Portal de Periódicos da CAPES

Comparison of EGFR Signaling Pathway Somatic DNA Mutations Derived From Peripheral Blood and Corresponding Tumor Tissue of Patients with Advanced Non-Small-Cell Lung Cancer Using Liquidchip Technology

2013; Elsevier BV; Volume: 15; Issue: 6 Linguagem: Inglês

10.1016/j.jmoldx.2013.06.006

1943-7811

Hui Zhang, Deruo Liu, Shanqing Li, Yong-Qing Zheng, Xinjie Yang, Xi Li, Quan Zhang, Na Qin, Jialin Lu, Lifen Ren‐Heidenreich, Huiyi Yang, Yuhua Wu, Xinyong Zhang, Jingying Nong, Sun Yi-fen, Shucai Zhang,

Cancer therapeutics and mechanisms

Somatic DNA mutations affecting the epidermal growth factor receptor (EGFR) signaling pathway are known to predict responsiveness to EGFR-tyrosine kinase inhibitor drugs in patients with advanced non–small-cell lung cancers. We evaluated a sensitive liquidchip platform for detecting EGFR, KRAS (alias Ki-ras), proto-oncogene B-Raf, and phosphatidylinositol 3-kinase CA mutations in plasma samples, which were highly correlated with matched tumor tissues from 86 patients with advanced non-small-cell lung cancers. Either EGFR exon 19 or 21 mutations were detected in 36 patients: 23 of whom had identical mutations in both their blood and tissue samples; whereas mutations in the remaining 13 were found only in their tumor samples. These EGFR mutations occurred at a significantly higher frequency in females, never-smokers, and in patients with adenocarcinomas (P ≤ 0.001). The EGFR exon 20 T790M mutation was detected in only one of the paired samples [100% (95% CI, 96% to 100%) agreement]. For KRAS, proto-oncogene B-Raf, and phosphatidylinositol 3-kinase CA mutations, the overall agreements were 97% (95% CI, 90% to 99%), 98% (95% CI, 92% to 99%), and 97% (95% CI, 90% to 99%), respectively, and these were not associated with age, sex, smoking history, or histopathologic type. In conclusion, mutations detected in plasma correlated strongly with mutation profiles in each respective tumor sample, suggesting that this liquidchip platform may offer a rapid and noninvasive method for predicting tumor responsiveness to EGFR-tyrosine kinase inhibitor drugs in patients with advanced non-small-cell lung cancers. Somatic DNA mutations affecting the epidermal growth factor receptor (EGFR) signaling pathway are known to predict responsiveness to EGFR-tyrosine kinase inhibitor drugs in patients with advanced non–small-cell lung cancers. We evaluated a sensitive liquidchip platform for detecting EGFR, KRAS (alias Ki-ras), proto-oncogene B-Raf, and phosphatidylinositol 3-kinase CA mutations in plasma samples, which were highly correlated with matched tumor tissues from 86 patients with advanced non-small-cell lung cancers. Either EGFR exon 19 or 21 mutations were detected in 36 patients: 23 of whom had identical mutations in both their blood and tissue samples; whereas mutations in the remaining 13 were found only in their tumor samples. These EGFR mutations occurred at a significantly higher frequency in females, never-smokers, and in patients with adenocarcinomas (P ≤ 0.001). The EGFR exon 20 T790M mutation was detected in only one of the paired samples [100% (95% CI, 96% to 100%) agreement]. For KRAS, proto-oncogene B-Raf, and phosphatidylinositol 3-kinase CA mutations, the overall agreements were 97% (95% CI, 90% to 99%), 98% (95% CI, 92% to 99%), and 97% (95% CI, 90% to 99%), respectively, and these were not associated with age, sex, smoking history, or histopathologic type. In conclusion, mutations detected in plasma correlated strongly with mutation profiles in each respective tumor sample, suggesting that this liquidchip platform may offer a rapid and noninvasive method for predicting tumor responsiveness to EGFR-tyrosine kinase inhibitor drugs in patients with advanced non-small-cell lung cancers. Lung cancer has the highest incidence among all cancers and is the leading cause of death worldwide. It was estimated that in 2012 lung cancer would account for 29% of all male and 26% of all female cancer deaths.1Siegel R. Naishadham D. Jemal A. Cancer statistics, 2012.CA Cancer J Clin. 2012; 62: 10-29Crossref PubMed Scopus (10422) Google Scholar Non-small-cell lung cancer (NSCLC) accounts for the vast majority of lung cancers (∼80%).2Govindan R. Page N. Morgensztern D. Read W. Tierney R. Vlahiotis A. Spitznagel E.L. Piccirillo J. Changing epidemiology of small-cell lung cancer in the United States over the last 30 years: analysis of the surveillance, epidemiologic, and end results database.J Clin Oncol. 2006; 24: 4539-4544Crossref PubMed Scopus (1449) Google Scholar Chemotherapy has long been the standard treatment suggested by the National Comprehensive Cancer Network as the first-line therapy for patients with advanced NSCLC, and in recent years the Food ad Drug Administration also has approved gefitinib or erlotinib as part of the first-line regimen for NSCLC. Recently, the North East Japan Study Group 002 Trial reported that quality of life was maintained much longer in advanced epidermal growth factor receptor (EGFR)-mutated NSCLC patients treated with gefitinib than in patients treated with the standard chemotherapy. Patients who received gefitinib had a significantly longer time to deterioration than patients who received carboplatin plus paclitaxel for pain and shortness of breath (hazard ratio, 0.28; 95% CI, 0.17 to 0.46; P < 0.0001) and daily functioning (hazard ratio, 0.32; 95% CI, 0.17 to 0.59; P < 0.0001), as well as anxiety (hazard ratio, 0.44; 95% CI, 0.22 to 0.87; P < 0.01).3Oizumi S. Kobayashi K. Inoue A. Maemondo M. Sugawara S. Yoshizawa H. Isobe H. Harada M. Kinoshita I. Okinaga S. Kato T. Harada T. Gemma A. Saijo Y. Yokomizo Y. Morita S. Hagiwara K. Nukiwa T. Quality of life with gefitinib in patients with EGFR-mutated non-small cell lung cancer: quality of life analysis of North East Japan Study Group 002 Trial.Oncologist. 2012; 17: 863-870Crossref PubMed Scopus (87) Google Scholar Accordingly, the importance of identifying EGFR signaling pathway gene mutations has been recognized for clinicians to select tyrosine kinase inhibitors (TKIs) as a treatment option for advanced NSCLC patients.4Bunn Jr., P.A. Doebele R.C. Genetic testing for lung cancer: reflex versus clinical selection.J Clin Oncol. 2011; 29: 1943-1945Crossref PubMed Scopus (16) Google Scholar Unfortunately, the difficulties of obtaining tumor tissue for evaluating NSCLC mutations presents significant limitations to this approach and underscores an unmet need for methods that allow for the testing of mutation states of released tumor DNA in peripheral blood samples. Because many patients with lung cancer are diagnosed with an advanced-stage disease and surgery is no longer an option, systemic treatment plays a critical role in clinical management. Progress has been made in the treatment of advanced NSCLC using the small-molecule TKIs, gefitinib and erlotinib, which target the EGFR.5Shigematsu H. Gazdar A.F. Somatic mutations of epidermal growth factor receptor signaling pathway in lung cancers.Int J Cancer. 2006; 118: 257-262Crossref PubMed Scopus (559) Google Scholar, 6Tsao M.S. Sakurada A. Cutz J.C. Zhu C.Q. Kamel-Reid S. Squire J. Lorimer I. Zhang T. Liu N. Daneshmand M. Marrano P. da Cunha Santos G. Lagarde A. Richardson F. Seymour L. Whitehead M. Ding K. Pater J. Shepherd F.A. Erlotinib in lung cancer-molecular and clinical predictors of outcome.N Engl J Med. 2005; 353: 133-144Crossref PubMed Scopus (1736) Google Scholar EGFR kinase domain mutations, in particular exon 19 deletions and exon 21 (L858R), have been identified as predictors of responsiveness to both gefitinib and erlotinib.7Sharma S.V. Bell D.W. Settleman J. Haber D.A. Epidermal growth factor receptor mutations in lung cancer.Nat Rev Cancer. 2007; 7: 169-181Crossref PubMed Scopus (2434) Google Scholar, 8Mitsudomi T. Yatabe Y. Mutations of the epidermal growth factor receptor gene and related genes as determinants of epidermal growth factor receptor tyrosine kinase inhibitors sensitivity in lung cancer.Cancer Sci. 2007; 98: 1817-1824Crossref PubMed Scopus (534) Google Scholar, 9Hirsch F.R. Varella-Garcia M. Bunn Jr., P.A. Franklin W.A. Dziadziuszko R. Thatcher N. Chang A. Parikh P. Pereira J.R. Ciuleanu T. von Pawel J. Watkins C. Flannery A. Ellison G. Donald E. Knight L. Parums D. Botwood N. Holloway B. Molecular predictors of outcome with gefitinib in a phase III placebo-controlled study in advanced non-small-cell lung cancer.J Clin Oncol. 2006; 24: 5034-5042Crossref PubMed Scopus (687) Google Scholar, 10Lynch T.J. Bell D.W. Sordella R. Gurubhagavatula S. Okimoto R.A. Brannigan B.W. Harris P.L. Haserlat S.M. Supko J.G. Haluska F.G. Louis D.N. Christiani D.C. Settleman J. Haber D.A. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib.N Engl J Med. 2004; 350: 2129-2139Crossref PubMed Scopus (9996) Google Scholar, 11Paez J.G. Janne P.A. Lee J.C. Tracy S. Greulich H. Gabriel S. Herman P. Kaye F.J. Lindeman N. Boggon T.J. Naoki K. Sasaki H. Fujii Y. Eck M.J. Sellers W.R. Johnson B.E. Meyerson M. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy.Science. 2004; 304: 1497-1500Crossref PubMed Scopus (8463) Google Scholar, 12Pao W. Miller V. Zakowski M. Doherty J. Politi K. Sarkaria I. Singh B. Heelan R. Rusch V. Fulton L. Mardis E. Kupfer D. Wilson R. Kris M. Varmus H. EGF receptor gene mutations are common in lung cancers from "never smokers" and are associated with sensitivity of tumors to gefitinib and erlotinib.Proc Natl Acad Sci U S A. 2004; 101: 13306-13311Crossref PubMed Scopus (3876) Google Scholar The T790M mutation resulted from an amino acid substitution at position 790 in EGFR exon 20, from threonine to methionine. T790M has been found to be associated with decreased sensitivity to TKIs. It now generally is considered an acquired resistance mutation to TKI treatment and therefore as a biomarker for poor clinical prognosis.13Engelman J.A. Mukohara T. Zejnullahu K. Lifshits E. Borras A.M. Gale C.M. Naumov G.N. Yeap B.Y. Jarrell E. Sun J. Tracy S. Zhao X. Heymach J.V. Johnson B.E. Cantley L.C. Janne P.A. Allelic dilution obscures detection of a biologically significant resistance mutation in EGFR-amplified lung cancer.J Clin Invest. 2006; 116: 2695-2706Crossref PubMed Scopus (397) Google Scholar, 14Oxnard G.R. Arcila M.E. Sima C.S. Riely G.J. Chmielecki J. Kris M.G. Pao W. Ladanyi M. Miller V.A. Acquired resistance to EGFR tyrosine kinase inhibitors in EGFR-mutant lung cancer: distinct natural history of patients with tumors harboring the T790M mutation.Clin Cancer Res. 2011; 17: 1616-1622Crossref PubMed Scopus (519) Google Scholar Additional studies have confirmed that EGFR signaling pathway gene mutations, such as KRAS (alias Ki-ras) and phosphatidylinositol 3-kinase (PI3K)CA, also are associated with the outcomes of patients treated with TKIs15Han S.W. Kim T.Y. Jeon Y.K. Hwang P.G. Im S.A. Lee K.H. Kim J.H. Kim D.W. Heo D.S. Kim N.K. Chung D.H. Bang Y.J. Optimization of patient selection for gefitinib in non-small cell lung cancer by combined analysis of epidermal growth factor receptor mutation, K-ras mutation, and Akt phosphorylation.Clin Cancer Res. 2006; 12: 2538-2544Crossref PubMed Scopus (226) Google Scholar, 16van Zandwijk N. Mathy A. Boerrigter L. Ruijter H. Tielen I. de Jong D. Baas P. Burgers S. Nederlof P. EGFR and KRAS mutations as criteria for treatment with tyrosine kinase inhibitors: retro- and prospective observations in non-small-cell lung cancer.Ann Oncol. 2007; 18: 99-103Crossref PubMed Scopus (134) Google Scholar, 17Eberhard D.A. Johnson B.E. Amler L.C. Goddard A.D. Heldens S.L. Herbst R.S. Ince W.L. Janne P.A. Januario T. Johnson D.H. Klein P. Miller V.A. Ostland M.A. Ramies D.A. Sebisanovic D. Stinson J.A. Zhang Y.R. Seshagiri S. Hillan K.J. Mutations in the epidermal growth factor receptor and in KRAS are predictive and prognostic indicators in patients with non-small-cell lung cancer treated with chemotherapy alone and in combination with erlotinib.J Clin Oncol. 2005; 23: 5900-5909Crossref PubMed Scopus (1342) Google Scholar, 18Mao C. Qiu L.X. Liao R.Y. Du F.B. Ding H. Yang W.C. Li J. Chen Q. KRAS mutations and resistance to EGFR-TKIs treatment in patients with non-small cell lung cancer: a meta-analysis of 22 studies.Lung Cancer. 2010; 69: 272-278Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 19Pao W. Wang T.Y. Riely G.J. Miller V.A. Pan Q. Ladanyi M. Zakowski M.F. Heelan R.T. Kris M.G. Varmus H.E. KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib.PLoS Med. 2005; 2: e17Crossref PubMed Scopus (1377) Google Scholar, 20Yousem S.A. Nikiforova M. Nikiforov Y. The histopathology of BRAF-V600E-mutated lung adenocarcinoma.Am J Surg Pathol. 2008; 32: 1317-1321Crossref PubMed Scopus (81) Google Scholar, 21Sasaki H. Kawano O. Endo K. Suzuki E. Haneda H. Yukiue H. Kobayashi Y. Yano M. Fujii Y. Uncommon V599E BRAF mutations in Japanese patients with lung cancer.J Surg Res. 2006; 133: 203-206Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 22Karakas B. Bachman K.E. Park B.H. Mutation of the PI3KCA oncogene in human cancers.Br J Cancer. 2006; 94: 455-459Crossref PubMed Scopus (375) Google Scholar; therefore, the detection of these mutations also has therapeutic predictive value. However, most of these patients are suffering from late-stage cancer, making it difficult to obtain tumor tissue for a DNA mutation assay. In recent years, it has been shown that tumor DNA released into plasma has the same genetic lesions as the primary tumor.23Gahan P.B. Swaminathan R. Circulating nucleic acids in plasma and serum. Recent developments.Ann N Y Acad Sci. 2008; 1137: 1-6Crossref PubMed Scopus (52) Google Scholar Several studies have shown that tumor DNA found in plasma, malignant pleural effusions, or bronchoalveolar lavage fluid have EGFR mutations that are identical to those in the corresponding tumors, and that circulating or free tumor DNA may be used as an alternative sample for mutation detection.24Kimura H. Kasahara K. Kawaishi M. Kunitoh H. Tamura T. Holloway B. Nishio K. Detection of epidermal growth factor receptor mutations in serum as a predictor of the response to gefitinib in patients with non-small-cell lung cancer.Clin Cancer Res. 2006; 12: 3915-3921Crossref PubMed Scopus (296) Google Scholar, 25He C. Liu M. Zhou C. Zhang J. Ouyang M. Zhong N. Xu J. Detection of epidermal growth factor receptor mutations in plasma by mutant-enriched PCR assay for prediction of the response to gefitinib in patients with non-small-cell lung cancer.Int J Cancer. 2009; 125: 2393-2399Crossref PubMed Scopus (92) Google Scholar, 26Bai H. Mao L. Wang H.S. Zhao J. Yang L. An T.T. Wang X. Duan C.J. Wu N.M. Guo Z.Q. Liu Y.X. Liu H.N. Wang Y.Y. Wang J. Epidermal growth factor receptor mutations in plasma DNA samples predict tumor response in Chinese patients with stages IIIB to IV non-small-cell lung cancer.J Clin Oncol. 2009; 27: 2653-2659Crossref PubMed Scopus (273) Google Scholar, 27Yung T.K. Chan K.C. Mok T.S. Tong J. To K.F. Lo Y.M. Single-molecule detection of epidermal growth factor receptor mutations in plasma by microfluidics digital PCR in non-small cell lung cancer patients.Clin Cancer Res. 2009; 15: 2076-2084Crossref PubMed Scopus (358) Google Scholar, 28Wu S.G. Gow C.H. Yu C.J. Chang Y.L. Yang C.H. Hsu Y.C. Shih J.Y. Lee Y.C. Yang P.C. Frequent epidermal growth factor receptor gene mutations in malignant pleural effusion of lung adenocarcinoma.Eur Respir J. 2008; 32: 924-930Crossref PubMed Scopus (105) Google Scholar, 29Zhang X. Zhao Y. Wang M. Yap W.S. Chang A.Y. Detection and comparison of epidermal growth factor receptor mutations in cells and fluid of malignant pleural effusion in non-small cell lung cancer.Lung Cancer. 2008; 60: 175-182Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 30Asano H. Toyooka S. Tokumo M. Ichimura K. Aoe K. Ito S. Tsukuda K. Ouchida M. Aoe M. Katayama H. Hiraki A. Sugi K. Kiura K. Date H. Shimizu N. Detection of EGFR gene mutation in lung cancer by mutant-enriched polymerase chain reaction assay.Clin Cancer Res. 2006; 12: 43-48Crossref PubMed Scopus (180) Google Scholar However, most of these studies were performed retrospectively, with a relatively small number of patients, and the studies overlooked downstream genes of the EGFR signaling pathway networks, which also may be associated with responsiveness to TKIs. SurExam Biotech, Inc (Guangzhou, China) has developed a novel technology: mutant enriched liquidchip (MEL). The analytical sensitivity of MEL for KRAS is 0.1% (mutant copies: wild type copies = 1:1000),31Wu S. Zhu Z. He J. Luo X. Xu J. Ren-Heidenreich L. A novel mutant-enriched liquidchip technology for the qualitative detection of somatic mutations in KRAS gene from both serum and tissue samples.Clin Chem Lab Med. 2010; 48: 1103-1106PubMed Google Scholar whereas the sensitivity of the 70plex liquidchip is 1%.32Li G. Luo X. He J. Zhu Z. Yu G. Qin H. Zeng T. Liu Z. Wu S. Xu J. Ren-Heidenreich L. A novel liquidchip platform for simultaneous detection of 70 alleles of DNA somatic mutations on EGFR, KRAS, BRAF and PI3KCA from formalin-fixed and paraffin-embedded slides containing tumor tissue.Clin Chem Lab Med. 2011; 49: 191-195PubMed Google Scholar The analytical sensitivities of MEL and 70plex for other genes tested were similar to KRAS (personal communication, L. Ren-Heidenreich). The 70plex liquidchip also was developed by SurExam and routinely used for somatic mutation testing of tumor tissue samples at the SurExam Clinical Laboratory. In this article, we report the feasibility of using MEL technology to detect the presence of EGFR signaling pathway gene somatic mutations in the plasma of patients with NSCLC. Our results indicate that the liquidchip platform is effective for identifying EGFR mutations in tumor DNA circulating in peripheral blood that correlate strongly with EGFR mutations obtained from the matched tumor tissues of patients. The correlation between gene mutations and patient characteristics also was analyzed. These findings suggest that this liquidchip technology may offer a rapid and noninvasive method for predicting NSCLC tumor responsiveness to EGFR tyrosine kinase inhibitor drugs in advanced NCSLC patients. To be eligible for the study, patients were required to have pathologically confirmed stage IIIB or IV NSCLC, a performance status of 0 to 2, and availability of both plasma and primary tumor tissue samples. Tumor staging was performed according to the Seventh Edition of TNM in Lung Cancer.33Sobin L.H. Gospodarowicz M.K. Wittekind C.Z. International Union Against Cancer (UICC): TNM Classification of Malignant Tumours. 7th Edition. Oxford, Wiley-Blackwell, 2009: 138-146Google Scholar Patients presenting with one or more metastatic sites to bone, liver, lung, brain, and pleura were classified as stage IV. Smoking status was based on medical records containing self-reported smoking history obtained during each patient's first clinical visit. All patients enrolled were treated at the Beijing Chest Hospital in Beijing, China. This study was conducted under an Institutional Review Board–approved protocol and monitored for assurance of human subjects' protection. All patients signed the informed consent form to participate in this study and granted permission for the use of their plasma and tumor tissue samples. Each enrolled patient contributed one plasma sample (1.5 mL) and one formalin-fixed paraffin-embedded tumor slice. Samples from each patient were collected before treatment. From these formalin-fixed paraffin-embedded samples, 36 were collected from the primary cancer via transbronchial lung biopsy, 28 were collected via pulmonary aspiration, 6 were resected intraoperatively, and 16 were obtained from metastatic sites (12 from lymph nodes and 4 from pleura). All specimens underwent examination by pathologists to confirm the diagnosis of NSCLC with advanced stages. Plasma samples were prepared as follows: 5 mL anticoagulated venous blood was collected from each patient before any treatment and then centrifuged at 1800 × g for 5 minutes at 4°C. Approximately 1.5 mL plasma was aspirated and transferred to a new centrifuge tube containing 15 μL of 20 mg/mL protease K and 50 μL of 20% SDS. Plasma samples then were shipped to the SurExam Clinical Laboratory for mutation analysis. At the laboratory, DNA from each tumor and plasma sample was extracted using the Maxwell system (Promega, Atlanta, GA) according to the manufacturer's instructions. A total of 39 mutation sites in exons 19 to 21 of EGFR, exons 2 and 3 of KRAS, exon 15 of proto-oncogene B-Raf (BRAF), and exons 9 and 20 of PI3KCA were analyzed in DNA from both tumor tissues and matched peripheral blood plasma samples (MPP). All of the mutational analyses were performed at the SurExam Clinical Laboratory (SurExam Biotech, Inc). The tissue samples were tested with SurPlex-xTAG70plex (70plex; SurExam Biotech, Inc).32Li G. Luo X. He J. Zhu Z. Yu G. Qin H. Zeng T. Liu Z. Wu S. Xu J. Ren-Heidenreich L. A novel liquidchip platform for simultaneous detection of 70 alleles of DNA somatic mutations on EGFR, KRAS, BRAF and PI3KCA from formalin-fixed and paraffin-embedded slides containing tumor tissue.Clin Chem Lab Med. 2011; 49: 191-195PubMed Google Scholar This technology simultaneously identifies 70 different mutations of the EGFR signaling pathway network and includes five major steps: i) multiplex PCR, to amplify the regions of target genes; ii) exonuclease I and shrimp alkaline phosphatase cleaning in which the PCR mixture was treated using exonuclease I and shrimp alkaline phosphatase to remove excess nucleotides and primers; iii) allele-specific primer extension in which the exonuclease I and shrimp alkaline phosphatase–cleaned PCR product was subjected to an allele-specific primer extension step in which each of 70 universal tags was linked to a specific primer sequence complementary to a specific gene of interest, the Tsp DNA polymerase then will extend only primers that are a 100% match to the templates; iv) hybridization to beads in which the allele-specific primer extension products were hybridized to specific anti-tag probes that were precoated on the polystyrene microspheres; and v) Luminex 200 (Luminex Co., Austin, TX) scan and data analysis. The beads then were applied to the Luminex 200 and median fluorescence intensity was read and analyzed. The detection of gene mutation in the MPP was conducted using SurPlex MEL as previously described.31Wu S. Zhu Z. He J. Luo X. Xu J. Ren-Heidenreich L. A novel mutant-enriched liquidchip technology for the qualitative detection of somatic mutations in KRAS gene from both serum and tissue samples.Clin Chem Lab Med. 2010; 48: 1103-1106PubMed Google Scholar Briefly, the MEL method includes the following six steps: i) extraction of DNA from the MPP; ii) introduction of a restriction site to the wild-type through PCR amplification; iii) elimination of the wild-type genes by restriction enzyme digestion; iv) selective amplification of the mutated DNA sequence; v) hybridization to a specific probe that was precoated on the polystyrene microspheres; and vi) data analysis using the multi-analyte profiling; Luminex Co.) analyzer. To make the standard curve of 0.0%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 5.0%, 10.0%, 20.0%, and 50.0% mutated DNA, different copy numbers of mutated plasmid DNA were mixed with 1.2 × 105 wild-type plasmid DNA in triplicate. The standard mixtures, along with samples, then underwent the liquidchip process. The results were analyzed statistically. Single-variable linear regression for the standard curve was performed to analyze the correlation between mutant DNA copy numbers and the median fluorescence intensity values, in which the logarithm of mutant DNA copy number to base 2 was the independent variable whereas the median fluorescence intensity value was the dependent variable. Regression line, 95% confidence interval and 95% prediction interval for linear regression were graphed. R-squared and P value were calculated. The percentage of mutant DNA then consequently was calculated. A two-sided P value of <0.05 was considered statistically significant. This analysis was performed with SAS software version 9.2 (SAS Institute Inc., Cary, NC). SPSS statistical software (version 18.0; SPSS, Inc., Chicago, IL) was used to analyze the data. The χ2 test and the Fisher exact test were used to assess the relationship between the mutation status of genes in the EGFR pathway and each of the clinical characteristics. A P value less than 0.05 with a 95% CI was considered statistically significant. A total of 86 eligible patients were enrolled in the study from March 2011 to January 2012 at the Beijing Thoracic Hospital. The patients consisted of 37 women and 49 men. There were 65 patients with lung adenocarcinomas, 20 with squamous cell carcinomas, and 1 with an unknown tumor type. A total of 44 patients were smokers or ever-smokers, and 42 patients were never-smokers. All 86 patients initially were diagnosed with stages IIIB to IV NSCLC. The patients' clinical and disease characteristics are listed in Table 1.Table 1Patient CharacteristicsPatients (N = 86)Percentage (%)Age, years Mean58 Range21–80 CGG) and Del E746-A750 (K745: AAA) in exon 19, which accounted for 44.4% (16 of 36) and 36.1% (13 of 36) of all EGFR-sensitive mutations, respectively. In KRAS and PI3KCA mutations, KRAS exon 2 G12C, G12S, G12A, G12D, PI3KCA exon 9 E545K, E542K, and exon 20 H1047R were detected. In BRAF, only V600E was detected. Twenty-two (25.6%) EGFR exon 19 mutations and 14 (16.3%) EGFR exon 21 mutations were detected in the 86 tumor tissues, whereas 15 (17.4%) EGFR exon 19 mutations and 8 (9.3%) EGFR exon 21 mutations were found in the MPP. Thirteen (15.1%) patients with tumor EGFR exons 19 or 21 mutations had no detectable mutation in the corresponding plasma DNA samples. EGFR T790M mutations were found in one of the paired samples. KRAS mutations were detected in three tissue samples and two plasma samples. In two cases, KRAS mutation states were positive in tissue but negative in plasma, whereas one case had the opposite findings. Similarly, a PI3KCA mutation was detected in six patients (7%), in which three were detected in paired samples; the others were either in tissue or plasma. Interestingly, two patients with plasma BRAF mutations had no detectable mutations in the corresponding primary tumors. The correlation between mutations detected in plasma DNA and tumor DNA is summarized in Table 2.Table 2Consistency of Gene Mutation Between Tumor Tissues and MPPTissueEGFR E19EGFR E21EGFR E20KRASBRAFPI3KCAMutationWtTotalMutationWtTotalMutationWtTotalMutationWtTotalMutationWtTotalMutationWtTotalPlasma Mutation15015808101112022314 Wt764716727808585282840848428082 Total22648614728618586383860868658186Agreement Negative100% (94%–100%)100% (95%–100%)100% (96%–100%)99% (93%–100%)98% (96%–100%)99% (93%–100%) Positive68% (47%–84%)57% (33%–79%)100% (21%–100%)33% (6%–79%)NA60% (23%–88%) Overall92% (84%–96%)93% (86%–98%)100% (96%–100%)97% (90%–99%)98% (92%–99%)97% (90%–99%)Correlation index∗Spearman rank correlation coefficient.0.784 (0.647−0.921)0.726 (0.544−0.909)1.000 (1.000–1.000)0.391 (−0.161−0.943)NA0.653 (0.289–1.000)P value∗Spearman rank correlation coefficient.<0.001<0.0010.0110.322NA0.070E, exon.∗ Spearman rank correlation coefficient. Open table in a new tab E, exon. We found that EGFR-sensitive mutation status in both plasma DNA samples and primary tumor DNA samples correlated with the patients' sex, smoking history, tumor histology, and disease stage. There were significantly higher mutation rates in females, never-smokers, adenocarcinomas, and stage IV disease. However, EGFR-sensitive mutation status in plasma DNA or tumor DNA did not correlate with patient age (Table 3).Table 3Correlation Between EGFR, KRAS, BRAF, and PI3KCA Mutations and Clinicopathologic CharacteristicsN = 86 n ( %)TissuePlasmaE19 n = 22E21 n = 14E19+E21 n = 36KRAS n = 3BRAF n = 0PI3KCA n = 5∗One patient harbored a PI3KCA mutation, but the tumor histology was not clear.E19 n = 15E21 n = 8E19+E21 n = 23KRAS n = 2BRAF n = 2PI3KCA n = 4∗One patient harbored a PI3KCA mutation, but the tumor histology was not clear.Age, year ≥6039 (45.35)107171037512212 <6047 (54.65)127192028311012 P–0.9910.7020.7671.000–0.6550.9100.4590.4420.2031.0001.000Sex Male49 (56.98)8210301516201 Female37 (43.02)14122600410717023 P–0.0240.0000.0000.256–0.1600.0420.0190.0000.5040.1820.310Smoking Smoker32 (37.21)516300213200 Ever-smoker12 (13.95)314002202002 Never-smoker42 (48.84)14122600311718022 P–0.2240.0100.0010.073–0.0960.0810.0680.0040.1780.3420.065Histologic Aden.65 (75.58)20143430313821122 Squa.20 (23.26)202001202101 P–0.0640.0330.0011.000–1.0000.5040.1890.0500.4171.0000.558Stage IIIB16 (18.60)123001000101 IV70 (81.40)21123330415823123 P–0.0601.0000.0381.000–1.0000.0630.3420.0050.3391.0000.568Aden., adenocarcinoma; Squa., squamous cell carcinoma.∗ One patient harbored a PI3KCA mutation, but the tumor histology was not clear. Open table in a new tab Aden.