|Year : 2015 | Volume
| Issue : 5 | Page : 63-67
A comparison of consistency of detecting c-MET gene amplification in peripheral blood and tumor tissue of nonsmall cell lung cancer patients
Daobao Chen1, Chunwei Xu2, Jie Wu1, Yuping Zhang3, Meiyu Fang4
1 Department of Surgery, Zhejiang Cancer Hospital, Hangzhou, Zhejiang 310021, P.R. China
2 Department of Pathology, The General Military Hospital of Beijing PLA, Beijing 100700, P.R. China
3 Department of Pathology, Shandong Weifang People's Hospital, Weifang, Shandong 261041, P.R. China
4 Department of Integrated Chinese Traditional Medicine and Western Medicine, Zhejiang Cancer Hospital, Hangzhou, Zhejiang 310021, P.R. China
|Date of Web Publication||31-Aug-2015|
Prof. Meiyu Fang
Department of Integrated Chinese Traditional Medicine and Western Medicine, Zhejiang Cancer Hospital, Hangzhou, Zhejiang 310021
Source of Support: None, Conflict of Interest: None
Objective: To detect the consistency of the c-MET gene amplification in peripheral blood and tumor tissue of patients with non-small cell lung cancer (NSCLC) and discuss the clinical application value of c-MET gene amplification in peripheral blood.
Materials and Methods: Real-time fluorescent quantitative polymerase chain reaction was used to test the tissues in 257 patients of NSCLCs and the peripheral blood samples in 318 patients of NSCLC, of which 185 cases of peripheral blood specimens could match the tissue samples, and detected the c-MET gene amplification in them by comparison of amplifications consistency in blood and tissue samples, and analyzed the correlation between c-MET gene amplification and clinical characteristics of patients.
Results: The c-MET gene amplification rate was 9.75% in peripheral blood of 31 patients with NSCLC, and was 8.95% in 23 cancer tissues, the amplification consistency, was 81.25% in peripheral blood-tumor tissue matched samples. The difference was statistically significant (P < 0.05).
Conclusion: The consistency of the c-MET gene amplification in peripheral blood and tissue is high. c-MET gene amplification of peripheral blood could be used for clinical diagnosis and treatment in cases when tissue specimen is hard to get.
Keywords: Amplification, cancer, c-MET gene, nonsmall cell lung
|How to cite this article:|
Chen D, Xu C, Wu J, Zhang Y, Fang M. A comparison of consistency of detecting c-MET gene amplification in peripheral blood and tumor tissue of nonsmall cell lung cancer patients. J Can Res Ther 2015;11, Suppl S1:63-7
|How to cite this URL:|
Chen D, Xu C, Wu J, Zhang Y, Fang M. A comparison of consistency of detecting c-MET gene amplification in peripheral blood and tumor tissue of nonsmall cell lung cancer patients. J Can Res Ther [serial online] 2015 [cited 2020 Jul 8];11:63-7. Available from: http://www.cancerjournal.net/text.asp?2015/11/5/63/163843
Daobao Chen and Chunwei Xu contribute equally to this work.
| > Introduction|| |
Lung cancer has been the most common cancer in terms of both incidence and mortality worldwide.  About 80-85% of all lung cancers are nonsmall-cell lung cancer (NSCLC) with two-thirds presenting with locally advanced or metastatic disease at diagnosis.  Treatment for these patients includes chemotherapy, radiotherapy and best supportive care,  which have been the cornerstone of treatment for NSCLC for many years. In recent years, due to rapid developments in targeted therapies, numerous small-molecule tyrosine kinase inhibitors (TKIs) drugs that target the epidermal growth factor receptor (EGFR), have been developed and applied clinically, such as gefitinib (Iressa, ZD1839; AstraZeneca, Wilmington, DE, USA) and erlotinib (Tarceva, OSI-774;OSIPharmaceuticals, Farmingdale, NY, USA), are the first targeted drugs to enter clinical use for the treatment of lung cancer. ,, Tumors harboring oncogenic driver mutations are significantly associated with the sensitivity of molecular treatment. ,, It was found that EGFR-mutant tumors with exon 19 deletions and L858R substitutions are highly sensitive initially to the TKIs. , Unfortunately, tumor cells eventually acquire resistance, with the progression of disease occurring in patients around 10-16 months after starting treatment.  Besides a second-site EGFR mutations such as T790M, , the amplification of the gene encoding an alternative kinase MET ,, plays a significant role in genetic mechanisms of resistance. The c-MET oncogene was isolated from a human osteosarcoma-derived cell line driven by a DNA rearrangement translocated promoter region (TPR)-MET, where the TPR locus on chromosome 1 fuses to the MET sequence on chromosome 7  and encodes for a prototype of the c-MET receptor tyrosine kinase subfamily. c-MET gene amplification causes protein overexpression and constitutive activation of the kinase domain  and has been observed both in primary tumors or as secondary events affecting therapy sensitivity in cancer cells.  Somatic mutations on the MET gene are rarely at 2-3%. , The most frequent genetic alteration is gene amplification, and as a consequence high c-MET protein expression and activation that has been reported as associated with a poor prognosis in NSCLC.  Recently, a study published in JAMA, revealed that treatment of targeted the oncogenic drivers in lung cancers lived longer.  A number of c-MET inhibitors, including Dacomitinib (ClinicalTrials.gov identifiers: NCT00548093  ) and Crizotinib (ClinicalTrials.gov identifiers: NCT00932893  ) are under clinical development. MET-targeted monoclonal antibody (onartuzumab [MetMAb]  ) and a MET-directed TKI (tivantinib)  are also in progress.
Thus, the detection of genetic driver abnormities in lung cancer patients has become the most important tool in clinical practice.
Specimens for genetic analyses are tumor tissue, surgical tissues, or biopsy specimens; however it is often difficult to obtain sufficient amounts of tumor samples from NSCLC patients due to reasons such as unrepresentative tumor cells, little amount cells, and genetic changes taken place.
It is necessary to find new surrogate sample types to detect driver genetic abnormity in NSCLC. Recent studies showed that genetic abnormities can be detected in peripheral blood DNA samples of patients with NSCLC and they were not only identical to those in the corresponding tumors, but could also predict the response of small-molecule TKIs treatment. 
AS the feasibility and availability of serum DNA detection methods have been confirmed, we performed a study to compare the consistency of c-MET amplification analyses in serum and tumor tissue.
| > Materials and Methods|| |
Samples collection and DNA extraction
Patients with pathologically confirmed NSCLC were recruited in our study between January 2007 and December 2013. Enrolled patients were from three institutions in China as follows: Zhejiang Cancer Center, Chinese people's liberation army general hospital, and Shandong Weifang People's Hospital. The diagnosis of NSCLC was based on the histological findings of the tumor tissue, and the histological type was determined according to World Health Organization criteria.  All the three institutions reviewed and approved the study by their Ethics Committees, respectively. All the patients signed informed consent to participate in this study and gave permission for the use of their plasma and tumor tissues. Patients did not receive any neoadjuvant treatment. The history of cigarette smoking was obtained from a patient interview by professional doctors. Smokers were defined as a lifetime smoking dose was more than 100 cigarettes.  Lifetime cigarette consumption was quantified by the number of packs smoked every day over the number of total smoking years (pack-years).
This study was performed on unselected and serially collected specimens of NSCLC. Tissues in 257 and the peripheral blood samples of 318 patients were collected and tested; of them, 185 cancerous tissues with matched preoperative peripheral blood samples from NSCLC patients were investigated the consistency of c-MET amplification status. Patients' blood samples were collected before the radical surgery, to avoid the contamination of skin cells, blood samples were taken through an intravenous catheter and discarded the first few milliliters of blood. Tumor tissues kept Formalin-fixed paraffin-embedded (FFPE) after the operation and stored in a freezer at −80°C until analysis. Genomic DNA was isolated using a proteinase-K digestion and phenol/chloroform extraction procedure by the QIAamp DNA FFPE Kit and Qiamp Blood Kit (Qiagen, Hilden, Germany) respectively according to the protocol described in the manufacturer's instructions. The extracted DNA was stored at −20°C until used. 
Real-time fluorescent quantitative polymerase chain reaction (PCR) was used to detect the c-MET gene amplification in tumor tissue and blood sample. The levels of MET was evaluated using the following primers and methods previously published.  MTHFR was selected as a control gene. MET-sense: 5'- CCA TCC AGT GTC TCC AGA AGT G-3'; MET-anti-sense: 5'- TTC CCA GTG ATA ACC AGT GTG TAG-3'; MTHFR-sense: 5'- CCA TCT TCC TGC TGC TGT AAC TG-3'; MTHFR-anti-sense: 5'- GCC TTC TCT GCC AAC TGT CC-3'. Genomic DNA (20 ng) was amplified for 45 cycles (10 s 94°C, 40 s 58°C) a Stratagene MX3000P real-time PCR system (Agilent Technologies, Santa Clara, USA), using the QuantiTect SYBR-Green PCR kit (Qiagen, Valencia, CA, USA) and 400 nM primers. Here, we chose a ratio of MET: MTHFR >1.5 to define MET amplification.
The c-MET gene amplifications were tested in blood and tissue samples and compared the consistency between them, and then the potential association between c-MET gene amplification and clinical parameters was evaluated statistically.
Statistical and database analysis
The Chi-square test was used to compare the association of c-MET gene amplification in peripheral blood and tumor tissues. The correlation between c-MET gene amplification and clinical characteristics of patients were analyzed by the Fisher's-exact test. A P < 0.05 was considered statistically significant. All statistical analyses were performed using the Statistical Package for the Social Sciences version 13.0 (SPSS, Inc., Chicago, IL, USA).
| > Results|| |
c-MET gene amplification in peripheral blood and cancer tissues
Of the 257 analyzed cancerous tissues, the c-MET gene amplification rate was 29 (11.28%), and was 31 (9.75%) in peripheral blood of 318 patients with NSCLC [Table 1]. Of the 185 paired cases, c-MET gene amplification was positive in 16 cancerous tissues and 34 peripheral bloods; of these patients, 13 were positive in the two kinds of samples, concurrently. The consistency was 81.25% in these peripheral blood-tumor tissue matched samples. The difference was statistically significant (κ =0.456, P = 0.000).
|Table 1: Comparison c-MET gene amplification (L) status analysis in peripheral blood and corresponding tumor tissue (n=185, ê=0.456) |
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Cancerous tissue c-MET gene amplification and clinical characteristics
In 257 cases of cancerous tissue samples, the active c-MET gene amplification status was detected. The c-MET gene amplification was identified in 23 samples, and the amplification rate was 8.95% [Table 2]. A significant association was found that the activated c-MET gene amplification rate was higher in people of smokers, P = 0.029.
|Table 2: Patient characteristics and c-MET gene amplification status in tumor tissue |
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It seemed that the c-MET amplification status has no significant relationship with the status of patients' age, gender, and pathological type (P > 0.5).
Peripheral blood c-MET gene amplification status and clinical characteristics
Of the 318 analyzed peripheral blood sample, c-MET gene amplification status was positive in 31 cases, and the positive rate was 9.75%. The correlation of clinical characteristics and peripheral blood c-MET gene amplification status are presented in [Table 3]. In this group, the c-MET gene amplification rate was significant higher in people of male gender and smokers. It seemed that patients of adenocarcinoma had a higher c-MET gene amplification rate (12.04%) compared with the nonadenocarcinoma (6.3%). However, the differences was insignificant trends, P = 0.091. The results showed that the c-MET amplification status has no significant relationship with the status of patients' age.
|Table 3: Patient characteristics and c-MET gene amplification status in blood |
Click here to view
| > Discussion|| |
NSCLC is currently segregated by the presence of actionable driver oncogenes. It is proven that lung cancer is highly associated with some genetic alterations, a lot of specific driver mutations, such as EGFR, KRAS, HER2, BRAF, PIK3CA and EML4-ALK contributed to the majority of lung cancer. As new oncogene aberrations in NSCLC are identified, the novel targeted therapies are developed and applied clinically. Somatic mutations in exons 19 and a single missense mutation that substitutes arginine for leucine at position 858 (L858R) are associated with increased sensitivity of lung adenocarcinomas to the selective EGFR kinase inhibitors, gefitinib (Iressa) and erlotinib (Tarceva). , However, lung cancers with drug-sensitive EGFR mutations that initially respond to TKIs eventually develop acquired resistance. , The c-MET gene amplification  plays an important role in resistance. Research by Engelman et al. revealed that MET activation can have profound effects on cell growth, survival, motility, invasion, and angiogenesis.  In addition, several studies have shown that increased c-MET copy number is an independent negative prognostic factor in surgically resected NSCLC. 
These findings show that the c-MET gene amplification identifying is crucial for therapy selection and prognosis predictor. As diversity in tumors and genomic instability induce the dynamically evolving entities both genetically and epigenetically, thus dynamic monitoring and analyzing the c-MET gene amplification status is necessary.
However, the detection of c-MET gene amplification, tumor tissue is the most common and standard sample source. In many cases, minimally invasive biopsies provide insufficient material and the size of tumors decreased after the treatment, these make it more difficult to obtain adequate tumor tissue for molecular studies. A blood sample can be obtained safely, with the option of repeat sampling from all NSCLC patients regardless of their characteristics; blood sampling was used in driver gene mutation detection recently. ,, The primary objective of this study was to compare the consistency of c-MET gene amplification detection in serum and tumor tissue. And the c-MET gene amplification status obtained from the tumor sample served as a standard to compare with the results obtain from the peripheral blood samples in our study.
We have shown that the c-MET gene amplification rates were 23 (8.95%) and 31 (9.75%) in tumor tissue and peripheral blood, respectively. The c-MET gene amplification status of blood samples showed strong coincidence with tumor tissues, there was a good consistency of about 81.25% (13/16) in c-MET gene amplification detection between tumor tissue and peripheral blood, (κ coefficient = 0.456, P = 0.000). The circulating c-MET gene amplification was readily identified in all patients. However, the positive rate was higher in blood than in tumor tissue, this might be due to the false positive of blood or false negative of tumor tissues.
In our study, the circulating c-MET gene amplification status of blood samples was significantly associated with patients' gender and smoke status; clinical characteristics, such as age and pathological types have no association with c-MET gene amplification status. However, the c-MET gene amplification rate in cancerous samples was significant higher in smokers (P = 0.029), but there is no significant difference between male and female. These results were not consistent with previously reported data.
A study by Cappuzzo et al.  found that c-MET gene amplifications were not associated with sex, smoking status, or histology. In contrast, MET FISH-positive status was significantly associated with grade 3 (P = 0.016) and with advanced stage (P = 0.01). Such in accordance may be due to the relatively low number of patient cases investigated in our study.
Our data revealed that c-MET gene amplifications detection in blood samples may allow for noninvasive genotyping in patients with NSCLC, which could be repeated at therapeutic decision-making points during a patient's course of therapy; although, our sample size, especially the matched sample, was limited. Further investigations with larger sample sizes to validate our results are warranted.
This study is supported by Medical Scientific Research Foundation of Zhejiang Province of China (grant no. 2013 KYB051) and Zhejiang Administration of Traditional Chinese Medicine Foundation (grant no. 2013ZQ005).
Financial support and sponsorship
This study is supported by Medical Scientific Research Foundation of Zhejiang Province of China (grant no. 2013 KYB051) and Zhejiang Administration of Traditional Chinese Medicine Foundation (grant no. 2013ZQ005).
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]