Exploration of resistance mechanisms for epidermal growth factor receptor‐tyrosine kinase inhibitors based on plasma analysis by digital polymerase chain reaction and next‐generation sequencing

Abstract

Liquid biopsy offers a potential alternative to tissue biopsy for detection of genetic alterations in cancer, and it has been introduced into clinical practice to detect the tyrosine kinase inhibitor (TKI) resistance‐conferring T790M mutation of epidermal growth factor receptor (EGFR) in patients with non‐small‐cell lung cancer (NSCLC). We prospectively collected tumor and plasma samples from 25 NSCLC patients who harbored activating mutations of EGFR and experienced failure of treatment with afatinib. The samples were analyzed by digital PCR (dPCR) and next‐generation sequencing (NGS). T790M was detected in plasma with a respective sensitivity and specificity of 83.3% and 70.0% by dPCR and 50.0% and 70.0% by NGS relative to analysis of corresponding tumor samples. Quantitation of T790M based on the ratio of the number of T790M alleles to that of activating mutation alleles (T/A ratio) improved the specificity of plasma analysis to 100% for both dPCR and NGS without a reduction in sensitivity. Although several afatinib resistance mechanisms other than T790M—including copy number gain of NRAS or MET—were identified in tumor samples, the corresponding genetic alterations were not detected in plasma. TP53 mutations were frequently identified in plasma and tumor samples, with most such mutations also having been detected before afatinib treatment. The presence of de novo TP53 mutations was associated with reduced progression‐free survival. Quantitation of T790M in plasma is thus a clinically relevant approach to determine the T790M status of tumors. In addition, genetic alterations coexisting with EGFR mutations can affect the efficacy of EGFR‐TKI treatment.

Longitudinal monitoring of somatic genetic alterations in circulating cell‐free DNA during treatment with epidermal growth factor receptor–tyrosine kinase inhibitors

“…NGS, the results for which are usually expressed as allele frequency, has the potential to become widely adopted for liquid biopsy analysis because it is able to simultaneously identify various genetic alterations including those responsible for resistance to targeted therapy. [24][25][26][27][28][29][30][31] Our findings suggest that the monitoring of allele frequency for EGFRactivating mutations in cfDNA by NGS may also be useful for the prediction of EGFR-TKI treatment efficacy.…”

“…Such a pattern was also evident for the allele frequency of EGFR ‐activating mutations (the ratio of the number of activating mutations to the total number of EGFR alleles) as determined by ddPCR (data not shown). NGS, the results for which are usually expressed as allele frequency, has the potential to become widely adopted for liquid biopsy analysis because it is able to simultaneously identify various genetic alterations including those responsible for resistance to targeted therapy . Our findings suggest that the monitoring of allele frequency for EGFR ‐activating mutations in cfDNA by NGS may also be useful for the prediction of EGFR‐TKI treatment efficacy.…”

“…Osimertinib manifested a robust efficacy even for patients with a high number of T790M alleles (n = 2), with these patients showing clearance of the mutation during treatment. These findings indicate that highly sensitive methods might detect a small population of T790M alleles in cfDNA that is not responsible for resistance to EGFR‐TKIs and that quantitative evaluation of T790M mutant alleles might be important …”

Prognostic value of TP53 concurrent mutations for EGFR- TKIs and ALK-TKIs based targeted therapy in advanced non-small cell lung cancer: a meta-analysis

“…Multiple studies have confirmed that it was reliable to identify guideline-recommended biomarkers in patients with mNSCLC through a comprehensive cfDNA test, especially with the highly sensitive and specific NGS-based detection. [67,68] For example, in the Non-invasive versus Invasive Lung Evaluation (NILE) study, plasma NGS tests in previously untreated mNSCLC showed high sensitivity and specificity with high tissue concordance, significantly faster return of results, and was even more rapidly and completely than the standard-of-care tissue genotyping [67]. The convenience, minimal invasiveness and repeatability of liquid biopsy enable the utility of cfDNA detection as a significant way not only for the detection of targetable driver alterations, but also for exploration of mechanisms of resistance to such drugs.…”

“…Previous studies have suggested that TP53 concurrent mutation might adversely impact the survival of patients with advanced NSCLC treated with EGFR-TKIs or ALK-TKIs for oncogenic EGFR or ALK mutations [27][28][29][30]. In addition, some preclinical studies indicated that TP53 might be one of the mechanisms that potentially confer resistance to EGFR-and ALK-TKIs treatment [31,32]. However, several other studies found no correlation between TP53 mutations and survival of patients with EGFR-mutated advanced NSCLC and treated with EGFR-TKIs [33][34][35].…”