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

Abstract

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. Ó2014 AACR.

The Importance of EGFR Mutation Detection Method for the Correct Clinical Management of Patients with Non-Small Cell Lung Cancer: A Case Report

“…Moreover, we agree with other author [8,16] suggesting the potential benefit of using of the new sensitive technologies to non-invasively screen of activating mutations in NSCLC patients, such as in plasma or other human fluid cell-free DNA. Thus, these findings may provide another aspect on future targeted molecular therapy.…”

Next generation sequencing techniques in liquid biopsy: focus on non-small cell lung cancer patients

“…By using focused gene panels the coverage can be narrowed down on clinically relevant targets so that each read is sequenced thousands of times, ensuring a high degree of sensitivity (8,11). As a general rule, this approach is named ultra-deep sequencing and enables to detect low abundant ctDNA in blood or other body fluids.…”

“…In particular, when all of the known coding exons in a genome are captured an 'exome sequencing' is obtained; this approach is usually carried out in research practice (7,10). Conversely, a target library composed by specific clinical relevant gene regions is obtained when narrow next generation panels are implemented (8,11). This latter approach better suits clinical practice and cfDNA applications, where a low limit of detection (LOD) is mandatory.…”

“…Today, the human reference genome version currently used is 19 th (hg19) (7-10); when differences occur between a base call and its aligned position to the hg19, variants are called. The implementation of different bioinformatics pipelines, either commercially available or developed in-house, can lead to a variability in the variant calling (7,8,11). Thus, the appropriate software settings should be optimized during the validation process taking into account the depth of coverage (number of reads covering a given base position), the average depth of coverage (average number of overlapping reads within the total sequenced area), the uniformity of coverage (distribution of coverage within specific targeted regions) and the specific parameter set for hotspots and non-hotspots variants ( Table 1) (10).…”

Is Circulating Tumor DNA (Ctdna) Use Ready For Prime Time? Applications and Challenges of Ctdna in the Era of Precision Oncology

“…Cell-free DNA consists of noncancerous nucleic acids and circulating tumor DNA (ctDNA). The proportion of ctDNA depends on the tumor cell of origin and stage of malignancy [2][3][4][5]. Peripheral blood biopsies can detect single nucleotide variants, indels, copy number variants, rearrangements and fusions, non-invasively, avoiding risk associated with repeat tissue biopsies.…”