Noninvasive Diagnosis of Actionable Mutations by Deep Sequencing of Circulating Free DNA in Lung Cancer from Never-Smokers: A Proof-of-Concept Study from BioCAST/IFCT-1002

Purpose: Tumor somatic mutation analysis is part of the standard management of metastatic lung cancer. However, physicians often have to deal with small biopsies and consequently with challenging mutation testing. Circulating free DNA (cfDNA) is a promising tool for accessing the tumor genome as a liquid biopsy. Here, we evaluated next-generation sequencing (NGS) on cfDNA samples obtained from a consecutive series of patients for the screening of a range of clinically relevant mutations. Experimental Design: A total of 107 plasma samples were collected from the BioCAST/IFCT-1002 lung cancer study (never-smokers cohort). Matched tumor DNA (tDNA) was obtained for 68 cases. Multiplex PCR-based assays were designed to target specific coding regions in EGFR, KRAS, BRAF, ERBB2, and PI3KCA genes, and amplicon sequencing was performed at deep coverage on the cfDNA/tDNA pairs using the NGS IonTorrent Personal Genome Machine Platform. Results: CfDNA concentration in plasma was significantly associated with both stage and number of metastatic sites. In tDNA, 50 mutations (36 EGFR, 5 ERBB2, 4 KRAS, 3 BRAF, and 2 PIK3CA) were identified, of which 26 were detected in cfDNA. Sensitivity of the test was 58% (95% confidence interval, 43%–71%) and the estimated specificity was 87% (62%–96%). Conclusion: These data demonstrate the feasibility and potential utility of mutation screening in cfDNA using IonTorrent NGS for the detection of a range of tumor biomarkers in patients with metastatic lung cancer. Clin Cancer Res; 20(17); 4613–24. ©2014 AACR.

[1]  C. Paweletz,et al.  Noninvasive Detection of Response and Resistance in EGFR-Mutant Lung Cancer Using Quantitative Next-Generation Genotyping of Cell-Free Plasma DNA , 2014, Clinical Cancer Research.

[2]  R. McCormack,et al.  First-line gefitinib in Caucasian EGFR mutation-positive NSCLC patients: a phase-IV, open-label, single-arm study , 2013, British Journal of Cancer.

[3]  Jaafar Bennouna,et al.  Plasma is a better source of tumor-derived circulating cell-free DNA than serum for the detection of EGFR alterations in lung tumor patients. , 2013, Lung cancer.

[4]  Philip Quirke,et al.  Accurately Identifying Low‐Allelic Fraction Variants in Single Samples with Next‐Generation Sequencing: Applications in Tumor Subclone Resolution , 2013, Human mutation.

[5]  H. Blanché,et al.  [Lung cancer in never smokers: a French national cohort (BioCAST/IFCT-1002)]. , 2013, Revue des maladies respiratoires.

[6]  K. Nishio,et al.  Detection of epidermal growth factor receptor T790M mutation in plasma DNA from patients refractory to epidermal growth factor receptor tyrosine kinase inhibitor , 2013, Cancer science.

[7]  B. Milleron,et al.  [Lung cancer in never smokers: a French national cohort (BioCAST/IFCT-1002)]. , 2013, Revue des maladies respiratoires.

[8]  Jae Cheol Lee,et al.  Detection of EGFR mutations in circulating free DNA by PNA-mediated PCR clamping , 2013, Journal of experimental & clinical cancer research : CR.

[9]  D. Planchard,et al.  Detection of circulating tumor cells harboring a unique ALK rearrangement in ALK-positive non-small-cell lung cancer. , 2013, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[10]  T. Mok,et al.  Detection of EGFR-activating mutations from plasma DNA as a potent predictor of survival outcomes in FASTACT 2: A randomized phase III study on intercalated combination of erlotinib (E) and chemotherapy (C). , 2013 .

[11]  N. Rosenfeld,et al.  Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA , 2013, Nature.

[12]  Philip Hugenholtz,et al.  Shining a Light on Dark Sequencing: Characterising Errors in Ion Torrent PGM Data , 2013, PLoS Comput. Biol..

[13]  Vincent Ferretti,et al.  Feasibility of real time next generation sequencing of cancer genes linked to drug response: Results from a clinical trial , 2013, International journal of cancer.

[14]  Carlos Caldas,et al.  Analysis of circulating tumor DNA to monitor metastatic breast cancer. , 2013, The New England journal of medicine.

[15]  A. Jakobsen,et al.  The prognostic value of KRAS mutated plasma DNA in advanced non-small cell lung cancer. , 2013, Lung cancer.

[16]  L. Pusztai,et al.  Cancer heterogeneity: implications for targeted therapeutics , 2013, British Journal of Cancer.

[17]  Bruce E Johnson,et al.  The Introduction of Systematic Genomic Testing for Patients with Non–Small-Cell Lung Cancer , 2012, Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer.

[18]  G. Parmigiani,et al.  Detection of Chromosomal Alterations in the Circulation of Cancer Patients with Whole-Genome Sequencing , 2012, Science Translational Medicine.

[19]  U. Banerji,et al.  Multi-Purpose Utility of Circulating Plasma DNA Testing in Patients with Advanced Cancers , 2012, PloS one.

[20]  Torunn I Yock,et al.  Ultrasensitive measurement of hotspot mutations in tumor DNA in blood using error-suppressed multiplexed deep sequencing. , 2012, Cancer research.

[21]  J. Soria,et al.  Tumour molecular profiling for deciding therapy—the French initiative , 2012, Nature Reviews Clinical Oncology.

[22]  Jun Wang,et al.  Comparison of Epidermal Growth Factor Receptor Mutation Statuses in Tissue and Plasma in Stage I–IV Non-Small Cell Lung Cancer Patients , 2012, Respiration.

[23]  J. Chung-Man Ho,et al.  EGFR Array: Uses in the Detection of Plasma EGFR Mutations in Non–Small Cell Lung Cancer Patients , 2012, Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer.

[24]  B. Faircloth,et al.  Primer3—new capabilities and interfaces , 2012, Nucleic acids research.

[25]  Sébastien Couraud,et al.  Lung cancer in never smokers--a review. , 2012, European journal of cancer.

[26]  J. Shih,et al.  Afatinib for patients with lung adenocarcinoma and epidermal growth factor receptor mutations (LUX-Lung 2): a phase 2 trial. , 2012, The Lancet. Oncology.

[27]  Helga Thorvaldsdóttir,et al.  Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration , 2012, Briefings Bioinform..

[28]  L. Bubendorf,et al.  The challenge of NSCLC diagnosis and predictive analysis on small samples. Practical approach of a working group. , 2012, Lung cancer.

[29]  P. A. Futreal,et al.  Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. , 2012, The New England journal of medicine.

[30]  E. Felip,et al.  Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial. , 2012, The Lancet. Oncology.

[31]  Koichi Goto,et al.  Epidermal Growth Factor Receptor Mutation Status in Circulating Free DNA in Serum: From IPASS, a Phase III Study of Gefitinib or Carboplatin/Paclitaxel in Non-small Cell Lung Cancer , 2012, Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer.

[32]  Kikuya Kato,et al.  Quantitative Detection of EGFR Mutations in Circulating Tumor DNA Derived from Lung Adenocarcinomas , 2011, Clinical Cancer Research.

[33]  S. Kimura,et al.  A Noninvasive System for Monitoring Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors with Plasma DNA , 2011, Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer.

[34]  K. Matsuo,et al.  Heterogeneous distribution of EGFR mutations is extremely rare in lung adenocarcinoma. , 2011, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[35]  Jun Ma,et al.  Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomised, phase 3 study. , 2011, The Lancet. Oncology.

[36]  Hyae-Young Kim,et al.  Circulating Cell-Free DNA in Plasma of Never Smokers with Advanced Lung Adenocarcinoma Receiving Gefitinib or Standard Chemotherapy as First-Line Therapy , 2011, Clinical Cancer Research.

[37]  Klaus Pantel,et al.  Cell-free nucleic acids as biomarkers in cancer patients , 2011, Nature Reviews Cancer.

[38]  H. Hakonarson,et al.  ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data , 2010, Nucleic acids research.

[39]  V. Rusch,et al.  Pathologic diagnosis of advanced lung cancer based on small biopsies and cytology: a paradigm shift. , 2010, Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer.

[40]  Caicun Zhou,et al.  Prediction of epidermal growth factor receptor mutations in the plasma/pleural effusion to efficacy of gefitinib treatment in advanced non-small cell lung cancer , 2010, Journal of Cancer Research and Clinical Oncology.

[41]  X. Wang,et al.  Potential Clinical Significance of a Plasma-Based KRAS Mutation Analysis in Patients with Advanced Non–Small Cell Lung Cancer , 2010, Clinical Cancer Research.

[42]  T. Mok,et al.  Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. , 2009, The New England journal of medicine.

[43]  P. Jänne,et al.  Noninvasive Detection of EGFR T790M in Gefitinib or Erlotinib Resistant Non–Small Cell Lung Cancer , 2009, Clinical Cancer Research.

[44]  Mehmet Toner,et al.  Detection of mutations in EGFR in circulating lung-cancer cells. , 2008, The New England journal of medicine.

[45]  K. Nishio,et al.  Evaluation of epidermal growth factor receptor mutation status in serum DNA as a predictor of response to gefitinib (IRESSA) , 2007, British Journal of Cancer.

[46]  R. Rosell,et al.  Methylation patterns and K-ras mutations in tumor and paired serum of resected non-small-cell lung cancer patients. , 2003, Cancer letters.

[47]  D. Saville.  Multiple Comparison Procedures: The Practical Solution , 1990 .

[48]  J. Pignon,et al.  A multicenter blinded study evaluating EGFR and KRAS mutation testing methods in the clinical non-small cell lung cancer setting--IFCT/ERMETIC2 Project Part 1: Comparison of testing methods in 20 French molecular genetic National Cancer Institute platforms. , 2014, The Journal of molecular diagnostics : JMD.

[49]  Peiyong Jiang,et al.  Cancer genome scanning in plasma: detection of tumor-associated copy number aberrations, single-nucleotide variants, and tumoral heterogeneity by massively parallel sequencing. , 2013, Clinical chemistry.

[50]  Lin Huan,et al.  Screening for Epidermal Growth Factor Receptor Mutations in Lung Cancer , 2010 .

[51]  S. Goodman,et al.  Circulating mutant DNA to assess tumor dynamics , 2008, Nature Medicine.