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

Abstract

The advent of genomic based personalized medicine has led to multiple advances in the molecular characterization of many tumor types, such as non-small cell lung cancer (NSCLC). NSCLC is diagnosed in most cases on small tissue samples that may be not always sufficient for EGFR mutational assessment to select patients for first and second generations' tyrosine kinase inhibitors (TKIs) therapy. In patients without tissue availability at presentation, the analysis of cell free DNA (cfDNA) derived from liquid biopsy samples, in particular from plasma, represent an established alternative to provide EGFR mutational testing for treatment decision making. In addition, a new paradigm for TKIs resistance management was recently approved by Food and Drug Administration, supporting the liquid biopsy based genotyping prior to tissue based genotyping for the detection of T790M mutation to select patients for third generation TKIs. In these settings, real time PCR (RT-PCR) and digital PCR 'targeted' methods, which detect known mutations by specific probes, have extensively been adopted. Taking into account the restricted reference range and the limited multiplexing power of these targeted methods, the performance of liquid biopsy analyses may be further improved by next generation sequencing (NGS). While most tissue based NGS genotyping is well established, liquid biopsy NGS application is challenging, requiring a careful validation of the whole process, from blood collection to variant calling. Here we review this evolving field, highlighting those methodological points that are crucial to accurately select NSCLC patients for TKIs treatment administration by NGS on cfDNA.

Technical Aspects for the Evaluation of Circulating Nucleic Acids (CNAs): Circulating Tumor DNA (ctDNA) and Circulating MicroRNAs

“…In the majority of clinical trails, EDTA containing tubes are used for blood collection (4-9 [9]). Using these tubes clotting is inhibited, and thus it is possible to recover plasma that represent the matrix of choice for ctDNA extraction.…”

“…Indeed, the more the time passes, the more is the risk of WBCs lysis, leading again to ctDNA contamination with wild-type background DNA. Moreover ctDNA is associated with a high turnover (15 min halflife), and therefore after blood collection, it is recommended to proceed with plasma preparation by centrifugation within 1 h [11]. Plasma can be stored for a long period at −20 °C or immediately processed for ctDNA extraction.…”

Cell free DNA analysis by SiRe® next generation sequencing panel in non small cell lung cancer patients: focus on basal setting

“…In particular for EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib, erlotinib and afatinib the identification of activating EGFR mutations in exon 18, 19 and 21 is mandatory before the first line treatment (3)(4)(5)(6)(7)(8). To date according to the European Medicines Agency guidelines, in patients without tissue availability, only for EGFR TKIs treatment decision making, cell-free DNA (cfDNA) can be used as a fast and non-invasive surrogate for EGFR mutational testing (9)(10)(11)(12)(13). However, the assessment of 1 gene mutations in cfDNA is challenging, in particular in basal setting, for the detection of first and second TKIs generation EGFR sensitizing mutations, due to the very low concentration of circulating tumor DNA, that represent only a small fraction of the total cfDNA (9,10,(12)(13)(14)(15).…”

“…To date according to the European Medicines Agency guidelines, in patients without tissue availability, only for EGFR TKIs treatment decision making, cell-free DNA (cfDNA) can be used as a fast and non-invasive surrogate for EGFR mutational testing (9)(10)(11)(12)(13). However, the assessment of 1 gene mutations in cfDNA is challenging, in particular in basal setting, for the detection of first and second TKIs generation EGFR sensitizing mutations, due to the very low concentration of circulating tumor DNA, that represent only a small fraction of the total cfDNA (9,10,(12)(13)(14)(15). Thus, the clinical implementation of next generation techniques, such as next generation sequencing (NGS) or digital PCR (dPCR) based assay is crucial (9,10,12,13,16,17).…”

“…However, the assessment of 1 gene mutations in cfDNA is challenging, in particular in basal setting, for the detection of first and second TKIs generation EGFR sensitizing mutations, due to the very low concentration of circulating tumor DNA, that represent only a small fraction of the total cfDNA (9,10,(12)(13)(14)(15). Thus, the clinical implementation of next generation techniques, such as next generation sequencing (NGS) or digital PCR (dPCR) based assay is crucial (9,10,12,13,16,17). In a recent study of ours, we validated the SiRe ® NGS panel for mutation detection in EGFR, KRAS, NRAS, BRAF, cKIT and PDGFR starting from cfDNA retrieved from patients with different solid tumors (NSCLC, metastatic colo-rectal cancer, melanoma and gastrointestinal stromal tumor) (13).…”