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ORIGINAL ARTICLE
Year : 2014  |  Volume : 10  |  Issue : 7  |  Page : 150-154

A comparison of consistency of detecting BRAF gene mutations in peripheral blood and tumor tissue of nonsmall-cell lung cancer patients


1 Department of Oncology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058; Department of Integrated Chinese Traditional Medicine and Wesern Medicine, Zhejiang Cancer hospital, Hangzhou, Zhejiang 310021, China
2 Department of Pathology, The General Military Hospital of Beijing PLA, Beijing 100700, China
3 Department of Surgery, Zhejiang Cancer hospital, Hangzhou, Zhejiang 310021, China
4 Department of Pathology, Shandong Weifang People's Hospital, Weifang, Shandong 261041, China
5 Department of Oncology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058, China

Date of Web Publication29-Nov-2014

Correspondence Address:
Chao He
Department of Oncology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.145847

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 > Abstract 

Objective: The aim was to detect the consistency of the BRAF gene mutation in peripheral blood and tumor tissue of patients with nonsmall-cell lung cancer and discuss the clinical application value of BRAF gene mutation in peripheral blood.
Materials and Methods: Real-time fluorescent quantitative polymerase chain reaction was used to test the tissues in 257 patients of nonsmall-cell lung cancer (NSCLC) 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 BRAF gene mutation in them by comparison of mutations consistency in blood and tissue samples, and analyzed the correlation between BRAF gene mutations and clinical characteristics of patients.
Results: The BRAF gene mutation rate was 7.23% in peripheral blood of 23 patients with nonsmall-cell lung cancer, and was 5.45% in 14 cancer tissues, the mutation consistency was 80.00% in peripheral blood-tumor tissue matched samples. The consistency was statistically significant (k =0.710, P = 0.000).
Conclusion: The consistency of the BRAF gene mutation in peripheral blood and tissue is high. BRAF gene mutations of peripheral blood could be used for clinical diagnosis and treatment in cases when tissue specimen is hard to get.

Keywords: Cancer, nonsmall-cell lung, BRAF gene, mutation


How to cite this article:
Fang M, Xu C, Wu J, Zhang Y, He C. A comparison of consistency of detecting BRAF gene mutations in peripheral blood and tumor tissue of nonsmall-cell lung cancer patients. J Can Res Ther 2014;10, Suppl S3:150-4

How to cite this URL:
Fang M, Xu C, Wu J, Zhang Y, He C. A comparison of consistency of detecting BRAF gene mutations in peripheral blood and tumor tissue of nonsmall-cell lung cancer patients. J Can Res Ther [serial online] 2014 [cited 2019 Dec 9];10:150-4. Available from: http://www.cancerjournal.net/text.asp?2014/10/7/150/145847


 > Introduction Top


Lung cancer has been the most common cancer in terms of both incidence and mortality worldwide. [1] 80-85% of all lung cancers are nonsmall-cell lung cancer (NSCLC) with two-thirds presenting with locally advanced or metastatic disease at diagnosis. [2] Treatment for these patients includes chemotherapy, radiotherapy and best supportive care, [3] which have been the cornerstone of treatment for nonsmall-cell lung cancer 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) and erlotinib (Tarceva, OSI-774; OSI Pharmaceuticals, Farmingdale, NY), are the first targeted drugs to enter clinical use for the treatment of lung cancer. [4],[5],[6] EGFR activating mutations can increase tumor sensitivity to EGFR TKIs. [7],[8] Yang Zhang et al. study revealed that NSCLC harbor oncogenic driver mutations in genes such as EGFR, anaplastic lymphoma kinase (ALK), human epidermal growth factor receptor-2 (HER-2), Kirsten rat sarcoma 2 viral oncogene homolog (K-RAS) and v-raf murine sarcoma viral oncogene homolog B (BRAF); [9],[10] Tumors harboring oncogenic driver mutations are significantly associated with the sensitivity of molecular treatment. [11],[12],[13] BRAF, one of the three members of the RAF kinase family, belongs to the group of serine-threonine kinases and plays a vital role in mitogen-activated protein kinase (MAPK) pathways. [14] Mutations of BRAF has been found in 0.5-3% of NSCLC. [15],[16] Among the different mutations occurring in the BRAF gene, BRAFV600E is the most common. A number of BRAF inhibitors, including sorafenib [17] (Nexavar, Bayer AG), vemurafenib [18] (ZELBORAF, Hoffmann-LaRoche Inc.) and dabrafenib [19] (formerly GSK2118436, which has received fast track development by the FDA for lung cancer), are under clinical development. Recently, a study published in JAMA, revealed that treatment of targeted the oncogenic drivers in lung cancers lived longer. [20] Thus, the detection of genetic driver mutation in lung cancer patients has become the most important tool in clinical practice.

Specimens for mutation analyses are tumor tissue, surgical tissues, or biopsy specimens; however it is often difficult to obtain sufficient amounts of tumor samples from NSCLC patients. It is necessary to find new surrogate sample types to detect driver mutation in NSCLC. Recent studies showed that mutations 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. [21]

AS the feasibility and availability of serum DNA detection methods have been confirmed, we performed a study to compare the consistence of BRAF mutation analyses in serum and tumor tissue.


 > Materials and methods Top


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 3 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. [22] 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. A history of cigarette smoking was obtained from the patient interview by professional doctors. Smokers were defined as a lifetime smoking dose was more than 100 cigarettes. [23] Lifetime cigarette consumption was quantified by the number of packs smoked everyday over the number of total smoking years (pack-years).

The study was performed on unselected and serially collected specimens of NSCLC. Tissues in 257 and the peripheral blood samples in 318 patients were gathered and tested, cancerous tissues with matched preoperative peripheral blood samples from 185 NSCLC patients were investigated the consistency of BRAF mutation. Patients' blood was 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. [24]

The BRAFV600E (exon 15) ADx - ARMS kit (Amoy ADx Ltd., Xiamen, CHN) was used to detect mutations by real-time polymerase chain reaction (PCR) following the user manual. The kit enabled us to detect the low-level mutant DNA in the background of wild-type DNA based on the allele-specific and real-time PCR technologies. The plate was sealed and loaded into a Stratagene MX3000P real-time PCR system (Agilent Technologies, Santa Clara, USA).

The BRAF gene mutations were detected in blood and tissue samples and compared the consistency between them, and then potential association between BRAFV600E and clinical parameters was evaluated statistically.

Statistical and database analysis

The Chi-square test was used to compare the association of BRAF mutation in peripheral blood and tumor tissues. The correlation between BRAF gene mutations and clinical characteristics of patients were analyzed using the Fisher's-exact test. A P < 0.05 was considered as statistically significant. All statistical analyses were performed using the Statistical Package for the Social Sciences version 13.0 (SPSS, Inc., Chicago, IL, USA).


 > Results Top


BRAF mutation in peripheral blood and cancer tissues

Of the 257 analyzed cancerous tissues, the BRAF gene mutation rate was 14 (5.45%), and was 23 (7.23%) in peripheral blood of 318 patients with NSCLC [Table 1]. Of the 185 paired cases, the BRAF mutation was positive in 10 cancerous tissues and 13 peripheral bloods; in these patients, 8 were positive in the two kinds of samples, concurrently. The consistency was 80.00% in these peripheral blood-tumor tissue matched samples. The difference was statistically significant (k =0.710, P = 0.000).
Table 1: Comparison BRAF mutation (L) status analysis in peripheral blood and corresponding tumor tissue (n=185, κ=0.710)


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Peripheral blood BRAF mutations and clinical characteristics

Of the 318 analyzed peripheral blood sample, BRAF mutation was positive in 23 cases, and the positive rate was 7.23%. The correlation of clinical characteristics and peripheral blood BRAF mutation status are presented in [Table 2]. In this group, it seemed that the BRAF mutation rate was higher in people of more than 60-year-old, female, never smoker and nonadenocarcinoma. However, these differences were insignificant trends, P > 0.5.
Table 2: Patient characteristics and BRAF mutation status in blood


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Cancerous tissue BRAF mutation and clinical characteristics

In 257 cases of cancerous tissue samples, the active BRAF mutation status was detected. The activation of BRAF mutation was identified in 14 samples and the mutation rate was 5.45% [Table 3]. A significant association was found that the activated BRAF mutation rate was higher in people of nonsmokers, P = 0.013.
Table 3: Patient characteristics and BRAF mutation status in tumor tissue


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It seemed that the BRAF mutation status has no significant relationship between the activated BRAF mutation and status of age, gender and pathological type (P > 0.5).


 > Discussion Top


Nonsmall-cell lung cancer 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, K-RAS, HER-2, 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.

Targeted therapy for molecularly selected populations with NSCLC is effective remarkably and show improved PFS compared to cytotoxic chemotherapy. [6],[7],[25]

BRAF, one of driver genes in NSCLC, is a serine-threonine kinase in the RAS/RAF/MEK/MAPK signaling pathway, which is downstream of EGFR and plays a significant role in tumorigenesis. BRAF-mutated tumors have been suggested to belong to an aggressive histotype, characterized by micropapillary features and shorter DFS and overall survival. [26] Many reports show that BRAF mutations are highly specific negative predictors of response to EGFR-TKIs and are associated with shorter DFS in advanced NSCLC. Warth A [27] et al. screened for recurrent mutations in K-RAS/NRAS/BRAF/MEK1 in nearly 200 tumor samples from patients with acquired resistance to EGFR TKIs. Among these samples, no K-RAS, NRAS, or MEK1 mutations were detected; however, they found one case with concurrent EGFR exon19 deletion and EGFR T790M and BRAFV600E mutations and another case with EGFR exon19 deletion and the BRAF G469A mutation (2/195, 1.0%). Antonio Marchetti et al.[28] retrospectively investigated 1046 cases of NSCLC, indicated that the BRAF mutations were associated with poor prognosis.

These findings show that the BRAF mutations 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 BRAF mutations status is necessary. However, the detection of BRAF mutation, 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. [29],[30],[31] The primary objective of the current study was to compare the consistency of BRAF mutation detection in serum and tumor tissue. And the BRAF mutation status obtained from the tumor sample served as a reference to compare with the results obtain from the peripheral blood samples in our study.

We have shown that the BRAF mutation rate was 14 (5.45%) and 23 (7.23%) in tumor tissue and peripheral blood, respectively. The BRAF mutation status of blood samples showed strong coincidence with tumor tissues, there was a good consistency of about 80% in BRAF mutation detection between tumor tissue and peripheral blood, (k =0.710, P = 0.000). The Circulating BRAF mutation were 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 BRAF mutation status of blood samples was not associated with patients' clinical characteristics, such as age, gender, smoke status and pathological types. However, the BRAF mutation rate of cancerous samples was higher in smokers, the difference was significant, P = 0.013. These results were not consistent with previously reported data. Study by Antonio Marchetti et al.[28] found BRAFV600E mutations were significantly more prevalent in females (approximately 9% of females with ADCs had V600E mutations) and were independent of smoking history. Such in accordance may be due to the relatively low number of patient cases investigated in our study.

Our data revealed that BRAF mutation 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.


 > Conclusion Top


We demonstrated the feasibility of measuring BRAF mutations in plasma. Notwithstanding its limitation, our study provides evidence to support that tumor tissue sample is still the best source for BRAF mutation analysis in NSCLC patients. The consistency of the BRAF gene mutation in peripheral blood and tissue is high. A peripheral blood sample may be used as an alternative source in special circumstances (for example, not enough tumor tissue samples available). Our results still need to be further confirmed by more investigation.


 > Acknowledgments Top


This study is supported by Medical Scientific Research Foundation of Zhejiang Province of China (grant no. 2013 KYB051).

 
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