Original Article

Clinical Outcome According to the Level of Preexisting Epidermal Growth Factor Receptor T790M Mutation in Patients With Lung Cancer Harboring Sensitive Epidermal Growth Factor Receptor Mutations Youngjoo Lee, MD1,2; Geon Kook Lee, MD, PhD1; Yeon-Su Lee, PhD3; Wenji Zhang, BS1; Jung-Ah Hwang, PhD3; Byung-Ho Nam, PhD4; Seok Hyun Kim, MD, PhD5; Joo-Hang Kim, MD, PhD6; Tak Yun, MD1; Ji-Youn Han, MD, PhD1; Heung Tae Kim, MD, PhD1; and Jin Soo Lee, MD, PhD1

BACKGROUND: Epidermal growth factor receptor (EGFR) T790M mutation drives acquired drug resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in patients with EGFR-mutant lung cancer. However, it was reported that this mutation may exist before drug exposure. The objective of the current study was to evaluate whether the clinical outcomes are affected by the percentage of preexisting T790M mutations within a tumor. METHODS: Pretreatment tissues were collected from 124 patients with advanced non-small cell lung cancer with sensitizing EGFR mutations that were detected by direct sequencing. Genotyping for EGFR T790M mutation was further performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Patients who were positive for the T790M mutation were divided to 2 subgroups according to T790M mutant signal frequency. RESULTS: The T790M mutation was found in 31 patients (25.0%). The T790M mutation frequency at which the risk of disease progression after therapy with EGFRTKIs begins to increase was estimated to be 3.2%. The patients with T790M-positive tumors had a shorter time to disease progression after treatment with EGFR-TKIs (median, 6.3 months vs 11.5 months; P CGG) in exon 21 (L858R) were considered sensitive EGFR mutations. Cases with the sensitive EGFR mutations and a wild-type EGFR gene, were genotyped further for the T790M mutation in exon 20. Genotyping for the T790M mutation was performed using matrix2091

Original Article

assisted laser desorption/ionization time-of-flight MS using the standard protocol of the MassARRAY System (Sequenom, San Diego, Calif). Sensitivity Analysis of the MS Assay

Serial DNA mixtures of mutant and wild-type DNA were genotyped by the MS assay to determine the detection limit and cutoff value for the T790M mutation (see online supporting information). The T790M-mutant signal calls were detectable in the cluster plot and mass spectrum of the DNA mixtures with a T790M mutation frequency of 2.5%. The mutant signal frequency was calculated as follows: mutant signal frequency (%) 5 (mutant peak height)/(mutant peak height 1 wildtype peak height) 3 100. The calculated mutant signal frequency in the 2.5% T790M DNA mixture was 2.95%. Thus, this value was used as the cutoff value for the T790M mutation in further analysis. The diluted mutant DNA percentage was linearly correlated with the mutant signal frequency (R2 5 0.9878) (see online supporting information). In Vitro Experiments for Estimating the Percentage of Resistant T790M Cells

In vitro sensitivity to gefitinib (LC Laboratories, Woburn, Mass) was measured in 2 pairs of cell line mixtures containing TKI-sensitive cells and TKI-resistant cells with the T790M mutation: HCC827/H1975 and PC9/PC9GRT790M.19 The HCC827, PC9, and H1975 cell lines were purchased from Korean Cell Line Bank (Seoul, Korea), RIKEN BioResource Center cell bank (Ibaraki, Japan), and American Type Culture Collection (Manassas, Va), respectively. The gefitinib-resistant PC9GRT790M cell line was generated by chronically exposing PC9 cells to stepwise increasing concentrations of gefitinib and isolation, followed by characterization of a single resistant clone in the study laboratory. TKI-sensitive cells and TKI-resistant cells with the T790M mutation were mixed at the indicated percentages and were treated with gefitinib for 72 hours. Growth inhibition was measured using the MTS assay (Promega, Madison, Wis) according to the manufacturer’s instructions.

was estimated using the Kaplan–Meier method and was compared between groups using the log-rank test. The Cox proportional hazards regression model was used for univariate and multivariate survival analyses to determine hazards ratios (HR) with 95% confidence intervals (95% CI). Two-sided values of P < .05 were considered to be statistically significant. RESULTS Patients

A total of 173 patients (124 with sensitive EGFR mutations and 49 with wild-type EGFR gene) were further genotyped by the MS-based assay. Patients’ characteristics were shown in Table 1. Survival data were followed until the end of April 2013, and the median follow-up duration was 35.6 months (range, 2.5 months-49.2 months). The median progression-free survival and OS of EGFR-mutant patients were 9.4 months (95% CI, 6.7 months-12.1 months) and 23.5 months (95% CI, 17.4 months-29.6 months), respectively. However, the median progressionfree survival and OS of patients with wild-type EGFR were 1.4 months (95% CI, 0.8 months-2.0 months) and 3.5 months (95% CI, 2.2 months-4.9 months), respectively. Frequency of the T790M Mutation

The tumor tissues were obtained by biopsy/aspiration (n 5 135; 78.0%) or by surgical resection (n 5 38; 22.0%). Surgical specimens included 27 primary lung tumors (15.6%) that were curatively resected at an early stage of disease. The majority of tumor sites were the lung and pleura (n 5 134; 77.5%). There was discordance between tissue samples for genotyping and direct sequencing in 57 patients (32.9%). The MS-based genotyping assay detected EGFR T790M mutations in 31 of 124 EGFR-mutant patients (25.0%) and in 3 of 49 patients with wild-type EGFR (6.1%). Patient characteristics and sampling methods did not differ between the patients with T790M-positive tumors and those with T790Mnegative tumors (data not shown).

Statistical Analysis

Effects of the T790M Mutation on EGFR-TKI Efficacy

Differences between pairs of categorical variables were analyzed using the chi-square test or Fisher exact test. Time to disease progression (TTP) was measured from the first day of therapy with EGFR-TKIs until the identification of disease progression. Overall survival (OS) was calculated from the first day of treatment with EGFR-TKIs until death or the most recent follow-up. Survival time

The response rate (RR) and disease control rate (DCR) were not significantly different between patients with T790Mpositive mutant tumors and T790M-negative mutant tumors (RR, 71.0% vs 83.9% [P 5 .115]; DCR, 83.9% vs 92.5% [P 5 .173]). However, the median TTP was significantly shorter in patients with T790M-positive mutant tumors compared with patients with T790M-negative

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De Novo EGFR T790M Mutations Frequency/Lee et al

TABLE 1. Patient and Treatment Characteristics Characteristics Total Age, y Sex Histology Stagea Smoking ECOG PS No. of metastatic organ Type of sensitive EGFR mutation EGFR TKI line Type of EGFR TKI Response to EGFR TKI

Category

EGFR MT No. (%)

65 >65 Female Male ADC Non-ADC IIIB IV Never Ever 1 >1 2 >2 Exon 19 del Exon 21 L858R First-line Second-line Gefitinib Erlotinib Yes No

124 87 (70.2) 37 (29.8) 74 (59.7) 50 (40.3) 120 (96.8) 4 (3.2) 4 (3.2) 120 (96.8) 82 (66.1) 42 (33.9) 79 (63.7) 45 (36.3) 57 (46.0) 67 (54.0) 78 (63.0) 46 (37.0) 50 (40.3) 74 (59.7) 59 (86.8) 9 (13.2) 100 (80.6) 24 (19.4)

EGFR WT No. (%) 49 (81.6) (19.6) (44.9) (55.1) (79.6) (20.4) (2.0) (98.0) (30.6) (69.4) (61.2) (38.8) (71.4) (28.6) — — 7 (14.3) 42 (85.7) 39 (79.6) 10 (20.4) 2 (4.1) 47 (95.9)

40 9 22 27 39 10 1 48 15 34 30 19 35 14

P

.124 .078 .675 1.000 .080 1.000 .120

.001 .750

Clinical outcome according to the level of preexisting epidermal growth factor receptor T790M mutation in patients with lung cancer harboring sensitive epidermal growth factor receptor mutations.

Epidermal growth factor receptor (EGFR) T790M mutation drives acquired drug resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in patients with...
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