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P3.02b-026 Association of EGFR Exon 19 Deletion and EGFR-TKI treatment duration with Frequency of T790M Mutation in EGFR-Mutant Lung Cancer Patients
January 2017
P3.02b-025 Rapid and Highly Sensitive EGFRdelEx19 and KRAS Exon 2 Mutation Detection in EBUS-TBNA Specimen of Lung Adenocarcinoma Topic: EGFR Biomarkers Filiz Oezkan,1 Thomas Herold,2 Kaid Darwiche,1 Wilfried Eberhardt,2 Daniel Christoph,2 Karl Worm,3 Lutz Freitag,4 Kurt Schmid,3 Thomas Hager,3 Frank Breitenbuecher,2 Martin Schuler2 1Department of Interventional Pneumology, Ruhrlandklinik, West German Lung Center, University Duisburg-Essen, Essen/ Germany, 2Department of Medical Oncology, University Hospital Essen, West German Cancer Centre, University Duisburg-Essen, Essen/Germany, 3Institute of Pathology, University Duisburg-Essen, University Hospital Essen, Essen/Germany, 4Department of Pulmonology, University of Zurich, Zürich/Switzerland Background: First-line treatment with afatinib prolongs overall survival in patients with metastatic non-smallcell lung cancer (NSCLC) harboring EGFR exon 19 deletion mutations. Conversely, somatic KRAS mutations are negative predictors for benefit from EGFR-targeting agents. Rapid availability of these biomarker results is mandatory to prevent delayed or inferior treatments. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is well-established for lung cancer diagnosis and staging. Next generation sequencing (NGS) via targeted resequencing allows simultaneous interrogation for multiple mutations, but has its limitations based on the amount of tumor tissue required and assay times. RT-PCR using Light-Cycler technology (LC-RTPCR) is a rapid and sensitive assay to detect somatic mutations in various tissues from NSCLC patients. The study’s aim was to analyze if LC-RTPCR is feasible for rapid EGFRdelE19 and KRAS Exon 2 mutation detection in EBUS-TBNA samples and to compare results with results obtained via standard NGS mutation analyses. Methods: 48 surplus EBUS-TBNA samples (38 lymph nodes, 10 primary tumor) from 47 patients with pulmonary adenocarcinoma were analyzed. Two samples were collected per lymph node. One was used for routine cytology, the other was freshly frozen (ff). DNA from ff-biopsies was extracted using Genomic DNA buffer set (QIAgen, Germany). Mutation analysis by LCRTPCR was conducted as previously described. NGS was performed on MiSeq (Illumina) via targeted resequencing using a customized multiplex-PCR panel covering 36 exons from 11 genes. Mutations were annotated with a
Abstracts
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minimum frequency of 2%. Processing time was approximately 4 days for NGS and 2 hours for LC-RTPCR analyses. Results: NGS of EBUS-TBNA samples was technically feasible for both markers in 22 (46%) samples, for EGFR testing in 32 (67%) samples, and for KRAS in 23 (48%) samples. EGFRdelEx19 mutations were detected in four (8.3% of intention-to-screen), and KRAS Exon 2 mutations in 15 cases (31%) cases. LC-RTPCR was technically feasible in all cases. All mutations detected by NGS were also detected by LC-RTPCR. LC-RTPCR detected three additional KRAS Exon 2 mutations and three additional EGFRdelEx19 mutations. NGS detected additional mutations in 4 cases (2 EGFR Exon 21, 1 KRAS Codon 61, 1 PIK3CA). Conclusion: LC-RTPCR is a rapid, highly sensitive method to detect mutations of immediate therapeutic relevance, such as EGFRdelEx19 and KRAS Exon 2 mutations, in limited EBUS-TBNA specimens from metastatic NSCLC patients. It is of value as rapid and sensitive initial assay in a two-step diagnostic process for first-line treatment decision, incorporating broader biomarker panels as second step. Keywords: EGFRdelEx19, KRAS Exon 2, NSCLC, EBUSTBNA
P3.02b-026 Association of EGFR Exon 19 Deletion and EGFR-TKI treatment duration with Frequency of T790M Mutation in EGFR-Mutant Lung Cancer Patients Topic: EGFR Biomarkers Norikazu Matsuo,1 Koichi Azuma,2 Kazuko Sakai,3 Akihiko Kawahara,2 Hidenobu Ishii,1 Takaaki Tokito,1 Takashi Kinoshita,1 Kazuhiko Yamada,1 Kazuto Nishio,3 Tomoaki Hoshino1 1Department of Internal Medicine, Division of Respirology, Neurology, and Rheumatology, Kurume University School of Medicine, Kurume/Japan, 2 Kurume University School of Medicine, Kurume/Japan, 3 Department of Genome Biology, Kinki University Faculty of Medicine, Osakasayama/Japan Background: The most common event responsible for resistance of epidermal growth factor receptor (EGFR)tyrosine kinase Inhibitor (TKI) is acquisition of the T790M mutation, which occurs in approximately 50% of patients who initially respond to EGFR-TKI. Recently, third-generation EGFR-TKIs have been shown to exert a
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remarkable effect against T790M resistance mutationpositive non-small cell lung cancer (NSCLC). T790M is an important predictive marker for third-generation EGFRTKIs; therefore, determining the clinicopathologic characteristics of T790M-harboring NSCLC showing relapse after EGFR-TKI therapy is of high clinical relevance. we evaluated whether T790M mutation is related to clinicopathologic or prognostic factors in patients with relapse of EGFR-mutant NSCLC after treatment with EGFR-TKIs. Methods: We retrospectively reviewed T790M status and clinical characteristics of advanced-stage or recurrent NSCLC patients who had received EGFR-TKI treatment and rebiopsy at Kurume University Hospital between March 2005 and December 2015. Results: we identified 193 patients with advanced or recurrent EGFR-mutant NSCLC who developed resistance to initial EGFR-TKI treatment. Of these patients, 105 (54%) underwent re-biopsy. Adequate histological specimens were available for 73 of these patients, who were enrolled in the study; 38 (52%) of them had T790M mutation and 35 (48%) did not. T790M mutation was present more frequently in patients with exon 19 deletion mutation (63%, 26 of 41) than in those with L858R mutation (38%, 12 of 32) (p¼0.035) and in patients who received EGFR-TKI treatment for at least 10 months in total (71%, 32 of 45) than in those who did not (21%, 6 of 28) (p<0.001). The median PFS after initial EGFR-TKI treatment was longer in the T790M mutation-positive group (13.6 months, 95% CI: 9.2-15.8) than in the negative group (7 months, 95% CI: 3.7-8.5) (p¼0.037). The median total duration of EGFR-TKI treatment in patients with T790M mutation was 15.3 months, which was significantly longer than that (8.1 months) in patients without T790M mutation (p<0.001). Multivariate analyze revealed that The type of EGFR mutation (exon 19 deletion mutation versus L858R point mutation, p¼0.011, OR¼0.21, 95% CI¼0.05-0.71) and total duration of EGFR-TKI treatment (10 months versus <10 months, p<0.001, OR¼0.09, 95% CI¼0.020.28) were significantly associated with the presence of T790M mutation. Conclusion: The patients with EGFR exon 19 deletion mutation and long-term treatment with EGFR-TKI exposure demonstrated a high prevalence of T790M mutation. These data are potentially important for clinical decision making in the NSCLC patients with EGFR mutation. Keywords: non-small cell lung cancer, EGFR, EGFR-TKI, T790M
Journal of Thoracic Oncology
Vol. 12 No. 1S
P3.02b-027 Detection of EGFR Mutations in Plasma of Lung Adenocarcinoma Patients Using Real-Time PCR and Mass Spectrometry Topic: EGFR Biomarkers Rossella Bruno,1 Elisabetta Macerola,1 Vincenzo Condello,1 Cristiana Lupi,2 Alessandro Ribechini,3 Antonio Chella,4 Greta Alì,2 Gabriella Fontanini1 1Department of Surgical, Medical, Molecular Pathology and Critical Area, University of Pisa, Pisa/Italy, 2Unit of Pathological Anatomy, University Hospital of Pisa, Pisa/Italy, 3Endoscopic Section of Pneumology, University Hospital of Pisa, Pisa/Italy, 4Unit of Pneumology, University Hospital of Pisa, Pisa/Italy Background: Lung adenocarcinoma patients harboring sensitizing EGFR mutations can benefit from treatment with tyrosine kinase inhibitors (TKI). Whenever tumor tissue is inadequate or unavailable, detection of EGFR mutations in circulating cell-free tumor (ct) DNA from plasma is crucial to predict and monitor response to therapy. In this study we compared EGFR status between tumor tissue and plasma, using real-time PCR. Moreover, we evaluated the adequacy of ctDNA for a multi-target mass spectrometry (MS) analysis. Methods: EGFR mutations were investigated in paired plasma and tumor tissues from a prospective series of 105 lung adenocarcinoma patients: 79 had no prior TKI treatment and 26 underwent re-biopsy for TKI-acquired resistance. Molecular analyses were performed on tissue by a routine MS test (evaluation of 307 hot-spots in 10 genes including EGFR) and on ctDNA by a validated Scorpion/LNA real-time PCR (evaluation of 30 EGFR mutations). In the 26 postTKI patients ctDNA analysis was performed also by MS. Results: 1-Plasma versus tissue by real-time PCR: overall sensitivity, specificity and concordance were 64.8%, 95.4% and 82%. In preTKI patients, 17 harboured EGFR sensitizing mutations on tissue, 10 detected also in plasma (sensitivity 58.8 %, specificity 100%, concordance 91%). All 26 postTKI patients preserved EGFR sensitizing mutations. Regarding the detection of EGFR T790M resistance mutation, sensitivity, specificity and concordance were 63.6%, 80% and 73%. Specifically, 11 patients were T790M-positive (42%): 7 on both specimens, 4 only on tissue and 3 only on ctDNA. 2-Plasma versus tissue by MS: sensitivity, specificity and concordance for T790M were 50%, 93.3% and 76%.
P3.02b-025 Rapid and Highly Sensitive EGFRdelEx19 and KRAS Exon 2 Mutation Detection in EBUS-TBNA Specimen of Lung Adenocarcinoma Topic: EGFR Biomarkers Filiz Oezkan,1 Thomas Herold,2 Kaid Darwiche,1 Wilfried Eberhardt,2 Daniel Christoph,2 Karl Worm,3 Lutz Freitag,4 Kurt Schmid,3 Thomas Hager,3 Frank Breitenbuecher,2 Martin Schuler2 1Department of Interventional Pneumology, Ruhrlandklinik, West German Lung Center, University Duisburg-Essen, Essen/ Germany, 2Department of Medical Oncology, University Hospital Essen, West German Cancer Centre, University Duisburg-Essen, Essen/Germany, 3Institute of Pathology, University Duisburg-Essen, University Hospital Essen, Essen/Germany, 4Department of Pulmonology, University of Zurich, Zürich/Switzerland Background: First-line treatment with afatinib prolongs overall survival in patients with metastatic non-smallcell lung cancer (NSCLC) harboring EGFR exon 19 deletion mutations. Conversely, somatic KRAS mutations are negative predictors for benefit from EGFR-targeting agents. Rapid availability of these biomarker results is mandatory to prevent delayed or inferior treatments. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is well-established for lung cancer diagnosis and staging. Next generation sequencing (NGS) via targeted resequencing allows simultaneous interrogation for multiple mutations, but has its limitations based on the amount of tumor tissue required and assay times. RT-PCR using Light-Cycler technology (LC-RTPCR) is a rapid and sensitive assay to detect somatic mutations in various tissues from NSCLC patients. The study’s aim was to analyze if LC-RTPCR is feasible for rapid EGFRdelE19 and KRAS Exon 2 mutation detection in EBUS-TBNA samples and to compare results with results obtained via standard NGS mutation analyses. Methods: 48 surplus EBUS-TBNA samples (38 lymph nodes, 10 primary tumor) from 47 patients with pulmonary adenocarcinoma were analyzed. Two samples were collected per lymph node. One was used for routine cytology, the other was freshly frozen (ff). DNA from ff-biopsies was extracted using Genomic DNA buffer set (QIAgen, Germany). Mutation analysis by LCRTPCR was conducted as previously described. NGS was performed on MiSeq (Illumina) via targeted resequencing using a customized multiplex-PCR panel covering 36 exons from 11 genes. Mutations were annotated with a
Abstracts
S1201
minimum frequency of 2%. Processing time was approximately 4 days for NGS and 2 hours for LC-RTPCR analyses. Results: NGS of EBUS-TBNA samples was technically feasible for both markers in 22 (46%) samples, for EGFR testing in 32 (67%) samples, and for KRAS in 23 (48%) samples. EGFRdelEx19 mutations were detected in four (8.3% of intention-to-screen), and KRAS Exon 2 mutations in 15 cases (31%) cases. LC-RTPCR was technically feasible in all cases. All mutations detected by NGS were also detected by LC-RTPCR. LC-RTPCR detected three additional KRAS Exon 2 mutations and three additional EGFRdelEx19 mutations. NGS detected additional mutations in 4 cases (2 EGFR Exon 21, 1 KRAS Codon 61, 1 PIK3CA). Conclusion: LC-RTPCR is a rapid, highly sensitive method to detect mutations of immediate therapeutic relevance, such as EGFRdelEx19 and KRAS Exon 2 mutations, in limited EBUS-TBNA specimens from metastatic NSCLC patients. It is of value as rapid and sensitive initial assay in a two-step diagnostic process for first-line treatment decision, incorporating broader biomarker panels as second step. Keywords: EGFRdelEx19, KRAS Exon 2, NSCLC, EBUSTBNA
P3.02b-026 Association of EGFR Exon 19 Deletion and EGFR-TKI treatment duration with Frequency of T790M Mutation in EGFR-Mutant Lung Cancer Patients Topic: EGFR Biomarkers Norikazu Matsuo,1 Koichi Azuma,2 Kazuko Sakai,3 Akihiko Kawahara,2 Hidenobu Ishii,1 Takaaki Tokito,1 Takashi Kinoshita,1 Kazuhiko Yamada,1 Kazuto Nishio,3 Tomoaki Hoshino1 1Department of Internal Medicine, Division of Respirology, Neurology, and Rheumatology, Kurume University School of Medicine, Kurume/Japan, 2 Kurume University School of Medicine, Kurume/Japan, 3 Department of Genome Biology, Kinki University Faculty of Medicine, Osakasayama/Japan Background: The most common event responsible for resistance of epidermal growth factor receptor (EGFR)tyrosine kinase Inhibitor (TKI) is acquisition of the T790M mutation, which occurs in approximately 50% of patients who initially respond to EGFR-TKI. Recently, third-generation EGFR-TKIs have been shown to exert a
S1202
remarkable effect against T790M resistance mutationpositive non-small cell lung cancer (NSCLC). T790M is an important predictive marker for third-generation EGFRTKIs; therefore, determining the clinicopathologic characteristics of T790M-harboring NSCLC showing relapse after EGFR-TKI therapy is of high clinical relevance. we evaluated whether T790M mutation is related to clinicopathologic or prognostic factors in patients with relapse of EGFR-mutant NSCLC after treatment with EGFR-TKIs. Methods: We retrospectively reviewed T790M status and clinical characteristics of advanced-stage or recurrent NSCLC patients who had received EGFR-TKI treatment and rebiopsy at Kurume University Hospital between March 2005 and December 2015. Results: we identified 193 patients with advanced or recurrent EGFR-mutant NSCLC who developed resistance to initial EGFR-TKI treatment. Of these patients, 105 (54%) underwent re-biopsy. Adequate histological specimens were available for 73 of these patients, who were enrolled in the study; 38 (52%) of them had T790M mutation and 35 (48%) did not. T790M mutation was present more frequently in patients with exon 19 deletion mutation (63%, 26 of 41) than in those with L858R mutation (38%, 12 of 32) (p¼0.035) and in patients who received EGFR-TKI treatment for at least 10 months in total (71%, 32 of 45) than in those who did not (21%, 6 of 28) (p<0.001). The median PFS after initial EGFR-TKI treatment was longer in the T790M mutation-positive group (13.6 months, 95% CI: 9.2-15.8) than in the negative group (7 months, 95% CI: 3.7-8.5) (p¼0.037). The median total duration of EGFR-TKI treatment in patients with T790M mutation was 15.3 months, which was significantly longer than that (8.1 months) in patients without T790M mutation (p<0.001). Multivariate analyze revealed that The type of EGFR mutation (exon 19 deletion mutation versus L858R point mutation, p¼0.011, OR¼0.21, 95% CI¼0.05-0.71) and total duration of EGFR-TKI treatment (10 months versus <10 months, p<0.001, OR¼0.09, 95% CI¼0.020.28) were significantly associated with the presence of T790M mutation. Conclusion: The patients with EGFR exon 19 deletion mutation and long-term treatment with EGFR-TKI exposure demonstrated a high prevalence of T790M mutation. These data are potentially important for clinical decision making in the NSCLC patients with EGFR mutation. Keywords: non-small cell lung cancer, EGFR, EGFR-TKI, T790M
Journal of Thoracic Oncology
Vol. 12 No. 1S
P3.02b-027 Detection of EGFR Mutations in Plasma of Lung Adenocarcinoma Patients Using Real-Time PCR and Mass Spectrometry Topic: EGFR Biomarkers Rossella Bruno,1 Elisabetta Macerola,1 Vincenzo Condello,1 Cristiana Lupi,2 Alessandro Ribechini,3 Antonio Chella,4 Greta Alì,2 Gabriella Fontanini1 1Department of Surgical, Medical, Molecular Pathology and Critical Area, University of Pisa, Pisa/Italy, 2Unit of Pathological Anatomy, University Hospital of Pisa, Pisa/Italy, 3Endoscopic Section of Pneumology, University Hospital of Pisa, Pisa/Italy, 4Unit of Pneumology, University Hospital of Pisa, Pisa/Italy Background: Lung adenocarcinoma patients harboring sensitizing EGFR mutations can benefit from treatment with tyrosine kinase inhibitors (TKI). Whenever tumor tissue is inadequate or unavailable, detection of EGFR mutations in circulating cell-free tumor (ct) DNA from plasma is crucial to predict and monitor response to therapy. In this study we compared EGFR status between tumor tissue and plasma, using real-time PCR. Moreover, we evaluated the adequacy of ctDNA for a multi-target mass spectrometry (MS) analysis. Methods: EGFR mutations were investigated in paired plasma and tumor tissues from a prospective series of 105 lung adenocarcinoma patients: 79 had no prior TKI treatment and 26 underwent re-biopsy for TKI-acquired resistance. Molecular analyses were performed on tissue by a routine MS test (evaluation of 307 hot-spots in 10 genes including EGFR) and on ctDNA by a validated Scorpion/LNA real-time PCR (evaluation of 30 EGFR mutations). In the 26 postTKI patients ctDNA analysis was performed also by MS. Results: 1-Plasma versus tissue by real-time PCR: overall sensitivity, specificity and concordance were 64.8%, 95.4% and 82%. In preTKI patients, 17 harboured EGFR sensitizing mutations on tissue, 10 detected also in plasma (sensitivity 58.8 %, specificity 100%, concordance 91%). All 26 postTKI patients preserved EGFR sensitizing mutations. Regarding the detection of EGFR T790M resistance mutation, sensitivity, specificity and concordance were 63.6%, 80% and 73%. Specifically, 11 patients were T790M-positive (42%): 7 on both specimens, 4 only on tissue and 3 only on ctDNA. 2-Plasma versus tissue by MS: sensitivity, specificity and concordance for T790M were 50%, 93.3% and 76%.