CLB-08913; No. of pages: 5; 4C: Clinical Biochemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Quantification of serum MET in non-small-cell lung cancer and its clinical significance Detao Li a,1, Fengzeng Li b,1, Yanfeng Wu a, Dandan Zhou a, Hui Chen a,⁎ a b
Clinical Laboratories, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China Department of dermatology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
a r t i c l e
i n f o
Article history: Received 5 September 2014 Received in revised form 1 November 2014 Accepted 29 November 2014 Available online xxxx Keywords: Serum MET Tumor biomarker Non-small-cell lung cancer Diagnostic value
a b s t r a c t Objectives: Mesenchymal–epithelial transition factor (MET) plays a critical role in the development and progression of lung cancer. We aimed to quantify the level of serum MET DNA, analyze its diagnostic value, and provide a novel biomarker for lung cancer. Methods: Serum MET DNA was extracted from 95 patients with lung cancer, 10 with benign lung diseases, and 34 healthy volunteers. MET DNA was quantified using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR). Data were analyzed using statistical software SPSS 17.0. Results: Serum MET DNA level in the lung cancer group was higher than in the healthy group and benign lung diseases group. Serum MET DNA level was higher in lung cancers patients with smoking, squamous cell carcinoma, advanced TNM stage, and increased tumor size. The difference in serum MET DNA level was not related to sex, age, and lymph node metastasis among the lung cancer patients. The receiver operating characteristic curve showed a sensitivity of 72.6% and specificity of 90.9% for the ability of serum MET DNA to detect lung cancer at the cutoff value of 1.30 × 104 copies/μL. The association of serum MET DNA level with existing clinical lung tumor markers was analyzed, including neuron-specific enolase, squamous cell carcinoma antigen, and cytokeratin fragment 21-1. With the combination of serum MET DNA, the sensitivity was raised from 39.1%, 24.9%, 66.1% to 83.3%, 79.4% and 90.7%, respectively. Conclusions: Quantification of serum MET DNA by FQ-PCR may serve as a novel accessible diagnostic tool for the clinical screening and detection of lung cancer. © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Lung cancer is the leading cause of human cancer-related death worldwide [1]. Non-small-cell lung cancer (NSCLC) accounts for ~80% of all lung cancers. The pathogenesis of lung cancer is concealed and most patients have lost the opportunity for surgical resection when they are diagnosed [2]. Although there are many biomarkers for lung cancer, they are of no use for accurate diagnosis of early stage disease. Therefore, highly sensitive and specific tumor biomarkers need to be found for the diagnosis, evaluation, and prognosis of lung cancer. Mesenchymal–epithelial transition factor (MET) is a member of the sub-family of receptor tyrosine kinases (RTKs) for hepatocyte growth factor (HGF) [3]. It is located on chromosome 7q21–31 [4] and plays an important role in the progression of various tumors through regulating cell proliferation, angiogenesis, apoptosis, altering cytoskeletal Abbreviations: FQ-PCR, real-time fluorescent quantitative polymerase chain reaction; NSCLC, non-small-cell lung cancer; MET, mesenchymal–epithelial transition factor; HGF, hepatocyte growth factor; NSE, neuron-specific enolase; SCC, squamous cell carcinoma antigen; CYFRA21-1, cytokeratin fragment 21-1. ⁎ Corresponding author. Fax: +86 23 68716215. E-mail addresses: [email protected], [email protected] (H. Chen). 1 Detao Li and Fengzeng Li contributed equally to this work.
function, and promoting metastasis [5–7]. The overexpression of MET has been found in various human cancers, such as gastric [8], lung [9], ovarian [10], and colorectal [11] cancer. Minuti et al. [12] found that increased MET gene copy numbers were associated with trastuzumab failure in HER2-positive metastatic primary breast cancer, by fluorescence in situ hybridization (FISH). Sun et al. [13] found that positive expression of MET occurred in 67.2% paraffin-embedded specimens of NSCLC, and the positive expression of MET was significantly associated with shortened overall survival. Cappuzzo et al. [14] reported that high MET gene copy number (defined as a mean of copies/cell) was observed in 11.1% of NSCLCs tumor tissue and affected patient survival negatively. Although a lot of research has focused on alteration of the MET gene and protein in tumor tissue, the level of serum MET DNA has not yet been reported. Serum DNA is an emerging noninvasive biomarker, which has been used widely for cancer, including diagnosis, assessment of tumor progression, evaluation, and monitoring of the efficacy of anticancer therapy [15]. Its potential value as a blood biomarker was highlighted in an editorial in Science [16]. The present study is believed to be the first to quantify serum MET DNA in lung cancer patients by fluorescent quantitative polymerase chain reaction (FQ-PCR). The relationship between serum MET level
http://dx.doi.org/10.1016/j.clinbiochem.2014.11.021 0009-9120/© 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Please cite this article as: Li D, et al, Quantification of serum MET in non-small-cell lung cancer and its clinical significance, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.11.021
2
D. Li et al. / Clinical Biochemistry xxx (2014) xxx–xxx
and clinicopathological characteristics was analyzed. We evaluated the diagnostic value of serum MET combined with or without existing tumor markers for lung cancer.
specific enolase (NSE), squamous cell carcinoma antigen (SCC), and cytokeratin fragment (CYFRA)21-1. Statistical analysis
Materials and methods Subjects From October 2012 to March 2014, 95 patients with lung cancer were selected as the cancer group, and 34 healthy volunteers and 10 patients with benign lung disease were selected as the control group (benign lung disease included: four patients with pulmonary fibrotic nodules, four with pulmonary granulomas, and two with pulmonary inflammatory pseudotumors). All the sera were collected before surgery and any other treatment. Eligible samples were identified at the Department of Cardiothoracic Surgery, The First Affiliated Hospital of Chongqing Medical University, China. Data on sex, age, primary tumor staging, smoking status, lymph node metastasis, and histopathological grades were collected. Pathological diagnosis was classified according to the WHO classification of lung cancer [17], and tumor–node– metastases (TNM) stage was classified by a cardiothoracic physician and an experienced pathologist. Informed consent was obtained from all patients and healthy volunteers, and the study was approved by the Ethics Committees of the First Affiliated Hospital of Chongqing Medical University. Sample collection and serum DNA extraction Venous blood (2.0 mL) was collected from each subject in tubes without anticoagulants. After incubation at 37 °C for 15 min, the serum was separated by centrifugation at 2 000 g for 10 min, followed by 12 000 g for 10 min. The serum samples were stored at −80 °C until use. Genomic DNA was extracted from serum using a QIA amp Blood Mini Kit based on affinity columns (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. Quantification of serum MET DNA by FQ-PCR MET primers were designed as previously described [18] as follows: forward, 5-GCT GGT GGT CCT ACC ATA CAT G-3; reverse, 5-CTG GCT TAC AGC TAG TTT GCC A-3. The product size was 112 bp. The MET plasmid was constructed using a TA cloning kit as follows: purified PCR products were ligated into pMD18-T vector (TaKaRa, Dalian, China), and the recombinant plasmids were transformed into DH5α competent Escherichia coli. Positive E. coli clones were screened and enriched, and recombinant plasmid DNA was extracted. A standard curve was generated with 10 dilutions of plasmid DNA (101–1010 copies/μL) using the cobas Z 480 (Roche Diagnostics, Mannheim, Germany). FQ-PCRs were conducted within a total volume of 20 μL/well and contained the following reagents: 10 μL SYBR Premix ExTaq TM II (2×), 0.8 μL forward primer, 0.8 μL reverse primer, 2 μL DNA, and 6.4 μL deionized distilled water. The PCR kits were purchased from TaKaRa (TaKaRa Bio, Osaka, Japan). PCRs were conducted under the following conditions: one cycle at 95 °C for 30 s; followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s; 95 °C for 5 s, one cycle at 60 °C for 1 min; one cycle at 50 °C for 30 s. Each sample and DNA standard was analyzed in duplicate, and the mean value was used for quantification. Only the standard curve with a coefficient of correlation N0.96 was accepted. Quantification of existing tumor markers Serum was collected in a similar manner to that described in “Sample collection and serum DNA extraction.” Existing tumor markers for lung cancer were analyzed using the chemiluminescence immunoassay E170 (Roche Diagnostics, Mannheim, Germany), including neuron-
Data were analyzed with Mann–Whitney U test for unpaired samples and correlation coefficients determined using Spearman's rank test. MET level was summarized as medium and quartile M (P25, P75). A receiver operating characteristic (ROC) curve was plotted to determine the area under the curve (AUC), sensitivity, and specificity of serum MET DNA. Statistical analysis was performed using SPSS 17.0 software (SPSS Inc, Chicago, IL, USA), and P b 0.05 was considered statistically significant. Results Methodological evaluation of FQ-PCR for serum MET DNA The within-group and between-day coefficients of variation were 0.83% (n = 10) and 1.08% (n = 10), respectively. The dissociation curves exhibited good specificity with a mean peak at 81.5 °C. A straight line was obtained with the negative control. Tenfold serial dilutions of plasmid MET DNA from 101 to 1010 copies/μL were used to generate a standard curve. The curve exhibited good linearity at 103–108 copies/μL, with a correlation coefficient of 0.999. Clinical characteristics The relevant clinical characteristics of lung cancer patients, including sex, age, smoking status, histopathological types, TNM stage, tumor size, and lymph node metastasis, are summarized in Table 1. Serum MET DNA levels in different groups Serum MET DNA levels M (P25, P75) were 2.03 × 104 (1.08 × 104, 6.06 × 104) copies/μL, 6.27 × 103 (4.47 × 103, 9.18 × 103) copies/μL, and 4.83 × 103 (2.46 × 103, 8.81 × 103) copies/μL in the lung cancer, benign lung disease, and healthy groups, respectively. Further analysis indicated that higher serum MET DNA level existed in the lung cancer group, compared to the benign disease (P b 0.0001) and healthy (P b 0.0001) groups. Moreover, the M (P25, P75) of TNM I was higher than that of benign lung disease (P b 0.01) and healthy control (P b 0.0001)
Table 1 clinical characteristics of study subjects. Clinical characteristics
Patients (n = 95)
Gender (F/M) Age (years) Smokers/nonsmokers
20/75 (21.1/78.9%) 3/7 (30.0/70.0%) 60 (55–66) 52 (48–59) 63/32 (66.3/33.7%) 2/8 (20.0/80.0%)
Histopathological types Squamous cell carcinoma Adenocarcinoma Others
45 (47.4%) 37 (38.9%) 13 (13.7%)
TNM stages I II III IV
15 (15.8%) 39 (41.1%) 17 (17.9%) 24 (25.2%)
Tumor size (cm) d≤4 dN4
55 (57.9%) 40 (42.1%)
Lymph node metastasis Positive Negative
70 (78.9%) 25 (21.1%)
Benign (n = 10) Healthy (n = 34) 8/26 (23.5/76.5%) 59 (55–66) 20/14 (58.8/41.2%)
Please cite this article as: Li D, et al, Quantification of serum MET in non-small-cell lung cancer and its clinical significance, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.11.021
D. Li et al. / Clinical Biochemistry xxx (2014) xxx–xxx
obviously. In contrast, there was no difference between the benign and healthy groups (P = 0.251) (Fig. 1).
Table 2 Serum MET DNA level in lung cancer group with different clinical characteristics. Clinical characteristics
Serum MET DNA levels among lung cancer patients Serum MET level was higher in squamous cell carcinoma than adenocarcinoma (P = 0.0117). Serum MET level was higher in smokers (P = 0.0396), advanced TNM stage tumors (P = 0.0304), and larger tumors (P = 0.0324). However, no significant differences were observed for sex (P = 0.2157), age (P = 0.3632), and lymph node metastasis (P = 0.5261).The data are shown in Table 2. Diagnostic value of serum MET DNA for lung cancer The ROC curve (Fig. 2) showed that the AUC of serum MET DNA was 0.893 (95% CI: 0.841–0.945), it was higher than that of existing markers (including NSE, SCC, and CYFRA21-1). In accordance with the rule of the maximum Youden index, 1.30 × 104 copies/μL was set as the optimum cutoff value with a sensitivity of 72.6% and specificity of 90.9% (Fig. 2). Diagnostic sensitivity was 39.1%, 24.9% and 66.1% for single NSE, SCC, and CYFRA21-1, respectively, which were lower than that of MET (72.6%). Upon combination of serum MET DNA with the existing markers, sensitivity increased to 83.3%, 79.4% and 90.7%, respectively, with a better specificity (Table 3). However, there was no evident correlation of serum MET DNA level with that of NSE, SCC, or CYFRA21-1 (P N 0.05, data not shown). Discussion Genomic alterations such as gene amplifications and deletions commonly contribute to the development and progression of tumors [19]. During oncogenesis, these changes lead to gains or losses of parts of the genome in tumor cells, and result in the functional disorder of genes that regulate cell proliferation, apoptosis, genomic stability, angiogenesis, invasion, and metastasis, negatively or positively. As an RTK for HGF, MET plays an important role in tumorigenesis and its overexpression disrupts the balance of cell proliferation and apoptosis. In recent years, the overexpression of MET has attracted increasing attention. The overexpression of MET gene has been analyzed in tumor tissues by various assays, including qRT-PCR, immunohistochemistry, and FISH in tumor tissues. By FISH, Pros et al. [20] found that the overexpression of MET was obvious in an NSCLC tissue
Fig. 1. Serum MET DNA levels in lung cancer and control groups.
3
Gender Male (n = 75) Female (n = 20) Age ≤60 years (n = 50) N60 years (n = 45) Smoke status Smoker(n = 63) Nonsmoke(n = 32) Histological types a Squamous cell carcinoma (n = 45) Adenocarcinoma (n = 37) TNM stages I + II (n = 54) III + IV (n = 41) Tumor size (cm) d ≤ 4 (n = 55) d N 4 (n = 40) Lymph node metastasis Positive (n = 70) Negative (n = 25)
Serum MET levels [M (P25, P75), copies/μL]
P value⁎ 0.2157
2.48 × 104 (1.48 × 104, 6.98 × 104) 1.73 × 104 (7.61 × 103, 6.65 × 104) 0.3632 2.90 × 104 (1.56 × 104, 7.28 × 104) 2.03 × 104 (9.63 × 103, 6.82 × 104) 0.0396# 2.93 × 104 (1.52 × 104, 8.97 × 104) 1.81 × 104 (8.04 × 103, 6.65 × 104) 0.0117# 3.05 × 104 (1.60 × 104, 9.04 × 104) 1.85 × 104 (8.44 × 103, 4.32 × 104) 0.0304# 2.00 × 104 (9.18 × 103, 6.32 × 104) 2.95 × 104 (1.68 × 104, 9.40 × 104) 0.0324# 1.89 × 104 (1.05 × 104, 4.06 × 104) 3.20 × 104 (1.65 × 104, 8.73 × 104) 0.5261 2.86 × 104 (1.29 × 104, 8.62 × 104) 2.03 × 104 (1.09 × 104, 7.18 × 104)
a The cases of others histological type is too little to be neglected. ⁎ Mann–Whitney U test. # Statistically significant difference.
microarray panel containing 302 samples. Kanteti et al. [18] observed gene amplification of MET in tumor tissues of lung cancer patients. These studies were based on tumor tissues, their drawback was obvious for its injured and hysteresis. Recent developments have shown that serum DNA offers a convenient, noninvasive, and repeatable “liquid biopsy,” and it can be used to assess tumor progression and evaluate prognosis and response to treatment [21]. In a study of gastric cancer, Kim et al. [22] found that increased serum DNA level was associated with tumor burden, such as larger tumor and advanced TNM stage. Based on these reports, in the present study, serum was chosen as the sample to quantify the level of MET DNA initially. The present study found that the level of serum MET DNA in the lung cancer group was significantly higher than that in the benign lung disease and healthy groups. Furthermore, the higher level of serum MET DNA in TNM I than that of control groups hinted that serum MET is a valuable biomarker for the early diagnosis of lung cancer. It has been demonstrated that expression of MET increased in gastric carcinoma, along with higher MET mRNA level and increased DNA copy number [23]. Dziadziuszko et al. [24] found that MET expressed in NSCLC tissues correlated with an increase in its gene copies. Gonzalez-Angulo et al. [25] found that MET gene copies increased significantly in early breast cancer tissue. We obtained a similar result to all the above studies. According to the overexpression of MET in lung tumor tissues and higher level of serum MET DNA in lung cancer, we conjectured that MET promises to be a biomarker for lung cancer screening. We found that the level of serum MET DNA in lung cancer patients with smoking, squamous cell carcinoma, advanced TNM stages and increased tumor sizes were higher than that of cancer patients with no smoking, adenocarcinoma, earlier TNM stages, and smaller tumor size significantly, respectively. It showed that MET was associated with the development and progression of lung cancer. The published research got the similar results in tissue. In NSCLC, Go et al. [4] found that the MET gene amplification was more frequent in tumor tissues of squamous cell carcinoma than that in adenocarcinoma, and it was also associated with advanced tumor stage by FISH. Sun et al. [26] found that the overexpression of MET was associated with advanced tumor stage, based on analyzing 61 NSCLC tissue specimens by FQ-PCR. Okuda et al. [27] found that the increasing expression of MET was significantly correlated with smoking status and tumor size by qPCR in tumor tissues of
Please cite this article as: Li D, et al, Quantification of serum MET in non-small-cell lung cancer and its clinical significance, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.11.021
4
D. Li et al. / Clinical Biochemistry xxx (2014) xxx–xxx
Fig. 2. ROC curve of serum MET DNA level and the existing markers for lung cancer.
Japanese NSCLC patients. Moreover, using silver in situ hybridization, Jin et al. [28] found that the increasing expression of MET was associated with smoking and higher pTNM stage in 143 Korean stage I lung cancer patients. All the above studies clearly showed that MET plays an important role in the development and progression of lung cancer. The ROC curve of serum MET DNA revealed a sensitivity of 72.6% and a specificity of 90.9% for lung cancer with 1.30 × 104 copies/μL as cutoff value. It has a better diagnostic value than that of existing tumor markers (NSE, SCC and CYFRA21-1), which are widely used for lung cancer diagnosis in clinical practice. Upon combination of serum MET DNA with any of the existing tumor markers (NSE, SCC, and CYFRA211) for lung cancer, sensitivity increased significantly. In the present study, the diagnostic value of serum MET DNA for lung cancer was evaluated, and the results indicated that serum MET DNA has potential as a tumor biomarker for the diagnosis of lung cancer, which deserves to be studied in a larger multicenter study. The present study clearly indicated that the level of serum MET DNA in the lung cancer group, especially in squamous cell carcinoma patients, was significantly higher than that in the benign lung disease and healthy groups. MET DNA had better diagnostic value than existing tumor markers. Therefore, serum MET DNA could be a superior candidate tumor biomarker for lung cancer diagnosis in clinical practice, and even used to prompt prognosis of lung cancer. Conflict of interest There is no actual and potential conflict of interest. Acknowledgements This research was supported by the Natural Science Foundation of CQ CSTC (grant no. cstc2012jjA10105), the National Clinical Key specialties Subject Construction Project [grant no. 2010 (305)] Table 3 Diagnostic sensitivity of serum MET DNA and existing tumor markers for lung cancer. Tumor marker
Sensitivity (%)
Specificity (%)
NSE SCC CYFRA21-1 MET MET + NSE MET + SCC MET + CYFRA21-1
39.1 24.9 66.1 72.6 83.3 79.4 90.7
79.5 84.1 86.4 90.9 72.3 76.4 78.5
Cutoff values: SCC 1.5 ng/mL; CYFRA21-1 3.0 ng/mL; NSE 13.0 ng/mL; MET 1.30 × 104 copies/μL.
References [1] Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin 2014;64(1): 9–29. [2] Navab R, Liu J, Seiden-Long I, Shih W, Li M, Bandarchi B, et al. Co-overexpression of Met and hepatocyte growth factor promotes systemic metastasis in NCI-H460 nonsmall cell lung carcinoma cells. Neoplasia 2009;11(12):1292–300. [3] Bottaro DP, Rubin JS, Faletto DL, Chan AM, Kmiecik TE, Vande Woude GF, et al. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science 1991;251:802–4. [4] Go H, Jeon YK, Park HJ, Sung SW, Seo JW, Chung DH. High MET gene copy number leads to shorter survival in patients with non-small cell lung cancer. J Thorac Oncol 2010;5(3):305–13. [5] Rees H, Williamson D, Papanastasiou A, Jina N, Nabarro S, Shipley J, et al. The MET receptor tyrosine kinase contributes to invasive tumour growth in rhabdomyosarcomas. Growth Factors 2006;24:197–208. [6] Cipriani NA, Abidoye OO, Vokes E, Salgia R. MET as a target for treatment of chest tumors. Lung Cancer 2009;63:169–79. [7] Benvenuti S, Comoglio PM. The MET receptor tyrosine kinase in invasion and metastasis. J Cell Physiol 2007;213:316–25. [8] Lee J, Seo JW, Jun HJ, Ki CS, Park SH, Park YS, et al. Impact of MET amplification on gastric cancer: possible roles as a novel prognostic marker and a potential therapeutic target. Oncol Rep 2011;25(6):1517–24. [9] Tsuta K, Kozu Y, Mimae T, Yoshida A, Kohno T, Sekine I, et al. c-MET/phospho-MET protein expression and MET gene copy number in non-small cell lung carcinomas. J Thorac Oncol 2012;7(2):331–9. [10] Yamamoto S, Tsuda H, Miyai K, Takano M, Tamai S, Matsubara O. Gene amplification and protein overexpression of MET are common events in ovarian clear-cell adenocarcinoma: their roles in tumor progression and prognostication of the patient. Mod Pathol 2011;24(8):1146–55. [11] Voutsina A, Tzardi M, Kalikaki A, Zafeiriou Z, Papadimitraki E, Papadakis M, et al. Combined analysis of KRAS and PIK3CA mutations, MET and PTEN expression in primary tumors and corresponding metastases in colorectal cancer. Mod Pathol 2013; 26(2):302–13. [12] Minuti G, Cappuzzo F, Duchnowska R, Jassem J, Fabi A, Fabi A, et al. Increased MET and HGF gene copy numbers are associated with trastuzumab failure in HER2positive metastatic breast cancer. Br J Cancer 2012;107(5):793–9. [13] Sun W, Ai T, Gao Y, Zhang Y, Cui J, Song L. Expression and prognostic relevance of MET and phospho-BaD in non-small cell lung cancer. OncolTargets Ther 2013;6: 1315–23. [14] Cappuzzo F, Marchetti A, Skokan M, Rossi E, Gajapathy S, Felicioni L, et al. Increased MET gene copy number negatively affects survival of surgically resected non-smallcell lung cancer patients. J Clin Oncol 2009;27:1667–74. [15] Marzese DM, Hirose H, Hoon DS. Diagnostic and prognostic value of circulating tumor-related DNA in cancer patients. Expert Rev Mol Diagn 2013;13(8): 827–44. [16] Medicine Kaiser J. Keeping tabs on tumor DNA. Science 2010;327(5969):1074. [17] Travis WD, Brambilla E, Mu¨ller-Hermelink HK, Harris CC. World health organization classification tumors of the lung, pleura, thymus and heart. Lyon. IARC Press; 2004 10–20. [18] Kanteti R, Yala S, Salgia Ferguson MK, MET R, HGF. EGFR, and PXN gene copy number in lung cancer using DNA extracts from FFPE archival samples and prognostic significance. J Environ Pathol Toxicol Oncol 2009;28(2):89–98. [19] Boberg DR, Batistela MS, Pecharki M, Ribeiro EM, Cavalli IJ, Lima RS, et al. Copy number variation in ACHE/EPHB4 (7q22) and in BCHE/MME (3q26) genes in sporadic breast cancer. Chem Biol Interact 2013;203(1):344–7. [20] Pros E, Lantuejoul S, Sanchez-Verde L, Castillo SD, Bonastre E, Suarez-Gauthier A, et al. Determining the profiles and parameters for gene amplification testing of growth factor receptors in lung cancer. Int J Cancer 2013;133(4):898–907.
Please cite this article as: Li D, et al, Quantification of serum MET in non-small-cell lung cancer and its clinical significance, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.11.021
D. Li et al. / Clinical Biochemistry xxx (2014) xxx–xxx [21] Fackler MJ, Lopez Bujanda Z, Umbricht C, Teo WW, Cho S, Zhang Z, et al. Novel methylated biomarkers and a robust assay to detect circulating tumor DNA in metastatic breast cancer. Cancer Res 2014;74(8):2160–70. [22] Kim K, Shin DG, Park MK, Baik SH, Kim TH, Kim S, et al. Circulating cell-free DNA as a promising biomarker in patients with gastric cancer: diagnostic validity and significant reduction of cfDNA after surgical resection. AnnSurgTreatRes 2014;86(3):136–42. [23] Ha SY, Lee J, Kang SY, Do IG, Ahn S, Park JO, et al. MET overexpression assessed by new interpretation method predicts gene amplification and poor survival in advanced gastric carcinomas. Mod Pathol 2013;26(12):1632–41. [24] Dziadziuszko R, Wynes MW, Singh S, Asuncion BR, Ranger-Moore J, Konopa K, et al. Correlation between MET gene copy number by silver in situ hybridization and protein expression by immunohistochemistry in non-small cell lung cancer. J Thorac Oncol 2012;7(2):340–7.
5
[25] Gonzalez-Angulo AM, Chen H, Karuturi MS, Chavez-MacGregor M, Tsavachidis S, Meric-Bernstam F, et al. Frequency of mesenchymal-epithelial transition factor gene (MET) and the catalytic subunit of phosphoinositide-3-kinase (PIK3CA) copy number elevation and correlation with outcome in patients with early stage breast cancer. Cancer 2013;119(1):7–15. [26] Sun W, Song L, Ai T, Zhang Y, Gao Y, Cui J. Prognostic value of MET, cyclin D1 and METgene copy number in non-small cell lung cancer. J Biomed Res 2013;27(3):220–30. [27] Okuda K, Sasaki H, Yukiue H, Yano M, Fujii Y. Met gene copy number predicts the prognosis for completely resected non-small cell lung cancer. Cancer Sci 2008; 99(11):2280–5. [28] Jin Y, Sun PL, Kim H, Seo AN, Jheon S, Lee CT, et al. MET gene copy number gain is an independent poor prognostic marker in Korean stage I lung adenocarcinomas. Ann Surg Oncol 2014;21(2):621–8.
Please cite this article as: Li D, et al, Quantification of serum MET in non-small-cell lung cancer and its clinical significance, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.11.021
Contents lists available at ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Quantification of serum MET in non-small-cell lung cancer and its clinical significance Detao Li a,1, Fengzeng Li b,1, Yanfeng Wu a, Dandan Zhou a, Hui Chen a,⁎ a b
Clinical Laboratories, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China Department of dermatology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
a r t i c l e
i n f o
Article history: Received 5 September 2014 Received in revised form 1 November 2014 Accepted 29 November 2014 Available online xxxx Keywords: Serum MET Tumor biomarker Non-small-cell lung cancer Diagnostic value
a b s t r a c t Objectives: Mesenchymal–epithelial transition factor (MET) plays a critical role in the development and progression of lung cancer. We aimed to quantify the level of serum MET DNA, analyze its diagnostic value, and provide a novel biomarker for lung cancer. Methods: Serum MET DNA was extracted from 95 patients with lung cancer, 10 with benign lung diseases, and 34 healthy volunteers. MET DNA was quantified using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR). Data were analyzed using statistical software SPSS 17.0. Results: Serum MET DNA level in the lung cancer group was higher than in the healthy group and benign lung diseases group. Serum MET DNA level was higher in lung cancers patients with smoking, squamous cell carcinoma, advanced TNM stage, and increased tumor size. The difference in serum MET DNA level was not related to sex, age, and lymph node metastasis among the lung cancer patients. The receiver operating characteristic curve showed a sensitivity of 72.6% and specificity of 90.9% for the ability of serum MET DNA to detect lung cancer at the cutoff value of 1.30 × 104 copies/μL. The association of serum MET DNA level with existing clinical lung tumor markers was analyzed, including neuron-specific enolase, squamous cell carcinoma antigen, and cytokeratin fragment 21-1. With the combination of serum MET DNA, the sensitivity was raised from 39.1%, 24.9%, 66.1% to 83.3%, 79.4% and 90.7%, respectively. Conclusions: Quantification of serum MET DNA by FQ-PCR may serve as a novel accessible diagnostic tool for the clinical screening and detection of lung cancer. © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Lung cancer is the leading cause of human cancer-related death worldwide [1]. Non-small-cell lung cancer (NSCLC) accounts for ~80% of all lung cancers. The pathogenesis of lung cancer is concealed and most patients have lost the opportunity for surgical resection when they are diagnosed [2]. Although there are many biomarkers for lung cancer, they are of no use for accurate diagnosis of early stage disease. Therefore, highly sensitive and specific tumor biomarkers need to be found for the diagnosis, evaluation, and prognosis of lung cancer. Mesenchymal–epithelial transition factor (MET) is a member of the sub-family of receptor tyrosine kinases (RTKs) for hepatocyte growth factor (HGF) [3]. It is located on chromosome 7q21–31 [4] and plays an important role in the progression of various tumors through regulating cell proliferation, angiogenesis, apoptosis, altering cytoskeletal Abbreviations: FQ-PCR, real-time fluorescent quantitative polymerase chain reaction; NSCLC, non-small-cell lung cancer; MET, mesenchymal–epithelial transition factor; HGF, hepatocyte growth factor; NSE, neuron-specific enolase; SCC, squamous cell carcinoma antigen; CYFRA21-1, cytokeratin fragment 21-1. ⁎ Corresponding author. Fax: +86 23 68716215. E-mail addresses: [email protected], [email protected] (H. Chen). 1 Detao Li and Fengzeng Li contributed equally to this work.
function, and promoting metastasis [5–7]. The overexpression of MET has been found in various human cancers, such as gastric [8], lung [9], ovarian [10], and colorectal [11] cancer. Minuti et al. [12] found that increased MET gene copy numbers were associated with trastuzumab failure in HER2-positive metastatic primary breast cancer, by fluorescence in situ hybridization (FISH). Sun et al. [13] found that positive expression of MET occurred in 67.2% paraffin-embedded specimens of NSCLC, and the positive expression of MET was significantly associated with shortened overall survival. Cappuzzo et al. [14] reported that high MET gene copy number (defined as a mean of copies/cell) was observed in 11.1% of NSCLCs tumor tissue and affected patient survival negatively. Although a lot of research has focused on alteration of the MET gene and protein in tumor tissue, the level of serum MET DNA has not yet been reported. Serum DNA is an emerging noninvasive biomarker, which has been used widely for cancer, including diagnosis, assessment of tumor progression, evaluation, and monitoring of the efficacy of anticancer therapy [15]. Its potential value as a blood biomarker was highlighted in an editorial in Science [16]. The present study is believed to be the first to quantify serum MET DNA in lung cancer patients by fluorescent quantitative polymerase chain reaction (FQ-PCR). The relationship between serum MET level
http://dx.doi.org/10.1016/j.clinbiochem.2014.11.021 0009-9120/© 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Please cite this article as: Li D, et al, Quantification of serum MET in non-small-cell lung cancer and its clinical significance, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.11.021
2
D. Li et al. / Clinical Biochemistry xxx (2014) xxx–xxx
and clinicopathological characteristics was analyzed. We evaluated the diagnostic value of serum MET combined with or without existing tumor markers for lung cancer.
specific enolase (NSE), squamous cell carcinoma antigen (SCC), and cytokeratin fragment (CYFRA)21-1. Statistical analysis
Materials and methods Subjects From October 2012 to March 2014, 95 patients with lung cancer were selected as the cancer group, and 34 healthy volunteers and 10 patients with benign lung disease were selected as the control group (benign lung disease included: four patients with pulmonary fibrotic nodules, four with pulmonary granulomas, and two with pulmonary inflammatory pseudotumors). All the sera were collected before surgery and any other treatment. Eligible samples were identified at the Department of Cardiothoracic Surgery, The First Affiliated Hospital of Chongqing Medical University, China. Data on sex, age, primary tumor staging, smoking status, lymph node metastasis, and histopathological grades were collected. Pathological diagnosis was classified according to the WHO classification of lung cancer [17], and tumor–node– metastases (TNM) stage was classified by a cardiothoracic physician and an experienced pathologist. Informed consent was obtained from all patients and healthy volunteers, and the study was approved by the Ethics Committees of the First Affiliated Hospital of Chongqing Medical University. Sample collection and serum DNA extraction Venous blood (2.0 mL) was collected from each subject in tubes without anticoagulants. After incubation at 37 °C for 15 min, the serum was separated by centrifugation at 2 000 g for 10 min, followed by 12 000 g for 10 min. The serum samples were stored at −80 °C until use. Genomic DNA was extracted from serum using a QIA amp Blood Mini Kit based on affinity columns (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. Quantification of serum MET DNA by FQ-PCR MET primers were designed as previously described [18] as follows: forward, 5-GCT GGT GGT CCT ACC ATA CAT G-3; reverse, 5-CTG GCT TAC AGC TAG TTT GCC A-3. The product size was 112 bp. The MET plasmid was constructed using a TA cloning kit as follows: purified PCR products were ligated into pMD18-T vector (TaKaRa, Dalian, China), and the recombinant plasmids were transformed into DH5α competent Escherichia coli. Positive E. coli clones were screened and enriched, and recombinant plasmid DNA was extracted. A standard curve was generated with 10 dilutions of plasmid DNA (101–1010 copies/μL) using the cobas Z 480 (Roche Diagnostics, Mannheim, Germany). FQ-PCRs were conducted within a total volume of 20 μL/well and contained the following reagents: 10 μL SYBR Premix ExTaq TM II (2×), 0.8 μL forward primer, 0.8 μL reverse primer, 2 μL DNA, and 6.4 μL deionized distilled water. The PCR kits were purchased from TaKaRa (TaKaRa Bio, Osaka, Japan). PCRs were conducted under the following conditions: one cycle at 95 °C for 30 s; followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s; 95 °C for 5 s, one cycle at 60 °C for 1 min; one cycle at 50 °C for 30 s. Each sample and DNA standard was analyzed in duplicate, and the mean value was used for quantification. Only the standard curve with a coefficient of correlation N0.96 was accepted. Quantification of existing tumor markers Serum was collected in a similar manner to that described in “Sample collection and serum DNA extraction.” Existing tumor markers for lung cancer were analyzed using the chemiluminescence immunoassay E170 (Roche Diagnostics, Mannheim, Germany), including neuron-
Data were analyzed with Mann–Whitney U test for unpaired samples and correlation coefficients determined using Spearman's rank test. MET level was summarized as medium and quartile M (P25, P75). A receiver operating characteristic (ROC) curve was plotted to determine the area under the curve (AUC), sensitivity, and specificity of serum MET DNA. Statistical analysis was performed using SPSS 17.0 software (SPSS Inc, Chicago, IL, USA), and P b 0.05 was considered statistically significant. Results Methodological evaluation of FQ-PCR for serum MET DNA The within-group and between-day coefficients of variation were 0.83% (n = 10) and 1.08% (n = 10), respectively. The dissociation curves exhibited good specificity with a mean peak at 81.5 °C. A straight line was obtained with the negative control. Tenfold serial dilutions of plasmid MET DNA from 101 to 1010 copies/μL were used to generate a standard curve. The curve exhibited good linearity at 103–108 copies/μL, with a correlation coefficient of 0.999. Clinical characteristics The relevant clinical characteristics of lung cancer patients, including sex, age, smoking status, histopathological types, TNM stage, tumor size, and lymph node metastasis, are summarized in Table 1. Serum MET DNA levels in different groups Serum MET DNA levels M (P25, P75) were 2.03 × 104 (1.08 × 104, 6.06 × 104) copies/μL, 6.27 × 103 (4.47 × 103, 9.18 × 103) copies/μL, and 4.83 × 103 (2.46 × 103, 8.81 × 103) copies/μL in the lung cancer, benign lung disease, and healthy groups, respectively. Further analysis indicated that higher serum MET DNA level existed in the lung cancer group, compared to the benign disease (P b 0.0001) and healthy (P b 0.0001) groups. Moreover, the M (P25, P75) of TNM I was higher than that of benign lung disease (P b 0.01) and healthy control (P b 0.0001)
Table 1 clinical characteristics of study subjects. Clinical characteristics
Patients (n = 95)
Gender (F/M) Age (years) Smokers/nonsmokers
20/75 (21.1/78.9%) 3/7 (30.0/70.0%) 60 (55–66) 52 (48–59) 63/32 (66.3/33.7%) 2/8 (20.0/80.0%)
Histopathological types Squamous cell carcinoma Adenocarcinoma Others
45 (47.4%) 37 (38.9%) 13 (13.7%)
TNM stages I II III IV
15 (15.8%) 39 (41.1%) 17 (17.9%) 24 (25.2%)
Tumor size (cm) d≤4 dN4
55 (57.9%) 40 (42.1%)
Lymph node metastasis Positive Negative
70 (78.9%) 25 (21.1%)
Benign (n = 10) Healthy (n = 34) 8/26 (23.5/76.5%) 59 (55–66) 20/14 (58.8/41.2%)
Please cite this article as: Li D, et al, Quantification of serum MET in non-small-cell lung cancer and its clinical significance, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.11.021
D. Li et al. / Clinical Biochemistry xxx (2014) xxx–xxx
obviously. In contrast, there was no difference between the benign and healthy groups (P = 0.251) (Fig. 1).
Table 2 Serum MET DNA level in lung cancer group with different clinical characteristics. Clinical characteristics
Serum MET DNA levels among lung cancer patients Serum MET level was higher in squamous cell carcinoma than adenocarcinoma (P = 0.0117). Serum MET level was higher in smokers (P = 0.0396), advanced TNM stage tumors (P = 0.0304), and larger tumors (P = 0.0324). However, no significant differences were observed for sex (P = 0.2157), age (P = 0.3632), and lymph node metastasis (P = 0.5261).The data are shown in Table 2. Diagnostic value of serum MET DNA for lung cancer The ROC curve (Fig. 2) showed that the AUC of serum MET DNA was 0.893 (95% CI: 0.841–0.945), it was higher than that of existing markers (including NSE, SCC, and CYFRA21-1). In accordance with the rule of the maximum Youden index, 1.30 × 104 copies/μL was set as the optimum cutoff value with a sensitivity of 72.6% and specificity of 90.9% (Fig. 2). Diagnostic sensitivity was 39.1%, 24.9% and 66.1% for single NSE, SCC, and CYFRA21-1, respectively, which were lower than that of MET (72.6%). Upon combination of serum MET DNA with the existing markers, sensitivity increased to 83.3%, 79.4% and 90.7%, respectively, with a better specificity (Table 3). However, there was no evident correlation of serum MET DNA level with that of NSE, SCC, or CYFRA21-1 (P N 0.05, data not shown). Discussion Genomic alterations such as gene amplifications and deletions commonly contribute to the development and progression of tumors [19]. During oncogenesis, these changes lead to gains or losses of parts of the genome in tumor cells, and result in the functional disorder of genes that regulate cell proliferation, apoptosis, genomic stability, angiogenesis, invasion, and metastasis, negatively or positively. As an RTK for HGF, MET plays an important role in tumorigenesis and its overexpression disrupts the balance of cell proliferation and apoptosis. In recent years, the overexpression of MET has attracted increasing attention. The overexpression of MET gene has been analyzed in tumor tissues by various assays, including qRT-PCR, immunohistochemistry, and FISH in tumor tissues. By FISH, Pros et al. [20] found that the overexpression of MET was obvious in an NSCLC tissue
Fig. 1. Serum MET DNA levels in lung cancer and control groups.
3
Gender Male (n = 75) Female (n = 20) Age ≤60 years (n = 50) N60 years (n = 45) Smoke status Smoker(n = 63) Nonsmoke(n = 32) Histological types a Squamous cell carcinoma (n = 45) Adenocarcinoma (n = 37) TNM stages I + II (n = 54) III + IV (n = 41) Tumor size (cm) d ≤ 4 (n = 55) d N 4 (n = 40) Lymph node metastasis Positive (n = 70) Negative (n = 25)
Serum MET levels [M (P25, P75), copies/μL]
P value⁎ 0.2157
2.48 × 104 (1.48 × 104, 6.98 × 104) 1.73 × 104 (7.61 × 103, 6.65 × 104) 0.3632 2.90 × 104 (1.56 × 104, 7.28 × 104) 2.03 × 104 (9.63 × 103, 6.82 × 104) 0.0396# 2.93 × 104 (1.52 × 104, 8.97 × 104) 1.81 × 104 (8.04 × 103, 6.65 × 104) 0.0117# 3.05 × 104 (1.60 × 104, 9.04 × 104) 1.85 × 104 (8.44 × 103, 4.32 × 104) 0.0304# 2.00 × 104 (9.18 × 103, 6.32 × 104) 2.95 × 104 (1.68 × 104, 9.40 × 104) 0.0324# 1.89 × 104 (1.05 × 104, 4.06 × 104) 3.20 × 104 (1.65 × 104, 8.73 × 104) 0.5261 2.86 × 104 (1.29 × 104, 8.62 × 104) 2.03 × 104 (1.09 × 104, 7.18 × 104)
a The cases of others histological type is too little to be neglected. ⁎ Mann–Whitney U test. # Statistically significant difference.
microarray panel containing 302 samples. Kanteti et al. [18] observed gene amplification of MET in tumor tissues of lung cancer patients. These studies were based on tumor tissues, their drawback was obvious for its injured and hysteresis. Recent developments have shown that serum DNA offers a convenient, noninvasive, and repeatable “liquid biopsy,” and it can be used to assess tumor progression and evaluate prognosis and response to treatment [21]. In a study of gastric cancer, Kim et al. [22] found that increased serum DNA level was associated with tumor burden, such as larger tumor and advanced TNM stage. Based on these reports, in the present study, serum was chosen as the sample to quantify the level of MET DNA initially. The present study found that the level of serum MET DNA in the lung cancer group was significantly higher than that in the benign lung disease and healthy groups. Furthermore, the higher level of serum MET DNA in TNM I than that of control groups hinted that serum MET is a valuable biomarker for the early diagnosis of lung cancer. It has been demonstrated that expression of MET increased in gastric carcinoma, along with higher MET mRNA level and increased DNA copy number [23]. Dziadziuszko et al. [24] found that MET expressed in NSCLC tissues correlated with an increase in its gene copies. Gonzalez-Angulo et al. [25] found that MET gene copies increased significantly in early breast cancer tissue. We obtained a similar result to all the above studies. According to the overexpression of MET in lung tumor tissues and higher level of serum MET DNA in lung cancer, we conjectured that MET promises to be a biomarker for lung cancer screening. We found that the level of serum MET DNA in lung cancer patients with smoking, squamous cell carcinoma, advanced TNM stages and increased tumor sizes were higher than that of cancer patients with no smoking, adenocarcinoma, earlier TNM stages, and smaller tumor size significantly, respectively. It showed that MET was associated with the development and progression of lung cancer. The published research got the similar results in tissue. In NSCLC, Go et al. [4] found that the MET gene amplification was more frequent in tumor tissues of squamous cell carcinoma than that in adenocarcinoma, and it was also associated with advanced tumor stage by FISH. Sun et al. [26] found that the overexpression of MET was associated with advanced tumor stage, based on analyzing 61 NSCLC tissue specimens by FQ-PCR. Okuda et al. [27] found that the increasing expression of MET was significantly correlated with smoking status and tumor size by qPCR in tumor tissues of
Please cite this article as: Li D, et al, Quantification of serum MET in non-small-cell lung cancer and its clinical significance, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.11.021
4
D. Li et al. / Clinical Biochemistry xxx (2014) xxx–xxx
Fig. 2. ROC curve of serum MET DNA level and the existing markers for lung cancer.
Japanese NSCLC patients. Moreover, using silver in situ hybridization, Jin et al. [28] found that the increasing expression of MET was associated with smoking and higher pTNM stage in 143 Korean stage I lung cancer patients. All the above studies clearly showed that MET plays an important role in the development and progression of lung cancer. The ROC curve of serum MET DNA revealed a sensitivity of 72.6% and a specificity of 90.9% for lung cancer with 1.30 × 104 copies/μL as cutoff value. It has a better diagnostic value than that of existing tumor markers (NSE, SCC and CYFRA21-1), which are widely used for lung cancer diagnosis in clinical practice. Upon combination of serum MET DNA with any of the existing tumor markers (NSE, SCC, and CYFRA211) for lung cancer, sensitivity increased significantly. In the present study, the diagnostic value of serum MET DNA for lung cancer was evaluated, and the results indicated that serum MET DNA has potential as a tumor biomarker for the diagnosis of lung cancer, which deserves to be studied in a larger multicenter study. The present study clearly indicated that the level of serum MET DNA in the lung cancer group, especially in squamous cell carcinoma patients, was significantly higher than that in the benign lung disease and healthy groups. MET DNA had better diagnostic value than existing tumor markers. Therefore, serum MET DNA could be a superior candidate tumor biomarker for lung cancer diagnosis in clinical practice, and even used to prompt prognosis of lung cancer. Conflict of interest There is no actual and potential conflict of interest. Acknowledgements This research was supported by the Natural Science Foundation of CQ CSTC (grant no. cstc2012jjA10105), the National Clinical Key specialties Subject Construction Project [grant no. 2010 (305)] Table 3 Diagnostic sensitivity of serum MET DNA and existing tumor markers for lung cancer. Tumor marker
Sensitivity (%)
Specificity (%)
NSE SCC CYFRA21-1 MET MET + NSE MET + SCC MET + CYFRA21-1
39.1 24.9 66.1 72.6 83.3 79.4 90.7
79.5 84.1 86.4 90.9 72.3 76.4 78.5
Cutoff values: SCC 1.5 ng/mL; CYFRA21-1 3.0 ng/mL; NSE 13.0 ng/mL; MET 1.30 × 104 copies/μL.
References [1] Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin 2014;64(1): 9–29. [2] Navab R, Liu J, Seiden-Long I, Shih W, Li M, Bandarchi B, et al. Co-overexpression of Met and hepatocyte growth factor promotes systemic metastasis in NCI-H460 nonsmall cell lung carcinoma cells. Neoplasia 2009;11(12):1292–300. [3] Bottaro DP, Rubin JS, Faletto DL, Chan AM, Kmiecik TE, Vande Woude GF, et al. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science 1991;251:802–4. [4] Go H, Jeon YK, Park HJ, Sung SW, Seo JW, Chung DH. High MET gene copy number leads to shorter survival in patients with non-small cell lung cancer. J Thorac Oncol 2010;5(3):305–13. [5] Rees H, Williamson D, Papanastasiou A, Jina N, Nabarro S, Shipley J, et al. The MET receptor tyrosine kinase contributes to invasive tumour growth in rhabdomyosarcomas. Growth Factors 2006;24:197–208. [6] Cipriani NA, Abidoye OO, Vokes E, Salgia R. MET as a target for treatment of chest tumors. Lung Cancer 2009;63:169–79. [7] Benvenuti S, Comoglio PM. The MET receptor tyrosine kinase in invasion and metastasis. J Cell Physiol 2007;213:316–25. [8] Lee J, Seo JW, Jun HJ, Ki CS, Park SH, Park YS, et al. Impact of MET amplification on gastric cancer: possible roles as a novel prognostic marker and a potential therapeutic target. Oncol Rep 2011;25(6):1517–24. [9] Tsuta K, Kozu Y, Mimae T, Yoshida A, Kohno T, Sekine I, et al. c-MET/phospho-MET protein expression and MET gene copy number in non-small cell lung carcinomas. J Thorac Oncol 2012;7(2):331–9. [10] Yamamoto S, Tsuda H, Miyai K, Takano M, Tamai S, Matsubara O. Gene amplification and protein overexpression of MET are common events in ovarian clear-cell adenocarcinoma: their roles in tumor progression and prognostication of the patient. Mod Pathol 2011;24(8):1146–55. [11] Voutsina A, Tzardi M, Kalikaki A, Zafeiriou Z, Papadimitraki E, Papadakis M, et al. Combined analysis of KRAS and PIK3CA mutations, MET and PTEN expression in primary tumors and corresponding metastases in colorectal cancer. Mod Pathol 2013; 26(2):302–13. [12] Minuti G, Cappuzzo F, Duchnowska R, Jassem J, Fabi A, Fabi A, et al. Increased MET and HGF gene copy numbers are associated with trastuzumab failure in HER2positive metastatic breast cancer. Br J Cancer 2012;107(5):793–9. [13] Sun W, Ai T, Gao Y, Zhang Y, Cui J, Song L. Expression and prognostic relevance of MET and phospho-BaD in non-small cell lung cancer. OncolTargets Ther 2013;6: 1315–23. [14] Cappuzzo F, Marchetti A, Skokan M, Rossi E, Gajapathy S, Felicioni L, et al. Increased MET gene copy number negatively affects survival of surgically resected non-smallcell lung cancer patients. J Clin Oncol 2009;27:1667–74. [15] Marzese DM, Hirose H, Hoon DS. Diagnostic and prognostic value of circulating tumor-related DNA in cancer patients. Expert Rev Mol Diagn 2013;13(8): 827–44. [16] Medicine Kaiser J. Keeping tabs on tumor DNA. Science 2010;327(5969):1074. [17] Travis WD, Brambilla E, Mu¨ller-Hermelink HK, Harris CC. World health organization classification tumors of the lung, pleura, thymus and heart. Lyon. IARC Press; 2004 10–20. [18] Kanteti R, Yala S, Salgia Ferguson MK, MET R, HGF. EGFR, and PXN gene copy number in lung cancer using DNA extracts from FFPE archival samples and prognostic significance. J Environ Pathol Toxicol Oncol 2009;28(2):89–98. [19] Boberg DR, Batistela MS, Pecharki M, Ribeiro EM, Cavalli IJ, Lima RS, et al. Copy number variation in ACHE/EPHB4 (7q22) and in BCHE/MME (3q26) genes in sporadic breast cancer. Chem Biol Interact 2013;203(1):344–7. [20] Pros E, Lantuejoul S, Sanchez-Verde L, Castillo SD, Bonastre E, Suarez-Gauthier A, et al. Determining the profiles and parameters for gene amplification testing of growth factor receptors in lung cancer. Int J Cancer 2013;133(4):898–907.
Please cite this article as: Li D, et al, Quantification of serum MET in non-small-cell lung cancer and its clinical significance, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.11.021
D. Li et al. / Clinical Biochemistry xxx (2014) xxx–xxx [21] Fackler MJ, Lopez Bujanda Z, Umbricht C, Teo WW, Cho S, Zhang Z, et al. Novel methylated biomarkers and a robust assay to detect circulating tumor DNA in metastatic breast cancer. Cancer Res 2014;74(8):2160–70. [22] Kim K, Shin DG, Park MK, Baik SH, Kim TH, Kim S, et al. Circulating cell-free DNA as a promising biomarker in patients with gastric cancer: diagnostic validity and significant reduction of cfDNA after surgical resection. AnnSurgTreatRes 2014;86(3):136–42. [23] Ha SY, Lee J, Kang SY, Do IG, Ahn S, Park JO, et al. MET overexpression assessed by new interpretation method predicts gene amplification and poor survival in advanced gastric carcinomas. Mod Pathol 2013;26(12):1632–41. [24] Dziadziuszko R, Wynes MW, Singh S, Asuncion BR, Ranger-Moore J, Konopa K, et al. Correlation between MET gene copy number by silver in situ hybridization and protein expression by immunohistochemistry in non-small cell lung cancer. J Thorac Oncol 2012;7(2):340–7.
5
[25] Gonzalez-Angulo AM, Chen H, Karuturi MS, Chavez-MacGregor M, Tsavachidis S, Meric-Bernstam F, et al. Frequency of mesenchymal-epithelial transition factor gene (MET) and the catalytic subunit of phosphoinositide-3-kinase (PIK3CA) copy number elevation and correlation with outcome in patients with early stage breast cancer. Cancer 2013;119(1):7–15. [26] Sun W, Song L, Ai T, Zhang Y, Gao Y, Cui J. Prognostic value of MET, cyclin D1 and METgene copy number in non-small cell lung cancer. J Biomed Res 2013;27(3):220–30. [27] Okuda K, Sasaki H, Yukiue H, Yano M, Fujii Y. Met gene copy number predicts the prognosis for completely resected non-small cell lung cancer. Cancer Sci 2008; 99(11):2280–5. [28] Jin Y, Sun PL, Kim H, Seo AN, Jheon S, Lee CT, et al. MET gene copy number gain is an independent poor prognostic marker in Korean stage I lung adenocarcinomas. Ann Surg Oncol 2014;21(2):621–8.
Please cite this article as: Li D, et al, Quantification of serum MET in non-small-cell lung cancer and its clinical significance, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.11.021
