Abstract
The clinical utility of plasma DNA for detecting cancer-specific mutations has rapidly achieved recognition, but reliability has not been established because of relatively low mutation-detection rates compared with those from tissue re-biopsy. To address this shortcoming we examined efficiency, in terms of mutation detection, of an automated DNA extraction system that uses cellulose magnetic beads. A fully automated, highly sensitive point-mutation-detection method, mutation-biased PCR and quenching probe (MBP-QP) system, was used for this study. Plasma DNA was extracted from 61 plasma samples collected from patients with advanced non-small cell lung cancer. Extraction was performed manually with 200 μl plasma (200-M) by using a silica membrane spin column system or an automated system using 200 μl (200-A) or 1000 μl (1000-A) plasma. Median DNA yield quantified by real-time PCR was 4.4, 4.5, and 17.3 ng with the three methods, respectively. Sensitivity for detecting epidermal growth factor receptor (EGFR) L858R point mutation was 36.6%, 58.5%, and 77.5%, and specificity was 93.3%, 100%, and 96.7%, respectively. Concordance rates were 60.6%, 76.1%, and 85.7%. The size distribution of plasma DNA with automated extraction was bimodal with modes at about 170 bp and 5 Kb, and plasma DNA of both sizes included tumor-derived DNA. In this report, we demonstrate that automated DNA extraction using cellulose magnetic beads can improve mutation-detection rates with plasma DNA in association with two overall sizes of DNA fragments recovered by this DNA isolation system. Examining the biological characteristics of these fragments will be the subject of further investigation.
Highlights
Clarifying the roles of oncogenes and their mutations can aid in the selection of individualized approaches to cancer treatment, including molecular targeted therapy
We demonstrate that automated DNA extraction using cellulose magnetic beads can improve mutation-detection rates with plasma DNA in association with two overall sizes of DNA fragments recovered by this DNA isolation system
Plasma collected from 61 patients with lung cancer and 10 healthy volunteers was divided into three different DNA extraction groups: 200-M, 200-A, and 1000-A. 200M refers to DNA isolated with manual extraction by a QIAamp DNA mini kit from 200 μl plasma. 200-A and 1000-A refer to DNA isolated with automated extraction by a Maxwell RSC ccfDNA plasma cartridge from 200 μl and 1000 μl plasma, respectively
Summary
Clarifying the roles of oncogenes and their mutations can aid in the selection of individualized approaches to cancer treatment, including molecular targeted therapy. In patients with non-small cell lung cancer (NSCLC) harboring epithelial growth factor receptor (EGFR) activating/sensitive mutations, such as L858R and exon 19 deletions, EGFR tyrosine kinase inhibitors (EGFR-TKIs) have been highly effective in approximately 70% of cases [1, 2]. Despite this encouraging initial response, the vast majority of patients treated with 1st generation EGFRTKIs acquire resistance about one year after treatment [2]. Performing invasive tissue re-biopsy of primary or metastatic tumors is not always practicable
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