Dynamic Plasma EGFR Mutation Status as a Predictor of EGFR-TKI Efficacy in Patients with EGFR-Mutant Lung Adenocarcinoma

Dynamic Plasma EGFR Mutation Status as a Predictor of EGFR-TKI Efficacy in Patients with EGFR-Mutant Lung Adenocarcinoma

Original Article Dynamic Plasma EGFR Mutation Status as a Predictor of EGFR-TKI Efficacy in Patients with EGFR-Mutant Lung Adenocarcinoma Jeng-Sen Ts...

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Original Article

Dynamic Plasma EGFR Mutation Status as a Predictor of EGFR-TKI Efficacy in Patients with EGFR-Mutant Lung Adenocarcinoma Jeng-Sen Tseng, MD,*† Tsung-Ying Yang, MD, PhD,†‡ Chi-Ren Tsai, MS,§║ Kun-Chieh Chen, MD,*† Kuo-Hsuan Hsu, MD,*¶ Meen-Hsin Tsai, MS,†# Sung-Liang Yu, PhD,**††‡‡§§ Kang-Yi Su, PhD,**║║ Jeremy J. W. Chen, PhD,* Gee-Chen Chang, MD, PhD,*†‡¶¶

Background: Epidermal growth factor receptor (EGFR) mutation status in lung cancer can effectively predict EGFR-tyrosine kinase inhibitor (TKI) efficacy. We evaluated the role of dynamic plasma cell-free DNA EGFR mutation status in outcome prediction. Methods: Advanced lung adenocarcinoma patients were enrolled and prospectively observed for outcomes of EGFR-TKI treatment. Peptide nucleic acid–zip nucleic acid polymerase chain reaction clamp method was developed to assess EGFR mutations in matched tumor and serial plasma cell-free DNA specimens. Results: A total of 72 patients were enrolled in this study, of which 62 patients (86.1%) had EGFR-mutant tumors (34 patients with exon 19 deletions, and 28 patients with L858R). Pretreatment plasma used for EGFR mutation testing showed a sensitivity of 59.7% and a specificity of 100%. Detection sensitivity was significantly higher in stage IV-M1b patients compared with stage IIIb and IV-M1a patients *Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan, ROC; †Division of Chest Medicine, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan, ROC; ‡Faculty of Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan, ROC; §Department of Pediatrics, Taichung Veterans General Hospital, Taichung, Taiwan, ROC; ║Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan, ROC; ¶Division of Critical Care and Respiratory Therapy, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan, ROC; #Institute of Statistical Science, Academia Sinica, Taipei, Taiwan, ROC; **Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC; ††Center for Optoelectronic Biomedicine, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC; ‡‡Graduate Institute of Pathology, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC; §§Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan, ROC; ║║Center of Genomic Medicine,National Taiwan University, Taipei, Taiwan, ROC; and ¶¶Comprehensive Cancer Center, Taichung Veterans General Hospital, Taichung, Taiwan, ROC. The authors Jeremy J. W. Chen and Gee-Chen Chang contributed equally to this study. Disclosure: The authors declare no conflict of interest. Authors for Correspondence: Gee-Chen Chang, MD, PhD, Faculty of Medicine, School of Medicine, National Yang-Ming University, No.155, Sec. 2, Linong Street, Taipei, 112 Taiwan, ROC. E-mail: [email protected] gov.tw; and Jeremy J. W. Chen, Institute of Biomedical Sciences, National Chung Hsing University, No.250, Kuo Kuang Rd., Taichung, 402 Taiwan, ROC. E-mail: [email protected] DOI: 10.1097/JTO.0000000000000443 Copyright © 2015 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/15/1004-0603

(78.0% versus 23.8%, p < 0.001). All patients who presented with EGFR-mutant tumors received first-line EGFR-TKI therapy. The objective response rate and disease control rate were 74.2% and 82.3%, respectively. Median progression-free survival and overall survival were 8.8 months (95% CI: 6.6–11.0) and 20.5 months (95% CI 15.1–26.0), respectively. Failure to clear plasma EGFR mutations after EGFR-TKI treatment was an independent predictor of lower disease control rate (odds ratio 5.26 [95% CI: 1.13–24.44]; p = 0.034), shorter progression-free survival (hazard ratio: 1.97 [95% CI: 1.33–2.91]; p = 0.001), and shorter overall survival (hazard ratio: 1.82 [95% CI: 1.04–3.18], p = 0.036). Conclusion: Changes in plasma EGFR mutation status can be successfully assessed using the peptide nucleic acid–zip nucleic acid polymerase chain reaction clamp method and can serve as an independent outcome predictor. Key Words: Peptide nucleic acid–zip nucleic acid polymerase chain reaction clamp, plasma cell-free DNA, Epidermal growth factor receptor mutations, Lung adenocarcinoma. (J Thorac Oncol. 2015;10: 603–610)

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ctivating mutations of epidermal growth factor receptor (EGFR) gene are the most common genetic alterations in lung adenocarcinoma in Eastern Asians,1 and serve as an important predictor of EGFR-tyrosine kinase inhibitor (TKI) efficacy. Compared with chemotherapy, EGFR-TKI has demonstrated a significantly higher response rate, longer progression-free survival (PFS), and better quality of life in non– small-cell lung cancer (NSCLC) patients harboring activating EGFR mutations.2,3 These encouraging results led clinicians to use EGFR-TKI as the first-line therapy for EGFR-mutant NSCLC patients.4,5 A recently published molecular testing guideline for EGFR mutations suggested that EGFR molecular testing should be mandatory in advanced stage disease as an aid to selecting suitable patients for EGFR-TKI treatment.6 However, adequate specimens are not always available because tissue sampling in lung cancer is associated with potential complications,7 and small specimens usually carry a higher molecular testing failure rate.8 It has also been shown that plasma specimens obtained from lung cancer patients contain a

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higher cell-free DNA (cfDNA) level than those from cancerfree patients,9 which may be because of the release of tumor DNA into the blood via tumor cell necrosis or apoptosis.10 Therefore, plasma cfDNA is considered a potential alternative specimen for EGFR mutation testing. Zip nucleic acids (ZNA), which are oligonucleotides conjugated with cationic spermine units that can increase the affinity for their target by decreasing the electrostatic repulsions, have been reported to be potent probes or primers for polymerase chain reaction (PCR) assay.11,12 Although several previous studies successfully detected EGFR mutations in plasma cfDNA, their reported accuracy rates varied widely,13 and few large cohort studies have investigated the dynamic changes of EGFR mutation status in plasma. In this study, we developed the peptide nucleic acid–ZNA PCR (PNA–ZNA PCR) clamp method to dynamically monitor the plasma EGFR mutation status in patients with advanced EGFR-mutant lung adenocarcinoma before and after the initiation of EGFR-TKI treatment as well as to evaluate its role in outcome prediction.

METHODS Patients This was a single-center prospective observational study. To be eligible for the study, patients were required to have pathologically confirmed lung adenocarcinoma, treatment-naïve and inoperable stage IIIb or IV diseases according to the 7th edition of the American Joint Committee for Cancer staging system,14 available tumor and serial plasma specimens for EGFR mutation testing, and clinically measurable disease. Patients were excluded if they had only evaluable disease, other active malignancy, and any prior history of treatment that could influence the tumor burden, such as an operation or administration of systemic antitumor medications. Ten patients with EGFR-wild-type (wt) tumors were recruited for this study to evaluate the specificity of plasma EGFR mutation status analysis. All other enrollees had to harbor common EGFR mutations (either exon 19 deletions or exon 21 L858R) in their tumor specimens. This study was approved by the Institutional Review Board of Taichung Veterans General Hospital. Written informed consents for genetic testing and clinical records were obtained from all patients.

EGFR-TKI Treatment and Outcome Evaluation All enrolled patients who presented with detectable EGFR mutations in their tumor specimens received EGFRTKI as the first-line treatment with either gefitinib or erlotinib. However, the first dose of EGFR-TKI was not prescribed until after baseline plasma specimens were collected. These patients were then prospectively surveyed for tumor responses and survival outcomes. In the case of patients with EGFR-wt tumors, the main treatment was chemotherapy, which was determined by each attending physician; hence, the treatment outcomes of these patients were not assessed in this study. Chest computed tomographies and other imaging studies required for response evaluation were performed every 8–12 weeks. Unidimensional measurement as defined by the Response Evaluation Criteria in Solid Tumors (Version 1.1)

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was used in this study.15 The objective response rate (ORR), disease control rate (DCR), PFS, and overall survival (OS) of EGFR-TKI treatment were assessed.

Specimens Collection and EGFR Mutation Tests For patients harboring EGFR mutations in tumor specimens, we collected plasma at baseline, 10 weeks after EGFRTKI treatment, and at the time of disease progression. For patients with EGFR-wt tumors, only baseline plasma specimens were collected. Tumor tissue DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) and plasma cfDNA was purified from 1 ml of ethylenediaminetetraacetic acid plasma using the same kit. The elution volumes of the DNeasy Mini Spin Column for tumor and plasma DNA extraction were 200 μl and 80 μl, respectively. EGFR mutation status in matched tumor and plasma specimens was assessed using PNA–ZNA PCR clamp method, which was modified from PNA-locked nucleic acid PCR clamp method developed by Nagai et al.,16 in which ZNA probes was substituted for locked nucleic acid probes to provide higher affinity for their targets and greater detection sensitivity (the detection sensitivity for both exon 19 deletions and exon 21 L858R detection could be up to 1:1000; Supplementary Figure 1, SDC 1, http://links.lww.com/JTO/ A759). PNA oligos were synthesized by PanaGene (Daejeon, Korea) and ZNA probes, oligonucleotides conjugated with four cationic spermine units at the 3′ end, were provided by Metabion (Steinkirchen, Germany). PNA–ZNA PCR clamp was performed in a 25 μl mixture, containing 0.5 units of Blend Taq DNA Polymerase (Toyobo Bio, Osaka, Japan), 1× Blend Taq Buffer, 400 μM of dNTP, 200 nM of forward and reverse PCR primers, 200 nM ZNA probes, 5 μM PNA clamp probes, and 5 μl of eluted DNA. The PCRs were performed using Rotorgene 6000 (Qiagen, Hilden, Germany) with the following cycling conditions: hold at 94°C for 3 minutes; complete 50 cycles of denaturation at 94°C for 20 seconds; anneal at 60°C for 30 seconds followed by extension at 72°C for 20 seconds.

Statistical Methods Univariate analyses of ORR, DCR, and detection sensitivity were performed using Fisher’s exact test. The Kaplan– Meier method was used to estimate PFS and OS. Differences in survival time were analyzed by log-rank test. Logistic regression model and Cox proportional hazard model were used to evaluate the impact of plasma EGFR mutation status on the outcome of EGFR-TKI treatment and for multivariate analyses of responses and survival outcomes. In the stepwise procedure, the significant level for entry and removal were 0.05 and 0.10, respectively. All statistical tests were carried out using SPSS 15.0 (SPSS Inc., Chicago, IL). Two-tailed tests and p values <0.05 for significance were used.

RESULTS Patient Characteristics A total of 72 lung adenocarcinoma patients met the enrollment criteria and participated in the study from May

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2012 to October 2013. This study was conducted at Taichung Veterans General Hospital. The baseline characteristics of patients are shown in Supplementary Table 1 (SDC 2, http:// links.lww.com/JTO/A760). In summary, the median age was 64.5 years, 40 patients (55.6%) were female, 49 patients (68.1%) were nonsmokers, 55 patients (76.4%) had Eastern Cooperative Oncology Group performance status (ECOG PS) 0–1, and 68 patients (94.4%) presented with stage IV disease (20 patients with M1a and 48 patients with M1b metastases).

Correlation of EGFR Mutation Status with Paired Tumor and Baseline Plasma cfDNA Specimens The EGFR mutation status in tumors was initially analyzed by PNA–ZNA PCR clamp method and the results were summarized in Supplementary Table 1 (SDC 2, http://links. lww.com/JTO/A760). Ten EGFR-wt patients (13.9%) were recruited for the purpose of evaluating the specificity of plasma EGFR mutation status. The remaining 62 patients (86.1%) had detectable EGFR mutations in their tumor specimens, including 34 patients with exon 19 deletions and 28 patients with exon 21 L858R. Tumor mutation status was verified using matrix-assisted-laser-desorption–ionization time-of-flight mass spectrometry, which is established as a standard EGFR detection method in Taiwan by National Taiwan University Center of Genomic Medicine (an ISO15189-certified medical laboratory); no discrepancies were noted between the mutation types assessed by either detection method in all patients. Supplementary Figure 2 (SDC 3, http://links.lww.com/JTO/ A761) shows the results of PNA–ZNA PCR clamp reactions in plasma EGFR mutation detection; both exon 19 deletions and L858R were successfully detected. The results of the correlation of EGFR mutation status with tumor and baseline plasma cfDNA specimens are shown in Table 1. Of the 62 patients with EGFR-mutant tumors, 37 patients had detectable EGFR mutations in baseline plasma specimens, yielding a sensitivity of 59.7%; all mutation types of these patients were consistent between the plasma and tumor specimens. Of 10 patients with EGFR-wt tumors, the plasma cfDNA specimens were all negative for EGFR mutations. Therefore, the detection specificities for each tumor genotype were 100%. As shown in Table 2, the only factor that correlated significantly with the detection sensitivity was tumor stage. Patients with stage IV-M1b diseases carried a significantly higher EGFR mutation detection sensitivity in baseline plasma

Dynamic Plasma EGFR Mutation Status

than those with stage IIIb and stage IV-M1a diseases (78.0% versus 23.8%, p < 0.001). There was no significant correlation between EGFR mutation types and the detection sensitivity.

Efficacy of EGFR-TKI Treatment in Patients with EGFR-Mutant Tumors All 62 patients with EGFR-mutant tumors received EGFR-TKI as the first-line treatment (58 with gefitinib and 4 with erlotinib). Forty-six patients achieved partial response and five patients had stable disease. No patient achieved complete response. The ORR and DCR were 74.2% and 82.3%, respectively. Results of univariate analysis of ORR and DCR are shown in Table 3. Patients with exon 19 deletions were associated with a higher ORR than those with L858R (85.3% versus 60.7%, p = 0.041). No other factors were significantly correlated with ORR and DCR. The median PFS was 8.8 months (95% CI: 6.4–11.2) and the median OS was 20.5 months (95% CI: 15.1–26.0). In the univariate analysis, patients of a relatively younger age (less than 65 years) and better ECOG PS (0–1) were associated with a longer OS (p = 0.016 and 0.002, respectively). No other factors were significantly correlated with PFS and OS (data not shown).

Correlation between Plasma EGFR Mutation Status and EGFR-TKI Treatment Outcomes Of the 62 patients with EGFR-mutant tumors, 37 patients (59.7%) had detectable EGFR mutations in baseline plasma cfDNA specimens. There was no significant difference in either ORR or DCR between patients with positive and negative baseline plasma EGFR mutations (75.7% versus 72.0%, p = 0.774, and 81.1% versus 84.0%, p = 1.000, respectively). Similar results were observed in PFS (8.7 months [95% CI: 8.1–9.2] versus 10.6 months [95% CI: 5.3–15.9], p = 0.365) and OS (20.5 months [95% CI: 15.6–25.5] versus not reached, p = 0.715) analyses (Supplementary Figure 3, SDC 4, http:// links.lww.com/JTO/A762). In addition, we evaluated the role of the dynamic changes of plasma EGFR mutation status in outcome prediction. We delineated three groups of patients according to their plasma EGFR mutation status. All 25 patients without detectable plasma EGFR mutations at baseline remained negative for plasma EGFR mutations after EGFR-TKI treatment and were classified as Group A. Of 37 patients with initially detectable EGFR mutations in plasma, 28 patients (75.7%) had clearance of baseline plasma EGFR mutations after treatment and were classified as Group B. The remaining

TABLE 1.  Sensitivities for Detecting Tumor EGFR Mutation Status Using Matched Plasma cfDNA Samples Tumor Genotype EGFR-mutanta  Exon 19 deletions  Exon 21 L858R EGFR-wild type

Stage III/IV-M1a % (n/N)

Stage IV-M1b %, (n/N)

Total % (n/N)

23.8 (5/21) 25.0 (3/12) 22.2 (2/9) 0.0 (0/3)

78.0 (32/41) 81.8 (18/22) 73.7 (14/19) 0.0 (0/7)

59.7 (37/62) 61.8 (21/34) 57.1 (16/28) 0.0 (0/10)

p < 0.001 for stage III/IV-M1a versus stage IV-M1b by Fisher’s exact test. EGFR, epidermal growth factor receptor; cfDNA, cell-free DNA; n/N, patients with compatible EGFR mutation status in matched plasma and tumor samples/patients been assessed.

a

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TABLE 2.  Univariate Analysis of Baseline Plasma cfDNA EGFR Mutation Status in Tumor EGFR-Mutant Patients Factor

Characteristics

Age

<65 years ≥65 years Male Female Nonsmokers Current/ex-smokers 0–1 ≥2 Exon 19 deletions Exon 21 L858R IIIb & IV-M1a IV-M1b

Gender Smoking status ECOG PS Mutation types Stage

Sensitivity, % (n/Na)

P Valueb

57.6 (19/33) 62.1 (18/29) 61.5 (16/26) 58.3 (21/36) 61.4 (27/44) 55.6 (10/18) 56.3 (27/48) 71.4 (10/14) 61.8 (21/34) 57.1 (16/28) 23.8 (5/21) 78.0 (32/41)

0.798 1.000 0.778 0.367 0.797 <0.001

n/N = plasma cfDNA/tumor with EGFR mutations. By Fisher’s exact test. cfDNA, cell-free DNA; EGFR, epidermal growth factor receptor; ECOG PS, Eastern Cooperative Oncology Group performance status.

a b

nine patients (24.3%), who stayed positive for EGFR mutations in plasma and whose mutation types were consistent with corresponding baseline plasma and tumor results, were classified as Group C. The results of outcome analyses are shown in Table 4 and Figure 1. The ORRs of Group A, B, and C patients were 72.0%, 85.7%, and 44.4%, respectively; the DCRs were 84.0%, 89.3%, and 55.6%, respectively. Group C patients with persistently detectable plasma EGFR mutations

were associated with a significantly lower ORR and DCR than Group A and B patients (p = 0.042 and 0.044, respectively). The PFSs of Group A, B, and C patients were 10.6 months (95% CI: 5.3–15.9), 10.9 months (95% CI: 7.5–14.2), and 4.8 months (95% CI: 0.0–12.8), respectively; OSs were not reached (OS probability was 0.62 at 22.2 months), 20.5 months (95% CI not applicable), and 10.8 months (95% CI: 4.9–16.8), respectively. Group C patients with persistently

TABLE 3.  Univariate Analysis of Objective Response Rate and Disease Control Rate in Patients Harboring EGFR Mutations (n = 62) Patient No. Age (years)  <65   ≥65 Gender  Male  Female Smoking  Nonsmokers  Current/ex-smokers ECOG PS  0–1   ≥2 Stage  IIIb & IV-M1a  IV-M1b Mutation types  Exon 19 deletions  Exon 21 L858R EGFR-TKI  Gefitinib  Erlotinib

ORR (%)

P Valuea

DCR (%)

P Valuea

33 29

72.7 75.9

1.000

81.8 82.8

1.000

26 36

65.4 80.6

0.242

76.9 86.1

0.502

44 18

79.5 61.1

0.200

84.1 77.8

0.715

48 14

72.9 78.6

1.000

81.3 85.7

1.000

21 41

81.0 70.7

0.542

90.5 78.0

0.305

34 28

85.3 60.7

0.041

88.2 75.0

0.200

58 4

74.1 75.0

1.000

81.0 100.0

1.000

By Fisher’s exact test. ORR, objective response rate; DCR, disease control rate; ECOG PS, Eastern Cooperative Oncology Group performance status; EGFR, epidermal growth factor receptor; TKI, tyrosine kinase inhibitors. a

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TABLE 4.  Impact of Plasma cfDNA EGFR Mutation Status on the Outcome of EGFR-TKI Treatment (n = 62) Best Responses Objective response rate  Group A vs. B  Group A vs. C  Group B vs. C Disease control rate  Group A vs. B  Group A vs. C  Group B vs. C Survival Time Progression-free survival  Group A vs. B  Group A vs. C  Group B vs. C Overall survival  Group A vs. B  Group A vs. C  Group B vs. C

Percentage (%)

Odds Ratio

95% CI

P1

P2

72.0 vs. 85.7 72.0 vs. 44.4 85.7 vs. 44.4

0.43 3.21 7.50

0.11–1.69 0.66–15.58 1.39–40.56

0.226 0.147 0.019

0.042

84.0 vs. 89.3 84.0 vs. 55.6 89.3 vs. 55.6

0.63 4.20 6.67

0.13–3.14 0.77–22.87 1.13–39.47

0.573 0.097 0.037

0.044

Time (Months)

Hazard Ratio

95% CI

P1

P2

10.6 vs. 10.9 10.6 vs. 4.8 10.9 vs. 4.8

0.97 1.85 4.42

0.49–1.91 1.18–2.90 1.85–10.57

0.933 0.007 0.001

<0.001

NR vs. 20.5 NR vs. 10.8 20.5 vs. 10.8

1.35 2.06 5.47

0.43–4.31 1.09–3.88 1.45–20.62

0.608 0.025 0.012

0.002

Group A: no detectable EGFR mutations in baseline plasma (n = 25). Group B: detectable EGFR mutations in baseline plasma and then clearance of plasma EGFR mutations after EGFR-TKI treatment (n = 28). Group C: persistence of detectable EGFR mutations in plasma (n = 9). P1: individual comparison between three groups by logistically regression model and Cox proportional hazard model for best responses and survival time, respectively. P2: comparison of group A and B vs. group C by Fisher’s exact test and log-rank test for best response and survival time, respectively. cfDNA, plasma cell-free DNA; EGFR, epidermal growth factor receptor; TKI, tyrosine kinase inhibitors; NR, not reached.

detectable plasma EGFR mutations were associated with a significantly shorter PFS and OS than Group A and B patients (p < 0.001 and p= 0.002, respectively). Because a significant portion of patients (30.6%) were still receiving EGFRTKI treatment, the EGFR mutation status in postprogression plasma was not mature enough to be analyzed. As shown in Figure 2, two patients who had detectable exon 19 deletions in baseline plasma received gefitinib as the first-line treatment. Although both of them initially had favorable responses, as shown by chest computed tomography, the patient whose post-EGFR-TKI plasma remained positive for EGFR mutations did have a shorter PFS. Of nine patients with persistently detectable plasma EGFR mutations, four patients

achieved partial response, one patient had stable disease, and four patients had progressive disease at the first post-EGFRTKI chest computed tomography follow-up. Therefore, post-EGFR-TKI plasma EGFR mutation status not only identified patients with primary resistance to EGFR-TKI but also patients who responded well initially but soon experienced disease progression. Because exon 19 deletions were predictor of better response, we further examined whether it would be easier to clear exon 19 deletions from plasma after EGFR-TKI treatment. In 37 patients with initially detectable plasma EGFR mutations, there was no significant difference in the clearance rate of EGFR mutations between these two mutations (p = 0.136).

FIGURE 1.  Kaplan–Meier plot showing progression-free survival (A) and overall survival (B) according to changes in plasma cfDNA EGFR mutation status (group A: no detectable EGFR mutations in baseline plasma [n = 25]; group B: detectable EGFR mutations in baseline plasma followed by clearance of plasma EGFR mutations after EGFR-TKI treatment [n = 28]; group C: persistent detectable EGFR mutations in plasma [n = 9]). EGFR, epidermal growth factor receptor; TKI, tyrosine kinase inhibitor; cfDNA, cell-free DNA. Copyright © 2015 by the International Association for the Study of Lung Cancer

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FIGURE 2.  Case presentation demonstrated predictive value of post-EGFR-TKI cfDNA EGFR mutation status. PR, partial response; 19Del, exon 19 deletions; Neg, negative; EGFR, epidermal growth factor receptor; TKI, tyrosine kinase ­inhibitor; cfDNA, cell-free DNA.

Failure to Clear Plasma EGFR Mutations after EGFR-TKI Treatment Predicts an Independently Poor Outcome Results of multivariate analysis are shown in Supplementary Table 2 (SDC 2, http://links.lww.com/JTO/ A760). In the multivariate logistic regression model for ORR analysis, no covariate reached a significant level in the model but we observed a trend toward a higher ORR in Group A and B than in Group C patients (odds ratio 3.94 [95% CI: 0.85–18.23]; p = 0.079). In the case of DCR analysis, dynamic change of plasma EGFR mutation status was the only factor independently associated with DCR (odds ratio 5.26 [95% CI: 1.13–24.44], p = 0.034) and Group C patients were less likely to experience disease control. Similarly, in the multivariate Cox proportional hazard model, Group C patients were found to be independently associated with a shorter PFS (hazard ratio [HR]: 1.97 [95% CI: 1.33–2.91]; p = 0.001) and OS (HR: 1.82 [95% CI: 1.04–3.18]; p = 0.036). Patients with ECOG PS 0–1 were more likely to experience a longer OS (HR: 1.84 [95% CI: 1.09–3.11], p = 0.022).

DISCUSSION Tumor EGFR mutation status is the key predictor of EGFR-TKI efficacy in lung cancer. Because adequate tumor specimens are not always available, many previous studies evaluated the potential use of blood as an alternative specimen for EGFR mutation testing.13,17 However, the baseline characteristics of these studies varied widely and the results were often inconsistent. By contrast, our study cohort was relatively homogeneous because all patients had advanced treatmentnaïve lung adenocarcinoma and were enrolled and observed prospectively. Although serum has been used in several studies,18–20 plasma was recently been reported to be a better source of cfDNA for the detection of EGFR mutations.21 Our results showed that EGFR mutations could be successfully detected in plasma specimens using the PNA–ZNA PCR clamp method

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and that dynamic plasma EGFR mutation status can effectively predict the outcome of EGFR-TKI treatment. Many studies have reported successful detection of EGFR mutations in plasma, but only a few have emphasized the predictive value of dynamic EGFR mutation status in plasma.22–24 More prospective studies with larger cohorts are still needed. In this study, we showed that failure to clear plasma EGFR mutations after EGFR-TKI treatment was an independent predictor of poor disease control and reduced survival time. Moreover, post-EGFR-TKI plasma EGFR mutation status could not only be used to identify patients with primary resistance to EGFR-TKI but also patients who responded well initially but soon experienced disease progression. Further studies are warranted to establish optimal treatment for such patients. The main reasons for inconsistent results in previous studies could be traced to the use of different detection methods.13 In addition, both tumor stage and proportion of EGFR-wt patients might account for the divergent results of previous studies. In this study, we disclose that EGFR mutations were more frequently detected in plasma obtained from patients with stage IV-M1b diseases than from those with stage IIIb or stage IV-M1a diseases, which suggests a correlation between tumor burden and detection sensitivity. Patients with more advanced diseases were more likely to have detectable plasma EGFR mutations. These results are similar to the observations of previous studies.25–27 In contrast with previous studies, this study assessed the tumor stage using the 7th edition of the American Joint Committee for Cancer staging system,14 which may lead to different patient sorting, such as those with malignant pleural effusion. Furthermore, it could be surmised that studies enrolling a higher number of EGFR-wt patients would show a higher consistency between tumor and blood specimens.28,29 Recently, Oxnard et al.30 demonstrated that plasma cfDNA genotyping by droplet digital PCR could be used to detect and monitor EGFR sensitizing and drug resistance mutations in NSCLC patients. However, the number of cases was small and the authors did not perform survival time analysis. In this study, we classified patients into three groups according to the dynamic changes in their plasma EGFR mutation status and identified patients with poor outcomes earlier in the course of treatment. The method can provide a chance to adjust the treatments for patients with potentially poorer outcome. In univariate analysis, patients harboring with exon 19 deletions were associated with a higher ORR than those with L858R, which is consistent with the observations of previous studies.31,32 However, there was no significant difference in the clearance rate of EGFR mutations between these two mutations. Furthermore, in multivariate analysis, neither responses nor survival time were significantly associated with mutation types. The dynamic status of plasma EGFR mutations was the only factor that predicted DCR and both PFS and OS independently. Similar to the finding of previous studies,33,34 ECOG PS also served as an independent predictor of OS. There are three major limitations of this study. First, we only assessed exon 19 deletions and L858R. However, our

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results could be applied to most EGFR-mutant patients because these two mutation types account for more than 90% of total EGFR mutations.1 Future investigations are warranted to expand the detection spectrum, including the T790M resistance mutation. Second, the EGFR mutation status in postprogression plasma was not mature enough to be analyzed. Herein, we emphasized the value of identifying patients with a relatively poor prognosis early in the course of treatment; this strategy allows us to potentially make adjustments to their treatment. Third, compared with digital PCR and next-generation technologies, our detection method is not superior in terms of detection sensitivity, and it lacks absolute quantification. However, realtime PCR-based methods should be easier to apply in clinical practice because of their availability and lower costs.35 In conclusion, we demonstrated that EGFR mutations can be successfully detected in plasma cfDNA using the PNA– ZNA PCR clamp method and showed that the detection sensitivity was higher in patients with more advanced diseases. Furthermore, our study demonstrated that dynamic changes in plasma EGFR mutation status can serve as an independent predictor of patients’ outcome and be used to help identify patients at risk of rapid disease progression. Further studies are warranted to determine how to adjust the treatments according to dynamic status of plasma EGFR mutations.

ACKNOWLEDGMENTS

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