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      PNA clamping-assisted fluorescence melting curve analysis for detecting EGFR and KRAS mutations in the circulating tumor DNA of patients with advanced non-small cell lung cancer

      research-article
      1 , 3 , , 2 , 1 , 1 , 1
      BMC Cancer
      BioMed Central
      EGFR, KRAS, Plasma, NSCLC, Tissue

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          Abstract

          Background

          Circulating cell-free DNA (cfDNA) is emerging as a surrogate sample type for mutation analyses. To improve the clinical utility of cfDNA, we developed a sensitive peptide nucleic acid (PNA)-based method for analyzing EGFR and KRAS mutations in the plasma cfDNA of patients with advanced non-small cell lung cancer (NSCLC).

          Methods

          Baseline tissue and plasma samples were collected from treatment-naïve advanced NSCLC patients participated in a randomized phase II study, which was registered with ClinicalTrials.gov at Feb. 2009 (NCT01003964). EGFR and KRAS mutations in the plasma cfDNA were analyzed retrospectively using a PNA clamping-assisted fluorescence melting curve analysis. The results were compared with those obtained from tissue analysis performed using the direct sequencing. Exploratory analyses were performed to determine survival predicted by the plasma and tissue mutation status.

          Results

          Mutation analyses in matched tissue and plasma samples were available for 194 patients for EGFR and 135 patients for KRAS. The mutation concordance rates were 82.0 % (95 % confidence interval [CI], 76.5–87.4) for EGFR and 85.9 % (95 % CI, 80.1–91.8) for KRAS. The plasma EGFR mutation test sensitivity and specificity were 66.7 % (95 % CI, 60.0–73.3) and 87.4 % (95 % CI, 82.7–92.1), respectively, and the plasma KRAS mutation test sensitivity and specificity were 50.0 % (95 % CI, 41.6–58.4) and 89.4 % (95 % CI, 84.2–94.6), respectively. The predictive value of the plasma EGFR and KRAS mutation status with respect to survival was comparable with that of the tissue mutation status.

          Conclusions

          These data suggest that plasma EGFR and KRAS mutations can be analyzed using PNA-based real-time PCR methods and used as an alternative to tumor genotyping for NSCLC patients when tumor tissue is not available.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s12885-016-2678-2) contains supplementary material, which is available to authorized users.

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          Most cited references18

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          Systemic Therapy for Stage IV Non-Small-Cell Lung Cancer: American Society of Clinical Oncology Clinical Practice Guideline Update.

          To provide evidence-based recommendations to update the American Society of Clinical Oncology guideline on systemic therapy for stage IV non-small-cell lung cancer (NSCLC).
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            Multiplex picodroplet digital PCR to detect KRAS mutations in circulating DNA from the plasma of colorectal cancer patients.

            Multiplex digital PCR (dPCR) enables noninvasive and sensitive detection of circulating tumor DNA with performance unachievable by current molecular-detection approaches. Furthermore, picodroplet dPCR facilitates simultaneous screening for multiple mutations from the same sample. We investigated the utility of multiplex dPCR to screen for the 7 most common mutations in codons 12 and 13 of the KRAS (Kirsten rat sarcoma viral oncogene homolog) oncogene from plasma samples of patients with metastatic colorectal cancer. Fifty plasma samples were tested from patients for whom the primary tumor biopsy tissue DNA had been characterized by quantitative PCR. Tumor characterization revealed that 19 patient tumors had KRAS mutations. Multiplex dPCR analysis of the plasma DNA prepared from these samples identified 14 samples that matched the mutation identified in the tumor, 1 sample contained a different KRAS mutation, and 4 samples had no detectable mutation. Among the tumor samples that were wild type for KRAS, 2 KRAS mutations were identified in the corresponding plasma samples. Duplex dPCR (i.e., wild-type and single-mutation assay) was also used to analyze plasma samples from patients with KRAS-mutated tumors and 5 samples expected to contain the BRAF (v-raf murine sarcoma viral oncogene homolog B) V600E mutation. The results for the duplex analysis matched those for the multiplex analysis for KRAS-mutated samples and, owing to its higher sensitivity, enabled detection of 2 additional samples with low levels of KRAS-mutated DNA. All 5 samples with BRAF mutations were detected. This work demonstrates the clinical utility of multiplex dPCR to screen for multiple mutations simultaneously with a sensitivity sufficient to detect mutations in circulating DNA obtained by noninvasive blood collection.
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              BEAMing: single-molecule PCR on microparticles in water-in-oil emulsions.

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                Author and article information

                Contributors
                +82-31-920-1154 , jymama@ncc.re.kr
                cjj96637@panagene.com
                jykim@ncc.re.kr
                73624@ncc.re.kr
                gklee@ncc.re.kr
                Journal
                BMC Cancer
                BMC Cancer
                BMC Cancer
                BioMed Central (London )
                1471-2407
                12 August 2016
                12 August 2016
                2016
                : 16
                : 627
                Affiliations
                [1 ]Lung Cancer Branch, Research Institute, National Cancer Center, Goyang, Korea
                [2 ]PANAGENE Inc., Daejeon, Korea
                [3 ]Center for Lung Cancer, Hospital, National Cancer Center, 323 Ilsan-ro, Ilsan-dong-gu, Goyang, Gyeonggi 10408 Korea
                Author information
                http://orcid.org/0000-0001-5033-0006
                Article
                2678
                10.1186/s12885-016-2678-2
                4983013
                27519791
                3b32c71c-3d50-4bb2-942a-0247097719d0
                © The Author(s). 2016

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 7 February 2016
                : 5 August 2016
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100003645, National Cancer Center;
                Award ID: 1410660
                Award ID: 1410682
                Award Recipient :
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2016

                Oncology & Radiotherapy
                egfr,kras,plasma,nsclc,tissue
                Oncology & Radiotherapy
                egfr, kras, plasma, nsclc, tissue

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