14%-99.12%), respectively, for L858R, with 93.02% concordance rate (κ=0.758; 95% CI, 0.571-0.945; P<0.0001). The reported highly sensitive and specific droplet digital PCR assays for EGFR mutation detection have potential in clinical blood testing.

Gefitinib Treatment in EGFR Mutated Caucasian NSCLC: Circulating-Free Tumor DNA as a Surrogate for Determination of EGFR Status

Introduction:In the phase IV, open-label, single-arm study NCT01203917, first-line gefitinib 250 mg/d was effective and well tolerated in Caucasian patients with epidermal growth factor receptor (EGFR) mutation-positive non–small-cell lung cancer (previously published). Here, we report EGFR mutation analyses of plasma-derived, circulating-free tumor DNA.Methods:Mandatory tumor and duplicate plasma (1 and 2) baseline samples were collected (all screened patients; n = 1060). Preplanned, exploratory analyses included EGFR mutation (and subtype) status of tumor versus plasma and between plasma samples. Post hoc, exploratory analyses included efficacy by tumor and plasma EGFR mutation (and subtype) status.Results:Available baseline tumor samples were 1033 of 1060 (118 positive of 859 mutation status known; mutation frequency, 13.7%). Available plasma 1 samples were 803 of 1060 (82 positive of 784 mutation status known; mutation frequency, 10.5%). Mutation status concordance between 652 matched tumor and plasma 1 samples was 94.3% (95% confidence interval [CI], 92.3–96.0) (comparable for mutation subtypes); test sensitivity was 65.7% (95% CI, 55.8–74.7); and test specificity was 99.8% (95% CI, 99.0–100.0). Twelve patients of unknown tumor mutation status were subsequently identified as plasma mutation-positive. Available plasma 2 samples were 803 of 1060 (65 positive of 224 mutation status-evaluable and -known). Mutation status concordance between 224 matched duplicate plasma 1 and 2 samples was 96.9% (95% CI, 93.7–98.7). Objective response rates are as follows: mutation-positive tumor, 70% (95% CI, 60.5–77.7); mutation-positive tumor and plasma 1, 76.9% (95% CI, 65.4–85.5); and mutation-positive tumor and mutation-negative plasma 1, 59.5% (95% CI, 43.5–73.7). Median progression-free survival (months) was 9.7 (95% CI, 8.5–11.0; 61 events) for mutation-positive tumor and 10.2 (95% CI, 8.5–12.5; 36 events) for mutation-positive tumor and plasma 1.Conclusion:The high concordance, specificity, and sensitivity demonstrate that EGFR mutation status can be accurately assessed using circulating-free tumor DNA. Although encouraging and suggesting that plasma is a suitable substitute for mutation analysis, tumor tissue should remain the preferred sample type when available.

EGFR, KRAS, BRAF, PTEN, and PIK3CA mutation in plasma of small cell lung cancer patients.

e18552 Background: Epidermal growth factor receptor (EGFR) exons 18, 19 and 21 mutation in NSCLC is related to response of tumors to EGFR tyrosine kinase inhibitor (TKI), while mutation with EGFR e...