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Sensitive Detection of EGFR Mutations in Cerebrospinal Fluid from Lung Adenocarcinoma Patients with Brain Metastases

2014; Elsevier BV; Volume: 16; Issue: 5 Linguagem: Inglês

10.1016/j.jmoldx.2014.04.008

1943-7811

Haihong Yang, Linbo Cai, Yalei Zhang, Hong-yu Tan, Qiuhua Deng, Meiling Zhao, Xin Xu,

Brain Metastases and Treatment

Epidermal growth factor receptor (EGFR) mutations in cerebrospinal fluid (CSF) might be useful predictive markers for EGFR tyrosine kinase inhibitor treatment of intracranial metastatic tumors. In this retrospective study, amplification refractory mutation system (ARMS)-PCR assays were used to investigate the EGFR gene status in 30 lung adenocarcinoma patients with brain metastases. A total of 16 patients tested positive for EGFR-activating mutations in CSF or tumor tissues. These included L858R mutation in exon 21 in six CSF samples and exon 19 deletions in seven CSF samples. EGFR mutations were detected between CSF and primary tumor samples with a 75% positive predictive value (95% CI, 0.45–1.00), 75% negative predictive value (95% CI, 0.51–0.99), 67% sensitivity (95% CI, 0.36–0.97), and 82% specificity (95% CI, 0.59–1.00). Most of the patients who had EGFR mutations in CSF achieved good responses with EGFR-tyrosine kinase inhibitor treatment. In conclusion, ARMS-PCR could be a sensitive method of detecting EGFR mutations in the CSF of patients with lung adenocarcinoma with brain metastases. As such, ARMS-PCR could play an important role in guiding EGFR-tyrosine kinase inhibitor treatments of intracranial tumors and for diagnosing brain metastases in patients with lung adenocarcinoma. Epidermal growth factor receptor (EGFR) mutations in cerebrospinal fluid (CSF) might be useful predictive markers for EGFR tyrosine kinase inhibitor treatment of intracranial metastatic tumors. In this retrospective study, amplification refractory mutation system (ARMS)-PCR assays were used to investigate the EGFR gene status in 30 lung adenocarcinoma patients with brain metastases. A total of 16 patients tested positive for EGFR-activating mutations in CSF or tumor tissues. These included L858R mutation in exon 21 in six CSF samples and exon 19 deletions in seven CSF samples. EGFR mutations were detected between CSF and primary tumor samples with a 75% positive predictive value (95% CI, 0.45–1.00), 75% negative predictive value (95% CI, 0.51–0.99), 67% sensitivity (95% CI, 0.36–0.97), and 82% specificity (95% CI, 0.59–1.00). Most of the patients who had EGFR mutations in CSF achieved good responses with EGFR-tyrosine kinase inhibitor treatment. In conclusion, ARMS-PCR could be a sensitive method of detecting EGFR mutations in the CSF of patients with lung adenocarcinoma with brain metastases. As such, ARMS-PCR could play an important role in guiding EGFR-tyrosine kinase inhibitor treatments of intracranial tumors and for diagnosing brain metastases in patients with lung adenocarcinoma. CME Accreditation Statement: This activity ("JMD 2014 CME Program in Molecular Diagnostics") has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity ("JMD 2014 CME Program in Molecular Diagnostics") for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity ("JMD 2014 CME Program in Molecular Diagnostics") has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity ("JMD 2014 CME Program in Molecular Diagnostics") for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Brain metastases are a frequent complication of non-small cell lung cancer (NSCLC), especially in patients with lung adenocarcinoma. Brain metastases are observed in 30% to 50% of patients at initial diagnosis with more patients developing metastases during treatment.1Hazard L.J. Jensen R.L. Shrieve D.C. Role of stereotactic radiosurgery in the treatment of brain metastases.Am J Clin Oncol. 2005; 28: 403-410Crossref PubMed Scopus (17) Google Scholar These patients are unable to undergo surgical resection of primary or cranial metastatic tumors to provide specimens for histopathological or biomarker studies. However, tumor-derived DNA could be secreted into body fluids surrounding the tumor. Therefore, the development of methods to identify potential molecular biomarkers from nonsurgical biopsy samples, such as cerebrospinal fluid (CSF), may facilitate the identification of clinically relevant gene signatures in patients with metastatic brain tumors. Brain metastases are diagnosed according to clinical presentation, primary malignant tumor, and radiological imaging. If the computed tomography or magnetic resonance imaging (MRI) aspect is atypical, tissue diagnosis, including brain tumor or CSF cytology, is necessary.2Soffietti R. Cornu P. Delattre J.Y. Grant R. Graus F. Grisold W. Heimans J. Hildebrand J. Hoskin P. Kalljo M. Krauseneck P. Marosi C. Siegal T. Vecht C. EFNS Guidelines on diagnosis and treatment of brain metastases: report of EFNS Task Force.Eur J Neurol. 2006; 13: 674-681Crossref PubMed Scopus (165) Google Scholar However, in certain clinical situations, MRI would not be helpful for patients with leptomeningeal metastases in which positive CSF cytology results are <40%.3Shingyoji M. Kageyama H. Sakaida T. Nakajima T. Matsui Y. Itakura M. Iuchi T. Yokoi S. Kimura H. Iizasa T. Detection of epithelial growth factor receptor mutations in cerebrospinal fluid from patients with lung adenocarcinoma suspected of neoplastic meningitis.J Thorac Oncol. 2011; 6: 1215-1220Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar The detection of oncogenes in CSF might facilitate the diagnosis of brain metastases in patients with lung adenocarcinoma, especially if the results are consistent with the corresponding results from the primary tumor. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) is a small-molecular agent capable of penetrating brain tissue and has been found to significantly improve survival rates and tumor responses in lung adenocarcinoma patients with metastatic brain tumors that harbor EGFR-activating mutations.4Kim J.E. Lee D.H. Choi Y. Yoon D.H. Kim S.W. Suh C. Lee J.S. Epidermal growth factor receptor tyrosine kinase inhibitors as a first-line therapy for never-smokers with adenocarcinoma of the lung having asymptomatic synchronous brain metastasis.Lung Cancer. 2009; 65: 351-354Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 5Porta R. Sánchez-Torres J.M. Paz-Ares L. Massutí B. Reguart N. Mayo C. Lianes P. Queralt C. Guillem V. Salinas P. Catot S. Isla D. Pradas A. Gúrpide A. de Castro J. Polo E. Puig T. Tarón M. Colomer R. Rosell R. Brain metastases from lung cancer responding to erlotinib: the importance of EGFR mutation.Eur Respir J. 2011; 37: 624-631Crossref PubMed Scopus (280) Google Scholar However, the most common target populations for treatment with EGFR-TKI remain females with adenocarcinomas, because these patients have a higher rate of EGFR mutation, and EGFR gene status can only be detected in approximately 10% of patients with advanced NSCLC in China.6Xue C. Hu Z. Jiang W. Zhao Y. Xu F. Huang Y. Wu J. Zhang Y. Zhao L. Zhang J. Chen L. Zhang L. National survey of the medical treatment status for non-small cell lung cancer (NSCLC) in China.Lung Cancer. 2012; 77: 371-375Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar The limited availability of testing technology and economic factors are the leading causes of such a low detection rate. The evaluation of the EGFR status in metastatic intracranial tumors may offer direct evidence to guide the clinical use of EGFR-TKI in treating these patients. Because positive rates of CSF cytology are low and readily available methods may not be suitable, the detection of EGFR gene status in tumor-derived free DNA in CSF might be a good clinical option.7Rhodes C.H. Honsinger C. Sorenson G.D. Detection of tumor-derived DNA in cerebrospinal fluid.J Neuropathol Exp Neurol. 1994; 53: 364-368Crossref PubMed Scopus (22) Google Scholar To test this hypothesis, we analyzed the EGFR status of tumor-derived free DNA in the CSF of lung adenocarcinoma patients with brain metastases by using amplification refractory mutation system (ARMS)-PCR assays. Thirty patients with pathologically confirmed diagnoses of lung adenocarcinoma with brain metastases were enrolled in this retrospective study. The patients had all been admitted to the First Affiliated Hospital of Guangzhou Medical University and Guangdong 999 Brain Hospital between November 2011 and December 2012. The inclusion criteria were as follows: they had not received prior brain radiotherapy, and patients with posterior fossa lesions or intracranial hypertension were excluded. Tumor responses were evaluated by radiological computed tomography imaging or MRI according to guidelines of Response Evaluation Criteria in Solid Tumors version 1.1.8Eisenhauer E.A. Therasse P. Bogaerts J. Schwartz L.H. Sargent D. Ford R. Dancey J. Arbuck S. Gwyther S. Mooney M. Rubinstein L. Shankar L. Dodd L. Kaplan R. Lacombe D. Verweij J. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1).Eur J Cancer. 2009; 45: 228-247Abstract Full Text Full Text PDF PubMed Scopus (18123) Google Scholar Written informed consent was obtained from all participants before the study. This study was approved by the institutional review board of the First Affiliated Hospital, Guangzhou Medical University (Guangzhou, China). A lumbar puncture was performed on each patient during which time 5 mL of CSF was aspirated. Formalin-fixed, paraffin-embedded primary tumor tissues were collected in fine-needle aspiration by bronchial fiberscopic or percutaneous transthoracic biopsy. Genomic DNA and cell-free DNA were extracted from formalin-fixed, paraffin-embedded lung tumor tissues and CSF samples, respectively, by using a QIAamp DNA FFPE Tissue Kit or a QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany) as appropriate. EGFR mutations were detected with the AmoyDx Human EGFR Gene 29 Mutations Detection kit with fluorescence PCR (Amoy Diagnostics, Xiamen, China), and assays were performed on an ABI7900 real-time PCR instrument (Applied Biosystems, Foster City, CA). Primers were labeled with 6-carboxyfluorescein and HEX/VIC. This EGFR kit detects 29 mutations in exons 18 to 21, including T790M, L858R, L861Q, S768I, G719S, G719A, and G719C; three insertions in exon 20; and 19 deletions in exon 19. DNA was amplified by PCR in a final volume of 25 μL that contained 5 μL of DNA, 25 mmol/L MgCl2, 25 mmol/L dNTP, 100 μmol/L of specific forward and reverse primers, 10 × Takara buffer, and 5 U/μL Takara HS-Taq (TaKaRa Biotechnology, Dalian, China). The first cycle of amplifications was performed with a 5-minute initial denaturation at 95°C, followed by 30 cycles of 45 seconds at 95°C, 45 seconds at 54°C, and 1 minute at 72°C, and a 6-minute final extension at 72°C. Products from the first cycle were amplified in the secondary cycle by using the same PCR conditions. Fisher's exact method was used to compare EGFR status between paired CSF and primary tumor tissues in patients with lung adenocarcinoma with brain metastases. Differences were considered to be statistically significant when P < 0.05. EGFR status in primary tumor tissue is considered to be a standard test method and was evaluated in CSF by calculating the positive predictive value (PPV), negative predictive value (NPV), sensitivity, and specificity as follows: i) PPV = number of true positives/(number of true positives + number of false positives); ii) NPV = number of true negatives/(number of true negatives + number of false negatives); iii) sensitivity = number of true positives/(number of true positives + number of false negatives); and iv) specificity = number of true negatives/(number of true negatives + number of false positives). SPSS version 13.0 (SPSS Inc., Chicago, IL) was used for the statistical analyses. Binomial confidence intervals were calculated with Stata version 10.0 (StataCorp LP, College Station, TX). Thirty patients with lung adenocarcinoma metastatic to the brain were enrolled in this retrospective study. They included 16 males and 14 females with a median age of 55 years (range, 28 to 82 years). Of these, 16 patients had received no prior treatment, 11 had received gefitinib with 8 showing good responses, and 7 patients had leptomeningeal metastases. The patient characteristics are summarized in Tables 1 and 2.Table 1Clinical Characteristics of Lung Adenocarcinoma Patients with Brain Metastases (n = 30)Characteristicn (%)Age (years) ≥6010 (33.3) <6020 (66.6)Sex Male16 (53.3) Female14 (46.7)Smoking status No17 (56.7) Yes13 (43.3)Brain metastases Only metastatic tumors23 (76.7) With meningeal lesions7 (23.3)Prior treatment Only chemotherapy3 (10) Only gefitinib4 (13.3) Gefitinib and chemotherapy7 (23.3) No treatment16 (53.3)CSF EGFR mutation Positive13 (43) Negative17 (57) Open table in a new tab Table 2Patient Characteristics and EGFR MutationsPatientSexAge, yearsBrain metastasisNeurological symptomsSmokingPrevious gefitinib treatmentCSF EGFR mutationPrimary tissues collectionTumor EGFR mutationFollowing treatmentFollowing TKI response (intracranial)1F80Single lesionNoNoPRWild-typeNANAGefitinibPD2F74Single lesionYesNoPDWild-typeNANAGefitinibPD3F71Multiple lesionsYesNoNAL858RBL858RGefitinib∗The following therapy was TKI combined with brain radiotherapy.PR4F61Single lesionNoNoNAL858RPWild-typeGefitinib∗The following therapy was TKI combined with brain radiotherapy.PR5F59Multiple, meningeal lesionsNoNoPR19-delB19-delErlotinibCR6F58Multiple lesionsNoNoPR19-delNANAErlotinibPR7F55Multiple lesionsNoNoPDWild-typePWild-typeErlotinibPD8F53Multiple lesionsYesNoNAWild-typeNANAGefitinibPD9F52Meningeal lesionsYesNoPRL858RPL858RErlotinibCR10F52Multiple lesionsYesNoNA19-delBWild-typeGefitinib∗The following therapy was TKI combined with brain radiotherapy.PR11F37Multiple lesionsNoNoNAWild-typeBWild-typeErlotinibSD12F34Multiple lesionsNoNoPRWild-typeNANAErlotinibPR13F32Multiple, meningeal lesionsYesNoPRWild-typeNANAErlotinibCR14F28Single lesionNoNoNAWild-typePWild-typeChemotherapySD15M82Single lesionNoNoPDWild-typeBWild-typeErlotinibPD16M76Single lesionNoNoSDWild-typeP19-delErlotinibPD17M75Meningeal lesionsYesNoNA19-delNANAChemotherapyNA18M68Meningeal lesionsYesYesNAWild-typePWild-typeErlotinibPD19M65Meningeal lesionsYesYesNAL858RPL858RErlotinibSD20M60Multiple lesionsNoYesNA19-delNANAChemotherapySD21M58Multiple lesionsYesNoPR19-delB19-delNANA22M57Multiple lesionsYesYesNAWild-typePWild-typeBrain radiotherapySD23M55Multiple lesionsYesYesNAWild-typePWild-typeBrain radiotherapyNA24M54Single lesionNoYesNAWild-typePWild-typeErlotinibPD25M53Multiple lesionsNoYesNAWild-typeB19-delErlotinibCR26M49Multiple lesionsNoNoNA19-delNANAGefitinibSD27M49Single lesionNoYesPDWild-typeBWild-typeBrain radiotherapyPD28M47Single lesionNoYesNAL858RNANAErlotinibPR29M42Multiple lesionsYesYesNAWild-typePL858RBrain radiotherapyNA30M36Meningeal lesionsYesNoPRL858RPL858RErlotinib∗The following therapy was TKI combined with brain radiotherapy.SDF, female; M, male; B, fine needle aspiration by bronchial fiberscope; CR, complete remission; CSF, cerebrospinal fluid; EGFR, epidermal growth factor receptor; NA, not applicable; P, percutaneous transthoracic biopsy; PD, progressive disease; PR, partial remission; SD, stable disease; TKI, tyrosine kinase inhibitor.∗ The following therapy was TKI combined with brain radiotherapy. Open table in a new tab F, female; M, male; B, fine needle aspiration by bronchial fiberscope; CR, complete remission; CSF, cerebrospinal fluid; EGFR, epidermal growth factor receptor; NA, not applicable; P, percutaneous transthoracic biopsy; PD, progressive disease; PR, partial remission; SD, stable disease; TKI, tyrosine kinase inhibitor. EGFR mutations were detected in either the CSF or primary tissues of 16 of 30 patients (53%) by ARMS-PCR assays (Table 2). The CSF samples of all of the patients were analyzed, and EGFR mutations were detected in the CSF of 13 of 30 patients (43%). The mutations included L858R in exon 21 in 6 of 30 CSF samples (20%) and exon 19 deletions in 7 of 30 samples (23%). Metastatic tumors in the brain parenchyma were diagnosed in eight of these patients, including two with a single intracranial lesion. The primary lung adenocarcinoma tissue samples of 20 patients were analyzed. L858R mutations were identified in the tissue samples from 5 of 20 patients (25%), and exon 19 deletions were detected in the samples from 4 of 20 patients (20%). EGFR status was compared between CSF samples from patients with brain metastases and paired primary lung tumor samples (Table 3). The PPV and NPV of the EGFR mutation test in CSF samples versus primary tumor samples were 6/8 (0.75, 95% CI: 0.45 to 1.00) and 9/12 (0.75; 95% CI, 0.51–0.99), respectively. The sensitivity and specificity of the CSF test were 6/9 (0.67; 95% CI, 0.36–0.97) and 9/11 (0.82; 95% CI, 0.59–1.00), respectively. We did not find a difference in EGFR status between CSF and primary tumor tissues (P = 0.065, κ = 0.49; 95% CI, 0.11–0.87).Table 3EGFR Mutations in CSF or Tumor TissuesEGFR MutationTumor EGFR mutation (n = 20)CSF EGFR mutation (n = 20)+− +62 −39P value0.065PPV (95% CI)0.75 (0.45–1.00)NPV (95% CI)0.75 (0.51–0.99)Sensitivity (95% CI)0.67 (0.36–0.97)Specificity (95% CI)0.82 (0.59–1.00)CSF, cerebrospinal fluid; EGFR, epidermal growth factor receptor; NPV, negative predictive value; PPV, positive predictive value. Open table in a new tab CSF, cerebrospinal fluid; EGFR, epidermal growth factor receptor; NPV, negative predictive value; PPV, positive predictive value. Of the 13 patients with EGFR mutations in their CSF, 6 only received EGFR-TKI treatment after a diagnosis of brain metastases. Four patients achieved a complete response or partial response of their intracranial tumor after erlotinib treatment, including three patients who showed an initial good response to gefitinib. Of these, two patients had L858R mutations and the other two had exon 19 deletions. The median overall survival after the beginning of treatment of brain metastases was 11.5 months (range, 4 to 21 months). A patient (patient 19) who had right lung cancer underwent radical resection and developed severe headaches, gait instability, and intellectual impairment after 2 years. A cranial MRI showed ventricle hydrops (Figure 1), and the CSF cytology was negative. However, L858R mutation was detected in both the CSF and primary tumor tissue, and he eventually received a diagnosis of leptomeningeal metastases. After receiving treatment with erlotinib there was rapid remission of the neurological symptoms. Traces of tumor-derived free DNA were extracted from the CSF of 30 patients with lung adenocarcinoma with brain metastases. We analyzed the traces of DNA by ARMS-PCR, one of the real-time PCR methods, which is more sensitive than Sanger DNA sequencing methods for low abundance of DNA.9Ellison G. Donald E. McWalter G. Knight L. Fletcher L. Sherwood J. Cantarini M. Orr M. Speake G. A comparison of ARMS and DNA sequencing for mutation analysis in clinical biopsy samples.J Exp Clin Cancer Res. 2010; 29: 132Crossref PubMed Scopus (135) Google Scholar, 10Angulo B. Conde E. Suárez-Gauthier A. Plaza C. Martínez R. Redondo P. Izquierdo E. Rubio-Viqueira B. Paz-Ares L. Hidalgo M. López-Ríos F. A comparison of EGFR mutation testing methods in lung carcinoma: direct sequencing, real-time PCR and immunohistochemistry.PLos One. 2012; 7: e43842Crossref PubMed Scopus (90) Google Scholar The use of ARMS-PCR was necessary because DNA levels in CSF are low and not solely derived from metastatic tumors. Our results indicate the ARMS-PCR method may be suitable for patients with a low abundance of mutant DNA in their CSF. With the use of ARMS-PCR, we detected EGFR mutations in the CSF of 53% of patients and achieved a 75% PPV (95% CI, 0.45–1.00) and 75% NPV (95% CI, 0.51–0.99), 67% sensitivity (95% CI, 0.36–0.97) and 82% specificity (95% CI, 0.59–1.00) correlation between CSF and primary tumor tissue samples despite no statistical difference between CSF and primary tumor tissue samples because of too small samples (P = 0.065). Indeed, between the paired primary tumor and tumor-derived DNA in CSF, 75% of samples had a concordant EGFR status. This is in close agreement with previous studies that reported EGFR mutation status in 70% to 80% of the metastatic primary tumors of NSCLC patients.11Monaco S.E. Nikiforova M.N. Cieply K. Teot L.A. Khalbuss W.E. Dacic S. A comparison of EGFR and KRAS status in primary lung carcinoma and matched metastases.Hum Pathol. 2010; 41: 94-102Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 12Han C. Ma J. Zhao J. Zhou Y. Jing W. Zou H. EGFR mutations, gene amplification, and protein expression and KRAS mutations in primary and metastatic tumors of nonsmall cell lung cancers and their clinical implications: a meta-analysis.Cancer Invest. 2011; 29: 626-634Crossref PubMed Scopus (15) Google Scholar Moreover, false-negative results in the CSF may stem from pretreatment with gefitinib, resulting in low tumor burden, and patients without gefitinib pretreatment might be expected to have increased amounts of mutated DNA in the CSF. In contrast, a study on the EGFR status in patients with neoplastic meningitis reported EGFR mutations by DNA sequencing in 45% of the patients with positive CSF cytology and 30% of the patients in CSF with negative CSF cytology.3Shingyoji M. Kageyama H. Sakaida T. Nakajima T. Matsui Y. Itakura M. Iuchi T. Yokoi S. Kimura H. Iizasa T. Detection of epithelial growth factor receptor mutations in cerebrospinal fluid from patients with lung adenocarcinoma suspected of neoplastic meningitis.J Thorac Oncol. 2011; 6: 1215-1220Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar Because of the difficulty in obtaining intracranial tumors, it was not possible for us to compare EGFR status between CSF samples and intracranial tumors. Although the EGFR status in CSF from patients with leptomeningeal metastases is relatively easy to detect because the tumor-derived DNA is secreted directly into the CSF from the tumor cells, there are few reports on EGFR status in tumor-derived DNA in CSF from patients with metastatic tumors in the brain parenchyma. In our study, most of the patients with EGFR mutations in CSF had metastatic tumors in the brain parenchyma, including two patients with a single intracranial lesion. EGFR-TKI has been found to significantly improve the response rates of metastatic brain tumors and survival rates of lung adenocarcinoma patients with asymptomatic synchronous brain metastasis, particularly in patients with EGFR-activating mutations in exons 19 or 21.4Kim J.E. Lee D.H. Choi Y. Yoon D.H. Kim S.W. Suh C. Lee J.S. Epidermal growth factor receptor tyrosine kinase inhibitors as a first-line therapy for never-smokers with adenocarcinoma of the lung having asymptomatic synchronous brain metastasis.Lung Cancer. 2009; 65: 351-354Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 5Porta R. Sánchez-Torres J.M. Paz-Ares L. Massutí B. Reguart N. Mayo C. Lianes P. Queralt C. Guillem V. Salinas P. Catot S. Isla D. Pradas A. Gúrpide A. de Castro J. Polo E. Puig T. Tarón M. Colomer R. Rosell R. Brain metastases from lung cancer responding to erlotinib: the importance of EGFR mutation.Eur Respir J. 2011; 37: 624-631Crossref PubMed Scopus (280) Google Scholar It has been reported that [11C]-erlotinib positron emission tomography/computed tomography accumulates in brain metastases,13Weber B. Winterdahl M. Memon A. Sorensen B.S. Keiding S. Sorensen L. Nexo E. Meldgaard P. Erlotinib accumulation in brain metastases from non-small cell lung cancer: visualization by positron emission tomography in a patient harboring a mutation in the epidermal growth factor receptor.J Thorac Oncol. 2011; 6: 1287-1289Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar and this was also found in brain metastases from patients with NSCLC. Therefore, we exploited the sensitivity of ARMS-PCR to determine the status of the EGFR oncogene in brain metastases. In our study, four of the six patients with EGFR-activating mutations in CSF, including three patients who had undergone pretreatment with gefitinib, achieved good intracranial tumor responses after receiving erlotinib for brain metastases. This result is in agreement with a report by Wu et al14Wu Y.L. Zhou C. Cheng Y. Lu S. Chen G.Y. Huang C. Huang Y.S. Yan H.H. Ren S. Liu Y. Yang J.J. Erlotinib as second-line treatment in patients with advanced non-small-cell lung cancer and asymptomatic brain metastases: a phase II study (CTONG-0803).Ann Oncol. 2013; 24: 993-999Crossref PubMed Scopus (200) Google Scholar on the treatment of asymptomatic brain metastases in NSCLC patients with EGFR mutations. We propose that a further study should be performed to observe the progress of intracranial diseases by following the EGFR gene mutation status in their CSF. However, the current methods of diagnosing brain metastases are limited by suboptimal sensitivity or specificity, especially in patients with neoplastic meningeal or metastatic tumors that are difficult to distinguish from manifestations of the primary neurological diseases only by brain imaging.15Chamberlain M.C. Neoplastic meningitis.Neurologist. 2006; 12: 179-187Crossref PubMed Scopus (61) Google Scholar Therefore, an alternative, more sensitive method of diagnosing brain metastases is required for clinical applications. Oncogenes are tumor specific and are not present in normal tissues; therefore, they can be detected in tumor-derived DNA in malignant pleural fluid or plasma from patients with lung cancer.16Brevet M. Johnson M.L. Azzoli C.G. Ladanyi M. Detection of EGFR mutations in plasma DNA from lung cancer patients by mass spectrometry genotyping is predictive of tumor EGFR status and response to EGFR inhibitors.Lung Cancer. 2011; 73: 96-102Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 17Zhang X. Zhao Y. Wang M. Yap W.S. Chang A.Y. Detection and comparison of epidermal growth factor receptor mutations in cells and fluid of malignant pleural effusion in non-small cell lung cancer.Lung Cancer. 2008; 60: 175-182Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar This suggests that oncogene detection in body fluids could contribute to the diagnosis of specific metastatic lesions. In the case of the patient 19 in our study, CSF examination was helpful for the diagnosis and treatment of his leptomeningeal metastases. In conclusion, ARMS-PCR could be a sensitive assay for detecting EGFR mutations in the CSF of lung adenocarcinoma patients with brain metastases. As such, it could be used as a guide in clinical EGFR-TKI therapies for patients with intracranial tumors, improve predictions of EGFR-TKI efficacy, and contribute to the diagnosis of brain metastases.