Original Article
A comparison of consistency of detecting BRAF gene mutations in peripheral blood and tumor tissue of nonsmall‑cell lung cancer patients 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 (κ =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. KEY WORDS: Cancer, nonsmall‑cell lung, BRAF gene, mutation
INTRODUCTION 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. [4‑6] 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 C150
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.[11‑13] 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.
Meiyu Fang1,2, Chunwei Xu3, Jie Wu4, Yuping Zhang5, Chao He1 Department of Oncology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058, 2Department of Integrated Chinese Traditional Medicine and Wesern Medicine, Zhejiang Cancer hospital, Hangzhou, Zhejiang 310021, 3 Department of Pathology, The General Military Hospital of Beijing PLA, Beijing 100700, 4 Department of Surgery, Zhejiang Cancer hospital, Hangzhou, Zhejiang 310021, 5Department of Pathology, Shandong Weifang People’s Hospital, Weifang, Shandong 261041, P.R. China 1
For correspondence: Prof. Chao He, Department of Oncology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058, China. E‑mail: fangjade2004@ icloud.com Access this article online Website: www.cancerjournal.net DOI: 10.4103/0973-1482.145847 PMID: *** Quick Response Code:
Journal of Cancer Research and Therapeutics - Volume 10 - Special Issue 2- 2014
Fang, et al.: Detect BRAF mutation in blood and tumor tissue
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 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 Journal of Cancer Research and Therapeutics - Volume 10 - Special Issue 2- 2014
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 0.5. 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 Table 1: Comparison BRAF mutation (L) status analysis in peripheral blood and corresponding tumor tissue (n=185, κ=0.710) BRAF mutation in B (+) (−) Total
BRAF mutation in T (+) 8 2 10
(−) 5 170 175
Total 13 172 185
BRAF=v‑raf murine sarcoma viral oncogene homolog B1, T=Tumor tissue, B=Peripheral blood, M+=Mutation positive, M−=Mutation negative
C151
Fang, et al.: Detect BRAF mutation in blood and tumor tissue
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. 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 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] Table 2: Patient characteristics and BRAF mutation status in blood Patient characteristics Age ≤60 >60 Gender Male Female Smoker Yes No Pathological pattern Adenocarcinoma Nonadenocarcinoma
Total blood saples (n=318) BRAF BRAF Percentage mutation (+) mutation (−)
P
8 15
118 177
6.35 7.81
0.622
20 3
275 20
6.78 13.04
0.484
16 7
215 80
6.93 8.05
0.808
17 6
229 66
6.91 8.33
0.682
BRAF=v‑raf murine sarcoma viral oncogene homolog B1, M+=Mutation positive, M−=Mutation negative
Table 3: Patient characteristics and BRAF mutation status in tumor tissue Patient characteristics Age ≤60 >60 Gender Male Female Smoker Yes No Pathological pattern Adenocarcinoma Nonadenocarcinoma
Total tumor tissue saples (n=257) BRAF BRAF Percentage mutation (+) mutation (−) 5 9
78 165
6.02 5.17
0.778
11 3
163 80
6.32 3.61
0.548
12 2
116 127
9.38 1.56
0.013
8 6
119 124
6.30 4.62
0.552
BRAF=v‑raf murine sarcoma viral oncogene homolog B1, M+=Mutation positive, M−=Mutation negative
C152
P
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.[29‑31] 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, (κ =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 Journal of Cancer Research and Therapeutics - Volume 10 - Special Issue 2- 2014
Fang, et al.: Detect BRAF mutation in blood and tumor tissue
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 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 This study is supported by Medical Scientific Research Foundation of Zhejiang Province of China (grant no. 2013 KYB051).
REFERENCES 1. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin 2014;64:9‑29. 2. Sher T, Dy GK, Adjei AA. Small cell lung cancer. Mayo Clin Proc 2008;83:355‑67. 3. Fang M, Wang S, Zheng Y, Kong X, Gong L, Qiu Y, et al. Maintenance therapy with oral etoposide following first‑line docetaxel‑cisplatin chemotherapy in metastatic non‑small cell lung cancer patients. Banglad J Pharmacol 2012;7:192‑8. 4. Fukuoka M, Yano S, Giaccone G, Tamura T, Nakagawa K, Douillard JY, et al. Multi‑institutional randomized phase II trial of gefitinib for previously treated patients with advanced non‑small‑cell lung cancer (The IDEAL 1 Trial) [corrected]. J Clin Oncol 2003;21:2237‑46. 5. Kris MG, Natale RB, Herbst RS, Lynch TJ Jr, Prager D, Belani CP, et al. Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non‑small cell lung cancer: A randomized trial. JAMA 2003;290:2149‑58. 6. Zhou C, Wu YL, Chen G, Feng J, Liu XQ, Wang C, et al. Erlotinib versus chemotherapy as first‑line treatment for patients with advanced EGFR mutation‑positive non‑small‑cell lung cancer (OPTIMAL, CTONG‑0802): A multicentre, open‑label, randomised, phase 3 study. Lancet Oncol 2011;12:735‑42. 7. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non‑small‑cell lung cancer to gefitinib. N Engl J Med 2004;350:2129‑39. 8. Pao W, Miller V, Zakowski M, Doherty J, Politi K, Sarkaria I, et al. EGF Journal of Cancer Research and Therapeutics - Volume 10 - Special Issue 2- 2014
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;101:13306‑11. 9. Li H, Pan Y, Li Y, Li C, Wang R, Hu H, et al. Frequency of well‑identified oncogenic driver mutations in lung adenocarcinoma of smokers varies with histological subtypes and graduated smoking dose. Lung Cancer 2013;79:8‑13. 10. Zhang Y, Sun Y, Pan Y, Li C, Shen L, Li Y, et al. Frequency of driver mutations in lung adenocarcinoma from female never‑smokers varies with histologic subtypes and age at diagnosis. Clin Cancer Res 2012;18:1947‑53. 11. De Greve J, Decoster L, De Mey J. Clinical activity of BIBW 2992, an irreversible inhibitor of EGFR/HER1 and HER2 in adenocarcinoma of the lung with mutations in the kinase domain of HER2neu. J Thorac Oncol 2010;5:S90. 12. Kwak EL, Bang YJ, Camidge DR, Shaw AT, Solomon B, Maki RG, et al. Anaplastic lymphoma kinase inhibition in non‑small‑cell lung cancer. N Engl J Med 2010;363:1693‑703. 13. Flaherty KT, Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman JA, et al. Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med 2010;363:809‑19. 14. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, et al. Mutations of the BRAF gene in human cancer. Nature 2002;417:949‑54. 15. Pao W, Girard N. New driver mutations in non‑small‑cell lung cancer. Lancet Oncol 2011;12:175‑80. 16. Kang YR, Park HY, Jeon K, Koh WJ, Suh GY, Chung MP, et al. EGFR and KRAS mutation analyses from specimens obtained by bronchoscopy and EBUS‑TBNA. Thorac Cancer 2013;4:264‑72. 17. Gridelli C, Morgillo F, Favaretto A, de Marinis F, Chella A, Cerea G, et al. Sorafenib in combination with erlotinib or with gemcitabine in elderly patients with advanced non‑small‑cell lung cancer: A randomized phase II study. Ann Oncol 2011;22:1528‑34. 18. Robinson SD, O’Shaughnessy JA, Cowey CL, Konduri K. BRAF V600E‑mutated lung adenocarcinoma with metastases to the brain responding to treatment with vemurafenib. Lung Cancer 2014;85:326‑30. 19. Rudin CM, Hong K, Streit M. Molecular characterization of acquired resistance to the BRAF inhibitor dabrafenib in a patient with BRAF‑mutant non‑small‑cell lung cancer. J Thorac Oncol 2013;8:e41‑2. 20. Kris MG, Johnson BE, Berry LD, Kwiatkowski DJ, Iafrate AJ, Wistuba II, et al. Using multiplexed assays of oncogenic drivers in lung cancers to select targeted drugs. JAMA 2014;311:1998‑2006. 21. Kimura H, Suminoe M, Kasahara K, Sone T, Araya T, Tamori S, et al. Evaluation of epidermal growth factor receptor mutation status in serum DNA as a predictor of response to gefitinib (IRESSA). Br J Cancer 2007;97:778‑84. 22. Travis W, Colby TV, Corrin B. Histologic Typing of Tumors of Lung and Pleura: World Health Organization International Classification of Tumors. 3rd ed. New York: Springer Verlag; 1999. 23. Carpenter CL, Morgenstern H, London SJ. Alcoholic beverage consumption and lung cancer risk among residents of Los Angeles County. J Nutr 1998;128:694‑700. 24. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. Vol. 6. New York: Cold Spring Harbor Laboratory; 1989. p. 22‑6, 34. 25. Shaw AT, Kim DW, Nakagawa K, Seto T, Crinó L, Ahn MJ, et al. Crizotinib versus chemotherapy in advanced ALK‑positive lung cancer. N Engl J Med 2013;368:2385‑94. 26. Paik PK, Arcila ME, Fara M, Sima CS, Miller VA, Kris MG, et al. Clinical characteristics of patients with lung adenocarcinomas harboring BRAF mutations. J Clin Oncol 2011;29:2046‑51. 27. Warth A, Penzel R, Lindenmaier H, Brandt R, Stenzinger A, Herpel E, et al. EGFR, KRAS, BRAF and ALK gene alterations in lung adenocarcinomas: Patient outcome, interplay with morphology and immunophenotype. Eur Respir J 2014;43:872‑83. 28. Marchetti A, Felicioni L, Malatesta S, Grazia Sciarrotta M, Guetti L, Chella A, et al. Clinical features and outcome of patients with C153
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non‑small‑cell lung cancer harboring BRAF mutations. J Clin Oncol 2011;29:3574‑9. 29. Zhao X, Han RB, Zhao J, Wang J, Yang F, Zhong W, et al. Comparison of epidermal growth factor receptor mutation statuses in tissue and plasma in stage I‑IV non‑small cell lung cancer patients. Respiration 2013;85:119‑25. 30. Bai H, Mao L, Wang HS, Zhao J, Yang L, An TT, et al. 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;27:2653‑9.
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31. Spindler KL, Pallisgaard N, Vogelius I, Jakobsen A. Quantitative cell‑free DNA, KRAS, and BRAF mutations in plasma from patients with metastatic colorectal cancer during treatment with cetuximab and irinotecan. Clin Cancer Res 2012;18:1177‑85. Cite this article as: 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;10:150-4.
Source of Support: Nil, Conflict of Interest: None declared.
Journal of Cancer Research and Therapeutics - Volume 10 - Special Issue 2- 2014
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A comparison of consistency of detecting BRAF gene mutations in peripheral blood and tumor tissue of nonsmall‑cell lung cancer patients 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 (κ =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. KEY WORDS: Cancer, nonsmall‑cell lung, BRAF gene, mutation
INTRODUCTION 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. [4‑6] 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 C150
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.[11‑13] 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.
Meiyu Fang1,2, Chunwei Xu3, Jie Wu4, Yuping Zhang5, Chao He1 Department of Oncology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058, 2Department of Integrated Chinese Traditional Medicine and Wesern Medicine, Zhejiang Cancer hospital, Hangzhou, Zhejiang 310021, 3 Department of Pathology, The General Military Hospital of Beijing PLA, Beijing 100700, 4 Department of Surgery, Zhejiang Cancer hospital, Hangzhou, Zhejiang 310021, 5Department of Pathology, Shandong Weifang People’s Hospital, Weifang, Shandong 261041, P.R. China 1
For correspondence: Prof. Chao He, Department of Oncology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058, China. E‑mail: fangjade2004@ icloud.com Access this article online Website: www.cancerjournal.net DOI: 10.4103/0973-1482.145847 PMID: *** Quick Response Code:
Journal of Cancer Research and Therapeutics - Volume 10 - Special Issue 2- 2014
Fang, et al.: Detect BRAF mutation in blood and tumor tissue
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 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 Journal of Cancer Research and Therapeutics - Volume 10 - Special Issue 2- 2014
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 0.5. 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 Table 1: Comparison BRAF mutation (L) status analysis in peripheral blood and corresponding tumor tissue (n=185, κ=0.710) BRAF mutation in B (+) (−) Total
BRAF mutation in T (+) 8 2 10
(−) 5 170 175
Total 13 172 185
BRAF=v‑raf murine sarcoma viral oncogene homolog B1, T=Tumor tissue, B=Peripheral blood, M+=Mutation positive, M−=Mutation negative
C151
Fang, et al.: Detect BRAF mutation in blood and tumor tissue
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. 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 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] Table 2: Patient characteristics and BRAF mutation status in blood Patient characteristics Age ≤60 >60 Gender Male Female Smoker Yes No Pathological pattern Adenocarcinoma Nonadenocarcinoma
Total blood saples (n=318) BRAF BRAF Percentage mutation (+) mutation (−)
P
8 15
118 177
6.35 7.81
0.622
20 3
275 20
6.78 13.04
0.484
16 7
215 80
6.93 8.05
0.808
17 6
229 66
6.91 8.33
0.682
BRAF=v‑raf murine sarcoma viral oncogene homolog B1, M+=Mutation positive, M−=Mutation negative
Table 3: Patient characteristics and BRAF mutation status in tumor tissue Patient characteristics Age ≤60 >60 Gender Male Female Smoker Yes No Pathological pattern Adenocarcinoma Nonadenocarcinoma
Total tumor tissue saples (n=257) BRAF BRAF Percentage mutation (+) mutation (−) 5 9
78 165
6.02 5.17
0.778
11 3
163 80
6.32 3.61
0.548
12 2
116 127
9.38 1.56
0.013
8 6
119 124
6.30 4.62
0.552
BRAF=v‑raf murine sarcoma viral oncogene homolog B1, M+=Mutation positive, M−=Mutation negative
C152
P
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.[29‑31] 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, (κ =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 Journal of Cancer Research and Therapeutics - Volume 10 - Special Issue 2- 2014
Fang, et al.: Detect BRAF mutation in blood and tumor tissue
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 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 This study is supported by Medical Scientific Research Foundation of Zhejiang Province of China (grant no. 2013 KYB051).
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Source of Support: Nil, Conflict of Interest: None declared.
Journal of Cancer Research and Therapeutics - Volume 10 - Special Issue 2- 2014
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