Therapy Monitoring of EGFR-Positive Non–Small-Cell Lung Cancer Patients Using ddPCR Multiplex Assays

Abstract

The detection of EGFR-sensitizing and EGFR-resistance mutations in advanced nonesmall-cell lung cancer patients is important for the selection and monitoring of EGFR tyrosine-kinase inhibitor therapy. Droplet digital PCR (ddPCR) multiplex assays allow for sensitive and simultaneous detection of multiple mutations in cell-free DNA (cfDNA) with a minimum of extract needed and at lower cost. Patients were screened for the EGFR tyrosine-kinase inhibitoresensitizing mutations Ex19Del, L858R, L861Q, G719S, and S768I using a novel ddPCR pentaplex assay. Patients who tested positive subsequently were monitored during treatment for the EGFR-sensitizing mutation and two EGFR-resistance mutations, T790M and C797S, using a ddPCR monitor triplex assay. The ddPCR multiplex assays enabled reliable detection of each mutation with a fractional abundance of at least 0.1%. For six patients, longitudinal data were analyzed and the ddPCR results provided a good reflection of the course of the disease and radiologic response. This study confirms that ddPCR on cfDNA supports the diagnosis and therapy selection, and shows that ddPCR multiplex assays on cfDNA could be a valuable additional diagnostic tool for therapy monitoring of nonesmall-cell lung cancer patients.

Liquid biopsy: a step closer to transform diagnosis, prognosis and future of cancer treatments

“…The target sequence is then amplified by end point PCR in each droplet and relative fractions of positive and negative droplets counted (fluorescent probes) that provide relative quantification of target samples [ 3 , 98 , 99 ]. Digital PCR has been shown to detect ctDNA in more than 75% of patients with advanced CRC, BC, and PC and to a good extent in patients with localized tumors [ 38 , 100 ]. BEAMing (beads, emulsions, amplification, and magnetics), on the other hand, is a modification of emulsion PCR where several different templates are amplified within a single tube, each in different compartments (or emulsion droplets) but along with primer bound beads that are recovered with the help of a magnetic field or centrifugal force [ 3 , 40 ].…”

“…Conversely, increased and shorter nuclear cfDNA content correlated with tumor recurrence [ 207 ]. cfDNA analysis, thus, offers promising breakthroughs in non-invasive liquid biopsy technologies [ 100 ]. Circulating cell-free miRNAs associated with EVs, apoptotic bodies, lipoproteins or AGOs (miRISC constituents) are known to be quite stable in a wide variety of body fluids like plasma or serum [ 208 ].…”

Resistance to TKIs in EGFR-Mutated Non-Small Cell Lung Cancer: From Mechanisms to New Therapeutic Strategies

“…As far as the clinician’s therapeutic strategy is concerned, ICIs combined with chemotherapy may consist of an alternative therapeutic approach, whereas it remains controversial whether rechallenging EGFR TKIs following a combination of chemotherapy and immunotherapy could be beneficial to some patients. Therapeutic prospects might improve as long as an early and precise diagnosis of EGFR TKIs resistance is achieved [ 178 ].…”

Investigating the Retained Inhibitory Effect of Cobimetinib against p.P124L Mutated MEK1: A Combined Liquid Biopsy and in Silico Approach

“…Notably, a different trend between the amount of ctDNA (copies/ml) and MAF was already reported, and cautiously interpreted as a sign of response. The authors recommended looking at both the MAF and the total amount of ctDNA for an overall view of the patient’s status [ 55 ]. As for our case, again, the availability of a single blood sample collected very close to the therapy switch makes the gathering of definite conclusions very difficult.…”