Patients were eligible if they had advanced NSCLC involving mutation(s), were clinically resistant to EGFR TKIs (gefitinib, erlotinib, icotinib, or afatinib), and were undergoing re-biopsy for genotyping as part of their routine clinical care. of the tests is necessary for the optimization of blood biopsy by ctDNA. Other factors that affect the tests, except technology, are still unclear. Here, we genotyped from either tissue or blood biopsy samples for detecting various mutations linked to TKI resistance in patients with NSCLC. In particular, we focused on the mutation T790M. DNA extracted from both types of biopsies was sequenced using the conventional ARMS. We chose this system for two reasons: first, it is still the standard system used for tissue biopsy at our hospital; second, we wanted to know whether there are other factors, except technology, affecting the results. The findings of this study may shed light on the optimization of the performance and reliability of blood biopsy, especially relative to tissue biopsy, for diagnosing acquired EGFR TKI resistance associated with T790M. Cops5 Materials and methods Study design This observational study was conducted on patients treated at West China Hospital (Chengdu, Peoples Republic of China) between 2014 and 2016. Patients were eligible if they had advanced NSCLC involving mutation(s), were clinically resistant to EGFR TKIs (gefitinib, erlotinib, icotinib, or afatinib), and were undergoing re-biopsy for genotyping as part of their routine clinical care. The study was approved by the Biomedical Research Ethics Committee of West China Hospital of Sichuan University, and all patients signed the informed consent form. Blood sampling and sequencing Peripheral blood was collected within 2 weeks of re-biopsy into a 10-mL vacutainer containing ethylenediaminetetraacetic acid. Blood samples were transported to our laboratory within 2 h of being drawn, plasma was isolated by centrifugation at 2,000 for 10 min, and the supernatant was further cleared at 8,000 for an additional 10 min. The Circulating Nucleic Acid Kit (AmoyDx, Xiamen, Peoples Republic of China) was used to isolate ctDNA according to the manufacturers protocol. genotyping was performed on tissue biopsies according to standard institutional procedures in a certified laboratory, with conventional ARMS sequencing carried out using the Human EGFR Gene Mutations Fluorescence PCR Diagnostic Kit (AmoyDx). The same kit was used to genotype from ctDNA. This kit has been approved for clinical use by the China Food and Drug Administration since 2010. Statistical analysis Diagnostic concordance between ARMS sequencing of tissue-derived DNA or ctDNA was assessed using Cohens , and the concordance was assessed for significance using the McNemar test. Percent agreement values were calculated based on the main diagonal in 22 tables. All statistical analyses were performed with SPSS 20.0 (IBM Corp., Armonk, NY, USA), and the significance threshold was set as mutation?19 Del30 (66.7)?L858R15 (33.3)?Other0 (0.0)Type of initial EGFR TKI?Gefitinib37 (82.2)?Erlotinib4 (8.9)?Icotinib4 (8.9)Line of initial EGFR TKI?First39 (86.7)?Second6 (13.3)Response to initial EFGR TKI?CR/PR/SD43 (93.3)?PD2 (6.7)Second biopsy?Tissue biopsy38 (84.4)?Blood biopsy31 (68.9)?Both24 (53.3) Open in a separate window Abbreviations: EGFR, epidermal growth factor receptor; TKI, tyrosine kinase inhibitor; CR, complete response; PR, partial response; SD, stable disease; PD progressive disease. EGFR genotyping in tissue biopsy and plasma ctDNA Among patients whose tissue biopsy was sequenced, 25 showed two mutations, 12 showed one mutation, and 1 had three mutations (Table 2). Among patients whose plasma ctDNA was sequenced, 14 had two types of mutation, 12 had no mutation, and 5 showed one mutation. Table 2 genotyping of initial and secondary biopsy genotypingmutation status based on tissue biopsy or plasma ctDNA with respect to (A) exon 19 deletion, (B) L858R mutation, or (C) T790M mutation. Abbreviation: ctDNA, circulating tumor DNA. Table 3 genotyping in patients who received both tissue and plasma ctDNA tests genotypinggene of patients with NSCLC for mutations PFK-158 associated with TKIs resistance from re-biopsied tumor DNA or from plasma PFK-158 ctDNA. Our results suggest that ctDNA can be a suitable template for genotyping in these patients, although the positive rate for T790M (37.5%) based on blood biopsy is lower than that based on tissue sequencing (70.8%). These findings support the usefulness of plasma ctDNA for mutation analysis as established in several other disease contexts9,12C17 and also suggest that there are other factors affecting the performance results of the test. We sequenced the gene from tissue and blood using conventional ARMS PFK-158 despite its lower sensitivity, 18 rather than using newer techniques such as ddPCR,12,19C21 because.
Patients were eligible if they had advanced NSCLC involving mutation(s), were clinically resistant to EGFR TKIs (gefitinib, erlotinib, icotinib, or afatinib), and were undergoing re-biopsy for genotyping as part of their routine clinical care
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