Epidermal Growth Factor Receptor Mutation Testing Research Articles

Detection of EGFR Mutations in Circulating Tumor DNA (ctDNA) Retrieved from Plasma - Interlaboratory Quality Assessment in the Czech Republic.

Detection of EGFR mutations in tumor tissue represents a standard testing procedure in patients with non-small cell lung cancer. Molecular testing of circulating tumor DNA (ctDNA) in plasma enables detection of mutations in cases where tumor specimens are unavailable or when monitoring of therapeutic responses is necessary. In addition, according to the recent literature, ctDNA better reflects the heterogeneity of the neoplastic cell population than isolated tumor lesion or metastasis. We report a national interlaboratory evaluation aimed at assessing the analytical quality of ctDNA EGFR testing in plasma across seven reference laboratories in the Czech Republic. Aliquots of 13 plasma samples were sent to 7 laboratories and consisted of commercially available 2ml plasma specimens of genomic DNA with mutant allelic frequencies of 5, 0.5, 0.05, and 0% of the most common sensitizing mutations (deletion in exon 19, L858R) and the resistance mutation T790M. DNA extraction and EGFR testing were performed according to standard procedures. In 6/7 laboratories the cobas® EGFR Mutation Test v2 was used. One laboratory employed the Super-ARMS® EGFR Mutation Detection Kit. In total, 91 genotypes were determined with an overall error rate of 24.2% (22/91). The overall error rates were 3.2% (2/63) for the 0.5% mutation frequency and 0% for the 5% mutation frequency (0/35), respectively. No false positive results were reported. The cobas® method achieved consistent results with the 0.05% mutation frequency for the exon 19 deletion. For L858R and T790M mutations, the threshold was above the 0.5% frequency. The results show that EGFR testing for ctDNA in plasma has limited sensitivity, especially for detection of the T790M mutation. Particularly, in ctDNA testing of very low mutated DNA plasma fractions (below 0.01%), emphasis should be placed on the use of highly sensitive molecular methods. The outcomes of this quality assessment confirm the need for rebiopsy in patients with negative plasma results because of a higher false negative rate in comparison to tissue testing. Key words: circulating DNA - liquid biopsy - epidermal growth factor receptor - non-small cell lung cancer - quality control This work was supported by grants of AstraZeneca and the project of the Ministry of Health number 00064203 (Motol University Hospital). The authors declare they have no potential confl icts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 4. 6. 2018 Accepted: 1. 8. 2018.

Clinical utility of a blood-based EGFR mutation test in patients receiving first-line erlotinib therapy in the ENSURE, FASTACT-2, and ASPIRATION studies

ObjectivePatients with advanced non-small-cell lung cancer (NSCLC) with an adenocarcinoma component are recommended to undergo epidermal growth factor receptor (EGFR) mutation testing when being considered for EGFR targeted therapy. We conducted an exploratory analysis to inform the clinical utility of EGFR mutation testing in blood cell-free DNA using the cobas®EGFR Mutation Test v2. Materials and methodsTwo EGFR mutation tests, a tissue-based assay (cobas® v1) and a tissue- and blood-based assay (cobas® v2) were used to analyze matched biopsy and blood samples (897 paired samples) from three Asian studies of first-line erlotinib with similar intent-to-treat populations. ENSURE was a phase III comparison of erlotinib and gemcitabine/platinum, FASTACT-2 was a phase III study of gemcitabine/platinum plus erlotinib or placebo, and ASPIRATION was a single-arm phase II study of erlotinib. Agreement statistics were evaluated, based on sensitivity and specificity between the two assays in subgroups of patients with increasing tumor burden. ResultsPatients with discordant EGFR (tissue+/plasma-) mutation status achieved longer progression-free and overall survival than those with concordant (tissue+/plasma+) mutation status. Tumor burden was significantly greater in patients with concordant versus discordant mutations. Pooled analyses of data from the three studies showed a sensitivity of 72.1% (95% confidence interval [CI] 67.8–76.1) and a specificity of 97.9% (95% CI 96.0–99.0) for blood-based testing; sensitivity was greatest in patients with larger baseline tumors. ConclusionsBlood-based EGFR mutation testing demonstrated high specificity and good sensitivity, and offers a convenient and easily accessible diagnostic method to complement tissue-based tests. Patients with a discordant mutation status in plasma and tissue, had improved survival outcomes compared with those with a concordant mutation status, which may be due to their lower tumor burden. These data help to inform the clinical utility of this blood-based assay for the detection of EGFR mutations.

P2.09-12 EGFR Mutation Status in Squamous Cell Carcinoma or Non-Small Cell Carcinoma Favor Squamous Cell Carcinoma Diagnosed from Small Lung Biopsies

Epidermal growth factor receptor (EGFR) mutation testing is strongly recommended in non-small cell lung cancer (NSCLC) patients with adenocarcinoma subtypes, or patients with mixed-type tumors containing adenocarcinomatous components. However, in clinical practice, patients showing squamous cell carcinoma (SqCC) morphology on small biopsies cannot be ruled out to have an adenocarcinomatous component elsewhere in the lesion due to sampling bias. Therefore, EGFR mutation testing may have been neglected in such circumstances, which would affect the clinical management of patients. In this study, we sought to examine the frequency of EGFR mutations in lung cancer patients with SqCC or Non-Small Cell Carcinoma (NSCC) favor SqCC diagnosed from small biopsies. The correlation between EGFR mutation status and clinicopathological characteristics was also evaluated. One hundred and forty-eight patients diagnosed as SqCC or NSCC favor SqCC from small lung biopsies were enrolled. All of these cases were histologically confirmed by evaluating cellular morphology using hematoxylin-eosin (HE) staining and TTF1, p40 or CK5/6 expression using immunohistochemistry. EGFR mutations were examined using real-time PCR based assay. Among 148 small biopsy cases, 135 (91.2%) were diagnosed as SqCC while 13 (8.8%) were NSCC favor SqCC. Approximately 8.8% (13/148) of all cases were found to have EGFR mutations, including 7 (53.8%) L858R mutation, 4 (30.8%) exon 19 deletion, and 2 (15.4%) cases with coexistent L858R and T790M mutations. EGFR mutation-positive cases accounted for 5.2% (7/135) in SqCC, 46.2% (6/13) in NSCC favor SqCC, and 83.3% (10/12) of all these cases were never smokers. The multivariate analysis showed that EGFR mutations were more prevalent in non-smokers than those in smokers (83.3% versus 16.7%, p = 0.03) and in patients diagnosed as NSCC favor SqCC than in those diagnosed as SqCC (46.2% versus 5.2%, p = 0.002). In addition, 4 out of 13 EGFR mutation-positive patients with SqCC or NSCC favor SqCC morphology were eventually diagnosed as adenosquamous carcinoma after further undergoing surgical resection. To the best of our knowledge, this is the first study directly addressing the importance of EGFR mutation testing in lung cancer patients with SqCC or NSCC favor SqCC diagnosed from small biopsies. Our findings suggest that EGFR mutation testing should not be excluded in such populations, especially in never-smokers, or patients with NSCC favor SqCC subtypes.

EGFR Mutation Testing of Nonsquamous NSCLC in New Zealand: Trends, Selectivity and Effects on the Prevalence of EGFR Mutation

Background: Given the benefits in using epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), clinical practice guidelines recommend EGFR mutation testing of nonsquamous non–small cell lung cancer (NSCLC). However, not all patients get tested, which may have an impact on the prevalence of EGFR mutation previously estimated. Aim: To determine the trends of EGFR mutation testing in patients with nonsquamous NSCLC in New Zealand, and to explore the possible associations between the proportions tested, selectivity and the prevalence of EGFR mutation. Methods: This population-based study involves all patients who were diagnosed with nonsquamous NSCLC in the four health regions of New Zealand between January 2010 and July 2016. We identified eligible patients from the New Zealand Cancer Registry and obtained information on EGFR testing from TestSafe, a clinical information sharing service. We then calculated the proportions of patients tested for EGFR mutation and computed selectivity indices for eleven periods. We used a log-linear model to assess the associations between the proportions tested, selectivity and the prevalence of EGFR mutation. Results: Of the 2986 patients involved in this analysis, 1280 (42.9%) were tested for EGFR mutation. The proportion tested increased from 3.7% in 2010 to 74.0% in 2016. Testing was more prevalent in younger age group, female, Asian and patients with adenocarcinoma, and when specimens for testing was available. Such selectivity, however, decreased from 2010 to 2016. The prevalence of EGFR mutation varied widely across the periods, ranging from 16.8% in January-June 2014 to 43.8% in 2010. It was negatively associated with the proportion tested ( P = 0.02), and positively associated with the selectivity of testing ( P = 0.03). The log linear models estimated that the prevalence of EGFR mutation would be at most 16.1% (95% CI: 9.5%–27.1%) if 100% of patients were tested. Conclusion: In New Zealand, the uptake of EGFR mutation testing has improved over time but there is still room for improvement. Incomplete and selective testing may result in an overestimation of the prevalence of EGFR mutation in patients with nonsquamous NSCLC.

P2.09-14 The Detection of EGFR Gene Mutation from Washing Solution of Devices used for the Bronchoscopic Examination

The detection of epidermal growth factor receptor (EGFR) gene mutation is necessary for selection of lung cancer treatment. Recently, biopsy specimens are used in many genomic and immunohistochemical examination methods to select the treatment policy, so it is important not to waste valuable cancer cell samples. For detection of EGFR gene mutation we examine the washing solution of devices used for the bronchoscopic examination in order to solve the issue. In this report, we investigate the detection ratio of EGFR mutation using this cytological sample. A total of 336 washing solution samples from lung cancer patients were examined for EGFR gene mutation testing in the period between November 2010 and October 2017. 328 cases of 336 excepting 8, which were carried out as re-biopsy, were surveyed in this examination. After the regular biopsy or scraping method, all devices were washed with 10ml saline liquid. Half volume of washing solution was used for regular cell examination to detect cancer cells. Only if a sufficient amount of non-small cancer cells were observed from it, the other volume was submitted for EGFR mutation examination. Final diagnosis was decided by histological specimen. Presence or absence of EGFR mutation and their types were observed from 328 samples retrospectively. Histological diagnosis of all 328 biopsy specimens are as follows; 200 Adenocarcinoma (AD), 100 Squamous cell carcinoma (SQ), 13 non-small cell lung carcinoma(NSCLC), 3 adenosquamous cell carcinoma(ADSQ), 7 high grade neuroendocrine carcinoma (HGNEC), and 5 others. The detection number of EGFR mutation from AD, SQ, ADSQ and NSCLC are 65, 4, 2, and 3 respectively. There is no EGFR mutation from remaining histology cases. In all of 74 EGFR mutation positive cases, Exon 19 deletion was detected in 33 cases (45%) and Exon 21 L858R was detected in 33 cases (45%). The detection rate of EGFR gene mutation from washing solution of devices is 33% (65/200) in AD and 4% (4/100) in SQ. These results are approximate to previous reports. Consequently, it is acceptable to use this method to investigate EGFR gene mutation of non-small lung cancer patients. Furthermore, it is an advantage that we can carry out an EGFR mutation test using this method with a cytology sample earlier than a method with a histology sample.

Molecular Testing in EBUS-TBNA Specimens of Lung Adenocarcinoma: A Study of Concordance Between Cell Block Method and Liquid-Based Cytology in Appraising Sample Cellularity and EGFR Mutations.

Cytological endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) specimens of mediastinal lymph node metastasis are frequently used to perform concomitant diagnosis, staging and genetic testing in non-small-cell lung cancer (NSCLC). The purposes of this single-center retrospective study were to evaluate EBUS-TBNA samples' adequacy for molecular testingof epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK), and to analyze the concordance between the cell block method and liquid-based cytology (LBC) in appraising the sample cellularity and in detecting EGFR mutation. We retrospectively examined 82 patients who underwent EBUS-TBNA from October 2012 to September 2015 and received a confirmed diagnosis of lymph node metastasis of lung adenocarcinoma. Each sample was processed using both cell block and LBC to carry out DNA analysis (adequacy criterion: tumor cell percentage >25%) and EGFR mutation testing. Fifty-four patients were male, 66 were current or former-smokers, and the median age was 67 years. The median size of sampled lymph nodes was 14.8 mm. Seventy-one and 66 samples were adequate to perform cell block and LBC, respectively. The κ-statistic (0.78) showed an excellent concordance. EGFR mutation was detected in eight patients using cell block and in seven using LBC, with a simple percentage agreement of 87.5%. ALK translocation was found in two patients. This study demonstrates the feasibility of EGFR mutation analysis with both cell block and LBC, with an excellent concordance between the two methods. Considering that the majority of advanced NSCLCs are diagnosed on cytology specimens, LBC is feasible and needs to be implemented for ancillary tests (immunocytochemistry, molecular analysis).

P2.01-62 Extracellular Vesicle-Based Egfr Genotyping in Bronchoalveolar Lavage Fluid from Non-Small Cell Lung Cancer Patients

Liquid biopsies for EGFR genotyping using plasma cell-free DNA (cfDNA) have limited value due to low sensitivity. Extracellular vesicles (EV) have been proven to contain mutant EGFR DNA in bronchoalveolar lavage fluid (BALF) from non-small cell lung cancer (NSCLC) patients. As an alternative to plasma liquid biopsies using cfDNA, we investigated the feasibility of EV-based liquid biopsy for EGFR genotyping using BALF from NSCLC patients, prospectively. EV DNA was extracted from the BALF of NSCLC patients with confirmed tissue/cytology-based EGFR genotyping at initial diagnostic work-up stage. (N=137) The number of patients with stage I, II, III, and IV are 37 (27%), 6 (4.4%), 23 (16.8%), 71 (51.8%), respectively. The patients were selected by favorable demographic data for EGFR mutation. EGFR mutation testing was done by peptide nucleic acid (PNA) clamping-assisted fluorescence melting curve analysis. The overall sensitivity and specificity of EGFR genotyping using BALF EV DNA were 69.5% and 83.3% and the concordance rate was 77.4%. (Kappa=0.534, P=0.000) The sensitivity was significantly increased along with the stage (42.1% in stage I, II, 60.0% in stage III, and 100% in stage IV). Especially, in stage IV disease, BALF EV-based EGFR typing did not miss all of tissue-proven 30 EGFR mutant cases and detected 9 additional mutant cases. Detection of EGFR mutation becomes significantly more sensitive as T stage increases. (T1 = 43.8%, T2 = 75%, T3 and T4 = 100%) Turn-around time (TAT) for EGFR genotyping using BALF EV DNA was 1-2 days, while it takes usually 2 weeks for tissue typing. BALF EV-based EGFR genotyping is a novel and highly promising method with rapid TAT and incredible accuracy in stage IV NSCLC patients. Further research in early stage disease is necessary and the matter of over-detection should be clinically validated by evaluating EGFR-TKI drug response.

P2.13-19 Prognostic Value Inferred from the Quantitative Measurement of EGFR Mutation Using PNA-Clamping Method in Advanced EGFR Mutant NSCLC Patients

Lung cancer is the leading cause of cancer death worldwide, with non-small cell lung cancer (NSCLC) accounting for the majority of cases. Recent advances in the understanding of the biology of tumors and in highly sensitive detection technologies for molecular analysis offer targeted therapies, such as epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). However, not all EGFR mutation positive NSCLC patients are equally responsive to EGFR-TKIs. Although many factors have been introduced to predict EGFR TKIs response, there seems to be no factor that can clearly explain the mechanism. The aim of this study was to evaluate whether the quantification of EGFR mutation predicts clinical outcomes of EGFR-TKI therapy. This study is retrospective analysis of prospectively collected data about all patients who underwent EGFR mutation test using peptide nucleic acid (PNA)-mediated clamping polymerase chain reaction (PCR) at the Pusan National University Hospital between October 2015 and December 2017. The efficiency of PCR clamping was determined by measuring the Ct value. The delta (Δ) Ct-1 value (standard Ct value minus sample Ct value) was calculated to measure the quantification of EGFR mutation. Among the total 552 patients tested for EGFR mutations and 142 (25.7%) were positive for EGFR mutations. Seventy-one patients received EGFR-TKIs. The patient population was subdivided using a mean ΔCt-1 value of 5.28. There was no clinically significant difference between the two groups. Patients with high ΔCt-1 value had significantly longer median overall survival (OS) than did patients with low ΔCt-1 value (24.5 vs 16.7 months, p=0.002). Patients with common EGFR mutation (exon 19 deletion or L858R) and high ΔCt-1 value showed a significantly longer median OS than did patients with common mutation and low ΔCt-1 value (24.1 vs 18.0 months, p=0.014). Multivariate analysis revealed only high ΔCt-1 value was significant prognostic factor of survival in advanced advanced EGFR mutant NSCLC. The ΔCt-1 value, which refers to EGFR quantification by PNA-clamping method, is a significant good prognostic factor of survival to EGFR-TKI therapy.

P3.13-03 Real-World Anaplastic Lymphoma Kinase Testing Practices: Results from a Survey in the United States

Anaplastic lymphoma kinase (ALK) translocation is a clinically validated predictive biomarker in multiple cancer types including non-small cell lung cancer, and widespread adoption of diagnostic assays permits a review of test performance. This analysis presents ALK and epidermal growth factor receptor (EGFR) testing rates, expediency, and results from 4 large national laboratories. Deidentified data from January 2013 to December 2015 were compiled from 40 states and Washington DC and included in the final analysis. ALK status was evaluated with the Vysis ALK Break Apart FISH Probe Kit; EGFR status was evaluated by therascreen EGFR RGQ PCR Kit, cobas EGFR Mutation Test, or Sanger sequencing. Results from the most recent conclusive test were used. Collection to order (time from date of biopsy to date of biomarker test order), order to results (time from date of biomarker test order to date of available results), and overall turnaround times (collection to order + order to results) were evaluated. Results were analyzed by laboratories and US regions (Northeast, Midwest, South, and West). Data were summarized with descriptive statistics. s of 59,058 ALK and 50,992 EGFR tests were collected; 50,023 patients were tested for both ALK and EGFR, 9035 had results for ALK only, and 969 for EGFR only. Mean patient ages at ALK-positive (ALK+) and EGFR-positive (EGFR+) diagnoses were 62.5 and 69.0 years, respectively. Overall ALK+ and inconclusive rates were 2.8% and 6.5%, respectively, vs 10.4% and 6.2% for EGFR testing. ALK+ (2.3–5.1%) and inconclusive (1.1–17.4%) rates varied between test centers. In the western US, the ALK+ rate (3.3%) was comparably greater than in other regions (2.4–2.7%). The median turnaround time was 15 days, with a median of 10 days for collection to order and 4 days for order to results; some regional variation was seen in turnaround time (14–18 days). No correlation was seen between age and turnaround time. EGFR+ and ALK+ rates and age distributions were consistent with prior reports. An interesting geographic difference in ALK rates was seen that warrants further investigation. The average turnaround time was longer than current guidelines recommend, suggesting an opportunity to improve current testing practices.

P1.09-31 Preliminary Experience with Liquid Biopsies in a Resource Constrained Setting and Its Impact on Treatment Decision Making

Liquid biopsies are potentially useful for molecular testing in the presence of inadequate tissue and for detection of resistance mechanisms in EGFR mutated NSCLC patients. Initial experience with liquid biopsies for advanced/metastatic NSCLC patients in a resource constrained setting is described herein. Retrospective audit of liquid biopsies performed over a 21-month period for NSCLC patients at a tertiary referral centre in North India. Testing methods included digital-droplet-PCR (ddPCR), Next-Generation-Sequencing (NGS) and Real-Time-PCR (RT-PCR). ddPCR was used to detect specific EGFR mutations (L858R exon21, E746_A750 exon19 deletions and T790M exon20) in ctDNA (detection limit based upon amplifiable DNA ranging from 0.5%-<0.01% of mutant allele). For NGS, cfDNA extracted from plasma was subjected to target enrichment by high multiplex PCR amplification and analyzed for mutations using gene panel (BRAF, EGFR, ERBB2/HER2, KRAS, MET) on the Ion Proton semiconductor sequencer (Life Technology, USA). RT-PCR assay using EGFR-RT52 kit (EntroGen USA) detects known somatic mutations in exons 18-21 of EGFR by amplifying mutant-specific sequences and relies on fluorescent probes for detection (detection limit of 1%). 42 patients [61.9% (n=26) males] underwent liquid biopsies. ddPCR, NGS and RT-PCR were performed in 31(73.8%), 7(16.7%) and 4(9.5%) patients respectively. Timing was prior to/during first-line treatment in 23(54.8%) and at progression on first-line treatment in 19(45.2%) patients. In the former, indication was inadequate tissue for molecular analysis (n=15), detection of other targetable mutation (after EGFR wild type status on tissue; n=3) and checking ctDNA status of T790M exon 20 detected in tissue (n=5). Among 19 patients with progression on first-line treatment, 17 were those with a sensitizing EGFR mutation at baseline who had progressed on EGFR-TKI of which 47.1% (n=8) were found to have T790M exon 20 mutation. The primary EGFR sensitizing mutation detected in tissue at baseline was also detected in ctDNA in 9 of 20 patients with a sensitivity of 45.0%. Agreement between results of tissue testing and liquid biopsy testing for EGFR mutations was 48.0% (12/25). Potential change in management with use of liquid biopsies was observed in 12 patients (28.6% overall and 52.6% if done at progression). Preliminary experience in our resource constrained setting indicates that greatest utility of liquid biopsies is for EGFR mutated patients with progression on EGFR-TKI wherein T790M detection rates are comparable to those reported in literature. However, the overall sensitivity and agreement is lower than that reported in literature and perhaps may be related to small number of patients.

P1.09-15 Preliminary Experience with Liquid Biopsies in a Resource Constrained Setting and its Impact on Treatment Decision Making

Liquid biopsies are potentially useful for molecular testing in the presence of inadequate tissue and for detection of resistance mechanisms in EGFR mutated NSCLC patients. Initial experience with liquid biopsies for advanced/metastatic NSCLC patients in a resource constrained setting is described herein. Retrospective audit of liquid biopsies performed over a 21-month period for NSCLC patients at a tertiary referral centre in North India. Testing methods included digital-droplet-PCR (ddPCR), Next-Generation-Sequencing (NGS) and Real-Time-PCR (RT-PCR). ddPCR was used to detect specific EGFR mutations (L858R exon21, E746_A750 exon19 deletions and T790M exon20) in ctDNA (detection limit based upon amplifiable DNA ranging from 0.5%-<0.01% of mutant allele). For NGS, cfDNA extracted from plasma was subjected to target enrichment by high multiplex PCR amplification and analyzed for mutations using gene panel (BRAF, EGFR, ERBB2/HER2, KRAS, MET) on the Ion Proton semiconductor sequencer (Life Technology, USA). RT-PCR assay using EGFR-RT52 kit (EntroGen USA) detects known somatic mutations in exons 18-21 of EGFR by amplifying mutant-specific sequences and relies on fluorescent probes for detection (detection limit of 1%). 42 patients [61.9% (n=26) males] underwent liquid biopsies. ddPCR, NGS and RT-PCR were performed in 31(73.8%), 7(16.7%) and 4(9.5%) patients respectively. Timing was prior to/during first-line treatment in 23(54.8%) and at progression on first-line treatment in 19(45.2%) patients. In the former, indication was inadequate tissue for molecular analysis (n=15), detection of other targetable mutation (after EGFR wild type status on tissue; n=3) and checking ctDNA status of T790M exon 20 detected in tissue (n=5). Among 19 patients with progression on first-line treatment, 17 were those with a sensitizing EGFR mutation at baseline who had progressed on EGFR-TKI of which 47.1% (n=8) were found to have T790M exon 20 mutation. The primary EGFR sensitizing mutation detected in tissue at baseline was also detected in ctDNA in 9 of 20 patients with a sensitivity of 45.0%. Agreement between results of tissue testing and liquid biopsy testing for EGFR mutations was 48.0% (12/25). Potential change in management with use of liquid biopsies was observed in 12 patients (28.6% overall and 52.6% if done at progression). Preliminary experience in our resource constrained setting indicates that greatest utility of liquid biopsies is for EGFR mutated patients with progression on EGFR-TKI wherein T790M detection rates are comparable to those reported in literature. However, the overall sensitivity and agreement is lower than that reported in literature and perhaps may be related to small number of patients.

P3.01-78 The Cytology Samples and Plasma Specimens were Feasible for the EGFR Molecular Testing.

EGFR mutation detection with real-time PCR is standard method to identify eligible patients for EGFR-TKI treatments in daily routine practice. Surgically resected tissues or biopsy specimens are mainly used as the sample materials for testing. However, the biopsy samples sometimes have a certain limitation in their volume and so the cytology specimens are chosen for EGFR testing instead. Plasma has also become an option especially in EGFR-TKI resistant cases in which often have difficulties to obtain the adequate tumor yield. In this study, we evaluated the feasibilities of using cytology samples and plasma specimens for the EGFR molecular testing. Patients provided written informed consent for use of the samples were participated in this prospective research. Cytology samples were obtained from biopsy and cells were suspended into liquid-based cytology (LBC) media. Tumor contents in the samples were confirmed with Papanicolaou stained slides. Plasma samples were also collected from patients shortly before the tissue biopsy. EGFR mutations in these samples were analyzed by cobas EGFR Mutation Test v2. Also, EGFR testing result of tissue specimens of the patients corresponded were collected from the medical records measured by cobas EGFR Mutation Test v2 as reference. The feasibilities of both cytology and plasma specimen were evaluated comparing with the tissue samples. One-hundred fifty-eight patients were registered to this study. Among those patients, 77 patients with matched set of samples were enrolled to this study. EGFR mutation rates in tissue, cytology, and plasma were 37.7, 29.9 and 16.9 %, respectively. Overall agreement rate of the cytology specimens and the plasma specimens against the tissue samples were 87.0 and 75.3%, respectively. All eightT790M mutation positive cases were perfectly matched between tissue and cytology specimens. The results suggested that tissue specimen is the most suitable sample for detecting mutations. Since high specificities were confirmed in both cytology and plasma specimens, the results are reliable as long as the call is positive. Choosing cytology or plasma specimens for EGFR testing can be the considerations for the patients who have difficulties in collecting tissue samples in the real world setting.

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.

Incomplete uptake of EGFR mutation testing and its impact on estimation of mutation prevalence in patients with non-squamous NSCLC: A population-based study in New Zealand

BackgroundEpidermal Growth Factor Receptor (EGFR) mutation testing is recommended for patients with non-squamous non-small cell lung cancer (NSCLC) but not all eligible patients get tested, which may bias the mutation prevalence estimated. This study aims to examine trends in the uptake of EGFR mutation testing in patients with non-squamous NSCLC in New Zealand; to develop a composite metric that quantifies the influences of demographic and clinico-pathological factors on the testing uptake; and to estimate the prevalence of EGFR mutation if all patients were tested. MethodsThis population-based study involved all patients who were diagnosed with non-squamous NSCLC in four health regions in New Zealand between January 2010 and December 2015. Eligible patients were identified from the New Zealand Cancer Registry and information on EGFR mutation testing was obtained through linkage to TestSafe, a clinical information sharing service, and laboratory records. ResultsOf 2701 eligible patients, 1059 (39.2%) were tested for EGFR mutation. The testing prevalence increased (3.7% in 2010 to 64.6% in 2014) and the influences of demographic and clinic-pathological factors decreased from 2010 to June 2014, and remained stable afterward. Of the tested patients, 229 (21.6%) were mutation positive with a decreasing trend observed from 2010 (43.8%) to June 2014 (16.8%). The best-fit log-linear model estimated the prevalence of EGFR mutation, if all patients were tested, as 15.5% (95% CI: 13.2%–18.0%). ConclusionThe methods described here allowed a more accurate estimation of the prevalence of EGFR mutation.

Real-world EGFR testing in patients with stage IIIB/IV non-small-cell lung cancer in North China: A multicenter, non-interventional study.

BackgroundBefore tyrosine kinase inhibitor (TKI) therapy can be administered in patients with advanced non‐small cell lung cancer (NSCLC), EGFR mutation testing is required. However, few studies have evaluated the extent of EGFR testing in real‐world practice in China.MethodsA multicenter, observational study of EGFR testing in NSCLC patients in North China was conducted. Treatment‐naïve patients or those with postoperative recurrent stage IIIB/IV NSCLC were enrolled. The primary objective was EGFR testing rate. Secondary objectives included EGFR mutation status, EGFR testing methods and specimens, factors associated with EGFR testing, and overall survival with or without EGFR testing.ResultsOverall, 2809 patients with stage IIIB/IV NSCLC were enrolled; 90.78% had adenocarcinoma. The EGFR screening rate was 42.54%. EGFR testing rates were higher in tumor samples obtained by lymph node puncture, and in patients with urban medical insurance, adenocarcinoma, non‐smokers, or those located in developed cities (all P < 0.001). The EGFR mutation rate was 46.44%. The most commonly used specimens for EGFR testing were biopsy tumor samples (67.53%). PCR‐based methods (72.05%), Sanger sequencing (5.36%), and Luminex liquid chip (5.10%) were the most frequently used testing platforms. Similar positive EGFR mutation rates were achieved with different platforms. TKI therapy was the first‐line treatment administered to most EGFR‐positive patients (56.22%), and chemotherapy in EGFR‐negative patients (84.88%). Overall survival was higher in EGFR‐tested than in untested patients (27.50 vs. 19.73 months; P = 0.007).ConclusionReal‐world EGFR testing rates for NSCLC in North China were relatively low because of clinical and social factors, including medical insurance coverage.

The Clinicopathological and Molecular Associations of PD-L1 Expression in Non-small Cell Lung Cancer: Analysis of a Series of 10,005 Cases Tested with the 22C3 Assay

PD-L1 expression testing is mandatory prior to pembrolizumab prescription in non-small cell lung cancer. Our service offers PD-L1 testing using the PD-L1 IHC 22C3 pharmDx assay, in parallel with EGFR, ALK, ROS1 and (in some cases) KRAS testing. We correlate PD-L1 expression in 10,005 tumours with patient age and sex, with tumour histological subtypes, with the sampling modality and type of tissue, and with the presence of other molecular alterations. PD-L1 expression testing was performed using the aforementioned assay; tumour proportion scores (TPS) of 1 and 50% were taken as cut-offs for low and high positivity, respectively. EGFR testing was performed using the cobas® EGFR Mutation Test v2. ALK testing was performed using the VENTANA ALK (D5F3) CDx Assay. KRAS testing was performed using pyrosequencing. TPS <1% was seen in 44.4% of tumours, 1-49% in 25.0% and ≥ 50% in 30.6%. We identified no significant relationship with age. Female patients were slightly more likely to express PD-L1. Poorly-differentiated tumour histology and ALK translocation were significantly associated with PD-L1 expression. Rare EGFR mutations tended to be associated with PD-L1 expression. Pleural and nodal metastases were more likely to express PD-L1 than primary tumours, but biopsy and cytological specimens did not show different PD-L1 expression rates. Our data show that the means of acquiring a tumour sample (biopsy versus cytology) does not have a significant impact on PD-L1 expression. However, we found that certain metastatic sites were associated with significantly higher expression rates, which has substantial implications for selection of tissue for testing.

Circulating tumour DNA as a cancer biomarker.

Measurement of genetically altered DNA shed from tumours into the circulation can potentially provide a new generation of blood-based cancer biomarkers. Compared with tissue DNA biomarkers which require surgery or biopsy, samples for circulating tumour DNA assays can be obtained with minimal inconvenience and at lower cost. Furthermore, in contrast to tissue, the use of circulating tumour DNA allows serial monitoring, faster delivery of results and potentially provides an integrative representation of genetic alterations across all tumour sites within a patient. In contrast to existing protein-based cancer biomarkers, all of which can be produced by benign disease, circulating tumour DNA biomarkers would be expected to be more specific for malignancy. Furthermore, unlike the available blood cancer biomarkers, circulating tumour DNA can be used to predict response to specific therapies, identify mechanisms of therapy resistance and detect potentially actionable mutations. One of the first circulating tumour DNA assays recommended for clinical use involves EGFR mutation testing for predicting response to EGFR tyrosine kinase inhibitors in patients with advanced non-small cell lung cancer, especially when tumour tissue is unavailable. In order to accelerate the introduction of circulating tumour DNA assays into routine clinical use, laboratory medicine staff will have to undergo training in the use of polymerase chain reaction and DNA sequencing. Furthermore, existing circulating tumour DNA assays will need to be simplified, standardized, shown to have clinical utility, be made available at reasonable costs and be reimbursable.

Abstract B32: RADIANCE: An open-label, nonrandomized, prospective biomarker study to assess analytic concordance between noninvasive testing and tissue testing for EGFR T790M mutation detection in patients with non-small cell lung cancer

Abstract Background: EGFR-TKIs are the recommended first-line therapy of patients (pts) with advanced non-small cell lung cancer (NSCLC) who have an EGFR-sensitizing mutation (EGFRm). However, most pts develop resistance to EGFR-TKIs, with the EGFR T790M resistance mutation observed in &amp;gt;50% of pts on first- and second-generation EGFR-TKIs. Osimertinib is a third-generation, CNS-active EGFR-TKI that potently and selectively inhibits both EGFRm and T790M resistance mutations, recommended for pts with T790M-positive advanced NSCLC who have progressed on first-line EGFR-TKI therapy. Tumor tissue is the preferred sample source for T790M testing; however, often tumor biopsy is not feasible following disease progression due to inaccessible sites and/or concerns about safety. Testing using plasma samples offers a less invasive, albeit less sensitive, alternative method for the detection of T790M-positive circulating tumor (ct) DNA. Additional noninvasive options, including urine ctDNA analyses, are also being investigated. The RADIANCE trial is designed to determine if urine and plasma tests can provide similar detection rates to tissue testing for T790M detection when combined. Pts identified as T790M-positive may be offered treatment with osimertinib. Trial Design: RADIANCE (NCT03137264) is an open-label, prospective biomarker study with a primary objective of assessing the analytic concordance between noninvasive testing (Guardant360 Plasma Assay and Trovera Liquid Biopsy Test using urine) and tissue testing (cobas® EGFR Mutation Test v2) for T790M in pts with NSCLC. Approximately 470 pts will be enrolled. The study consists of two parts, Diagnostic Analytic Validity (Part 1) and Clinical Outcomes (Part 2). In Part 1, eligible pts will provide a tumor biopsy (cobas tissue test), plasma (cobas plasma and Guardant360 tests) and urine sample (Trovera test) for T790M testing. Eligibility criteria include a primary diagnosis of EGFRm-positive NSCLC with evidence of disease progression during or following treatment with a first- or second-generation EGFR-TKI. Pts must not be enrolled on another clinical trial, or have previously received osimertinib or another T790M-directed therapy. In Part 2, pts with T790M-positive status by cobas tissue and/or cobas plasma test will be treated according to standard of care, and may receive osimertinib. Pts will be followed for clinical outcomes for up to 18 months, and evaluated for objective response, progression-free survival, and duration of response (investigator-assessed using RECIST v1.1) and safety. Pts who are T790M negative or do not receive osimertinib will have completed the study after Part 1. Citation Format: Hatim Husain, Martin Dietrich, Steven Dubinett, David E. Gerber, Jeffrey Gregg, Michelle Shiller, Howard West, Kim Thai, Alvin Milner, Nabil Chehab, Christina Baik. RADIANCE: An open-label, nonrandomized, prospective biomarker study to assess analytic concordance between noninvasive testing and tissue testing for EGFR T790M mutation detection in patients with non-small cell lung cancer [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B32.

P29 Fast Response to Osimertinib in Patients with EGFR Mutated NSCLC with Brain Metastasis and Carcinomatous Meningitis

Approximately 25% of the patients with lung adenocarcinoma stage IV with EGFR mutation show CNS compromise when diagnosed. Osimertinib is a third generation TKI (tyrocinkinasa inhibitor) active against sensitivity and resistant mutations of EGFR (Del Ex19/L858R) & (T790M) with high penetration into the CNS. Here we show two cases of lung adenocarcinoma patients with CNS compromise who received first line osimertinib treatment. Case 1: 42 year old, non-smoker female with lung adenocarcinoma who underwent surgical resection in 2007. In 2017 developed endocraneal hypertension syndrome and craneal VI left par palsy. CSF: high spinal fluid protein concentration with normal glucose level, non celular (x 3) CT: solid nodule in the lower left lobe LII (36 mm) and ground glass multiple opacities. BRAIN MRI: hyperintensity of signal in FLAIR in the interior side of the encephalic trunk, enhanced with gadolinium. Lung biopsy: Moderately differentiated adenocarcinoma (T2aNxM1b), with EGFR exon 19 deletion. Suspected carcinomatous meningitis. Initial treatment: Erlotinib 150 mg Qd, switching to osimertinib 80 mg Qd due to symptom progression. She showed a fast response within 5 days with resolution of neurological symptoms and partial response to lung lesions 90 days after initiation of treatment. Case 2: 47 year old female, mild smoker (7 pack/y). Pericardic tamponade at diagnosis. PET/CT: bone hyperuptake, multiple ganglionar and ground glass opacities mostly in the upper right lobe. Brain MRI: with cortical and subcortical, brain and cerebellum sub centimetric multiple lesions. Supraclavicular lymph node biopsy: Poorly differentiated adenocarcinoma (TXN3M1c). with EGFR L861Q mutation. It also was detected with in serum and pericardial fluid (Cobas EGFR Mutation test V2). She start Osimertinib 80 mg qd, CT control showed a decrease of the ground glass opacities and partial response in the CNS. Not applicable Not applicable Both cases showed fast intracraneal responses to Osimertinib for the classic and the less frequent mutation (Del 19 & L861Q) supporting its use in first line treatment in this patient.

Large-Scale EGFR Mutation Testing in Clinical Practice: Analysis of a Series of 18,920 Non-Small Cell Lung Cancer Cases.

We make use of a very large dataset of non-small cell lung cancer specimens to examine the molecular epidemiology of EGFR mutations, particularly with respect to rare and compound mutations, and to non-adenocarcinoma histological subtypes. We also demonstrate the feasibility of large-scale EGFR mutation screening using the full range of specimens encountered in routine practice. We retrospectively reviewed 18,920 unselected EGFR mutation results from our centre between July 2009 and October 2016, using Qiagen's therascreen EGFR RGQ PCR Kit. Mutation rates were correlated with patient demographics and tumour histology. Our testing success rate was 93.9%, with similar success rates using histological and cytological specimens. Rare, potentially-targetable mutations accounted for 9.5% of all mutations detected. We identified a 2.5% mutation rate in tumours diagnosed as squamous cell carcinomas. There was a trend towards increasing EGFR mutation rates with increasing age, and while Del19 was the commonest mutation in the young, L858R predominated in the elderly. We found that EGFR mutation heterogeneity is rare within tumours and between primary and metastatic deposits. Our data demonstrate that large-scale, reflex EGFR mutation testing is feasible and affordable in the context of a publicly-funded health system. Furthermore, we have shown that the use of techniques sensitive only to classical mutations and selection of patients on the grounds of age, sex and histology denies patients access to potentially beneficial TKI therapy.