Ultra-deep next-generation sequencing of plasma cell-free DNA in patients with advanced lung cancers: results from the Actionable Genome Consortium

Abstract

Background: Noninvasive genotyping using plasma cell-free DNA (cfDNA) has the potential to obviate the need for some invasive biopsies in cancer patients while also elucidating disease heterogeneity. We sought to develop an ultra-deep plasma next-generation sequencing (NGS) assay for patients with non-small-cell lung cancers (NSCLC) that could detect targetable oncogenic drivers and resistance mutations in patients where tissue biopsy failed to identify an actionable alteration.Patients and methods: Plasma was prospectively collected from patients with advanced, progressive NSCLC. We carried out ultra-deep NGS using cfDNA extracted from plasma and matched white blood cells using a hybrid capture panel covering 37 lung cancer-related genes sequenced to 50 000Â raw target coverage filtering somatic mutations attributable to clonal hematopoiesis. Clinical sensitivity and specificity for plasma detection of known oncogenic drivers were calculated and compared with tissue genotyping results. Orthogonal ddPCR validation was carried out in a subset of cases.Results: In 127 assessable patients, plasma NGS detected driver mutations with variant allele fractions ranging from 0.14% to 52%. Plasma ddPCR for EGFR or KRAS mutations revealed findings nearly identical to those of plasma NGS in 21 of 22 patients, with high concordance of variant allele fraction (r ¼ 0.98). Blinded to tissue genotype, plasma NGS sensitivity for de novo plasma detection of known oncogenic drivers was 75% (68/91). Specificity of plasma NGS in those who were driver-negative by tissue NGS was 100% (19/19). In 17 patients with tumor tissue deemed insufficient for genotyping, plasma NGS identified four KRAS mutations. In 23 EGFR mutant cases with acquired resistance to targeted therapy, plasma NGS detected potential resistance mechanisms, including EGFR T790M and C797S mutations and ERBB2 amplification.Conclusions: Ultra-deep plasma NGS with clonal hematopoiesis filtering resulted in de novo detection of targetable oncogenic drivers and resistance mechanisms in patients with NSCLC, including when tissue biopsy was inadequate for genotyping.

Two‐point‐NGS analysis of cancer genes in cell‐free DNA of metastatic cancer patients

“…Regarding the frequency of mutation in cfDNA, our data confirmed the literature evidences with SNVs in TP53 , PIK3CA and KRAS and CNVs in FGFR3 as the most commonly observed in breast and lung . Our data demonstrate the absence of a correlation with mutation type and primary tumor organ since we identified mutation in 22 different genes among 13 different solid tumors without specific prevalence (Figure ).…”

“…Regarding the frequency of mutation in cfDNA, our data confirmed the literature evidences with SNVs in TP53, PIK3CA and KRAS and CNVs in FGFR3 as the most commonly observed in breast and lung. 18,19 Our data demonstrate the absence of a correlation with mutation type and primary tumor organ since we identified mutation in 22 different genes among 13 different solid tumors without specific prevalence ( Figure 4). The sniper clones leading to disease progression can be distinguished by cfDNA two-point-NGS analysis pinpointing the needs of grouping patients on truly growing clones instead of on primary tumor organ in clinical trials.…”

Assessing the Concordance of Genomic Alterations between Circulating-Free DNA and Tumour Tissue in Cancer Patients

“…Release of genomic DNA from white blood cells, resulting in contamination of cfDNA, can be a consequence of delayed processing. The presence of contamination in cfDNA can, however, be accounted for by sequencing white blood cells and filtering somatic mutations attributable to clonal haematopoiesis [73], although this approach will not neutralise the diluting effect of such contamination on the tumour fraction of cfDNA. The necessity of extracting cfDNA from plasma was challenged by a study conducted by Sefrioui et al, The group compared concordance rates in a cohort of 17 mCRC patients and established 93% and 88% mutation detection rates for cfDNA isolated from plasma and crude plasma samples, respectively, suggesting that extraction of cfDNA from plasma may enhance detection by increasing tumour purity [74].…”

Prognostic potential of liquid biopsy tracking in the posttreatment surveillance of patients with nonmetastatic nasopharyngeal carcinoma

“…Liquid biopsy via circulating tumor cells or cell‐free DNA analysis has value as a more cost‐effective method that is easier to perform and less invasive than currently available approaches (eg, tissue biopsies or imaging scans) for the diagnosis of early cancer, monitoring treatment response, and detecting tumor recurrence . It is important to note that liquid biopsy can be repeated easily when following the patient during or after treatment, allowing for the early detection of minimal residual disease, locoregional recurrence (LRR), and distant micrometastasis (DM) .…”