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

Abstract

Background Although the efficacy of molecularly target agents in vitro, their use in routine setting is limited mainly to the use of anti‐HER2 and antiEGFR agents in vivo. Moreover, core biopsy of a single cancer site may not be representative of the whole expanding clones and cancer molecular profile at relapse may differ with respect to the primary tumor. Methods We assessed the status of a large panel of cancer driver genes by cell‐free DNA (cfDNA) analysis in a cohort of 68 patients with 13 different solid tumors at disease progression. Whenever possible, a second cfDNA analysis was performed after a mean of 2.5 months, in order to confirm the identified clone(s) and to check the correlation with clinical evolution. Results The approach was able to identify clones plausibly involved in the disease progression mechanism in about 65% of cases. A mean of 1.4 mutated genes (range 1‐3) for each tumor was found. Point mutations in TP53, PIK3CA, and KRAS and copy number variations in FGFR3 were the gene alterations more commonly observed, with a rate of 48%, 20%, 16%, and 20%, respectively. Two‐points‐Next‐Generation Sequencing (NGS) analysis demonstrated statistically significant correlation between allele frequency variation and clinical outcome (P = .026). Conclusions Irrespective of the primary tumor mutational burden, few mutated genes are present at disease progression. Clinical outcome is consistent with variation of allele frequency of specific clones indicating that cfDNA two‐point‐NGS analysis of cancer driver genes could be an efficacy tool for precision oncology.

Private somatic mutations identified with liquid biopsy lead tumor progression in solid cancers

“…In comparison with data from our previous study [5] , showing that at the beginning of tumor expansion there was a consistent, although variable, mutational burden from tens to hundreds, disease relapse is generally characterized by a "driver" clone that is responsible for metastases. The curve in Figure 2, simulating a Gaussian bell shape (similar to the typical population curve), underlines this concept.…”

“…cfDNA was extracted from 4 mL of plasma using AVENIO ctDNA Expanded Kit according to the manufacturer's instructions. cfDNA quality and quantity were verified as described in Palmieri et al [5] . cfDNA sequencing was performed using AVENIO circulating tumor DNA (ctDNA) Expanded Kit (Roche, Basel, Switzerland) on Illumina NextSeq 550 (Illumina, San Diego, CA, USA).…”

“…New technologies such as the liquid biopsy of cell-free DNA (cfDNA) facilitate proper detection of somatic tumor mutations, which are the key mutation of specific cancer driver genes. The technique is non-invasive, is a valuable alternative to physical biopsies, and opens new avenues for personalized medicine [5] .…”

A pilot study of next generation sequencing–liquid biopsy on cell-free DNA as a novel non-invasive diagnostic tool for Klippel–Trenaunay syndrome

“…Plasma sample from patient 2 was obtained as described in Palmieri et al. 9 Briefly, at least 10 ml of whole blood was collected in EDTA tubes. Plasma was separated within 5 h through two different centrifugation steps (10’ × 1600 g and 10’ × 3000 g at RT), obtaining at least 3 ml of plasma.…”

“…7,8 During the last years, next generation sequencing (NGS)–liquid biopsy has emerged as an innovative non-invasive technique for the identification of key mutations that are responsible for tumor growth allowing to optimize diagnosis, monitoring, and therapeutic choice. 9–11 Therefore, cell-free DNA (cfDNA) analysis overcomes the space-time profile constraint of physical biopsies and opens a new scenario for vascular malformations where tissue biopsy represents an invasive high risky, sometimes life-threatening, diagnostic procedure. The use of liquid biopsy would also improve the opportunities to check illness evolution on a molecular basis.…”