Lung Cancer 89 (2015) 337–342

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Lung Cancer journal homepage: www.elsevier.com/locate/lungcan

Concurrence of EGFR amplification and sensitizing mutations indicate a better survival benefit from EGFR-TKI therapy in lung adenocarcinoma patients Ling Shan a,1 , Ziping Wang b,1 , Lei Guo a , Hongyan Sun b , Tian Qiu a , Yun Ling a , Wenbin Li a , Lin Li a , Xiuyun Liu a , Bo Zheng a , Ning Lu a,∗∗ , Jianming Ying a,∗ a b

Departments of Pathology, Cancer Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China Departments of Medical Oncology, Cancer Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China

a r t i c l e

i n f o

Article history: Received 24 January 2015 Received in revised form 26 May 2015 Accepted 11 June 2015 Keywords: Lung adenocarcinoma Epidermal growth factor receptor Mutation Gene amplification Tyrosine kinase inhibitor Tumor heterogeneity

a b s t r a c t Objectives: Tumor heterogeneity, which causes different EGFR mutation abundance, is believed to be responsible for varied progression-free survival (PFS) in lung adenocarcinoma (ADC) patients receiving EGFR-TKI treatment. Frequent EGFR amplification and its common affection in EGFR mutant allele promote the hypothesis that EGFR mutant abundance might be determined by EGFR copy number variation and therefore examination of EGFR amplification status in EGFR mutant patients could predict the efficacy of EGFR-TKI treatment. Materials and methods: In this study, 86 lung ADC patients, who harbored EGFR activating mutations and received EGFR-TKI treatment, were examined for EGFR amplification and expression by Dual-color Silver in situ Hybridization (DISH) and immunohistochemistry analysis, respectively. Results and conclusion: Forty-one of 86 (47.7%) samples with EGFR activating mutations were identified with EGFR amplification. Patients with EGFR gene amplification had a significantly longer PFS than those without (16.3 vs. 9.1 months, p = 0.004). The EGFR expression was then examined by immunohistochemistry analysis. Thirty-nine of 86 (45%) tumors had EGFR overexpression, which was significantly correlated with EGFR amplification (p = 0.000). However, patients with EGFR overexpression exhibited no difference in PFS (14.1 vs. 13.3 months, p = 0.797). In conclusion, EGFR amplification occurs frequently in lung ADC patients harboring EGFR activating mutations, and could serve as an indicator for better response from EGFR-TKI treatment. © 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The discovery of epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) has allowed the identification of a subset of patients who displayed objective responses to EGFR tyrosine kinase inhibitors (TKIs) [1–4]. EGFR mutations occur in approximately 10% of NSCLC patients of Caucasian ethnicity and an estimated 50% of lung adenocarcinoma

∗ Corresponding author at: Department of Pathology, Cancer Hospital, Chinese Academy of Medical Sciences, Beijing 100021, China. Tel.: +86 10 87787515; fax: +86 10 87787515. ∗∗ Corresponding author at: Department of Pathology, Cancer Hospital, Chinese Academy of Medical Sciences, Beijing 100021, China. Tel.: +86 10 87788435; fax: +86 10 87788435. E-mail addresses: [email protected] (N. Lu), [email protected] (J. Ying). 1 These author contributed equally to this work. http://dx.doi.org/10.1016/j.lungcan.2015.06.008 0169-5002/© 2015 Elsevier Ireland Ltd. All rights reserved.

(ADC) patients of East Asian [5]. EGFR mutations affect the EGFR tyrosine kinase domain within exons 18–21. More than 90% of all EGFR mutations are in-frame deletions in exon 19 and the L858R substitution in exon 21. Apart from these two hotspot mutations, rare mutations include insertions in exon 20 and substitutions in exon 18 or 20 [6]. Mutations in EGFR exons 19 and 21 are clustered near the ATP cleft of the TK domain, which is targeted by EGFR-TKIs, and force EGFR into an active conformation, resulting in constitutive activation independent of ligand binding. EGFR mutant cells are exquisitely sensitive to inhibition by TKIs. In these cells, EGFR-TKIs decrease activation of EGFR, PI3K/Akt and MAPK pathways, and inhibit cell proliferation and induce apoptosis [7,8] The median progression-free survival (PFS) is 9–13 months for the patients harboring EGFR-activating mutation treated with EGFR-TKIs [1–3,9,10]. However, it remains unclear why the patients carrying the same EGFR-activating mutations experienced the PFS varying from fewer than 6 months to over 3 years.

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Coexistent genetic alterations in cancer-driving genes, i.e. KRAS mutations, MET amplification, PTEN loss and BIM polymorphism, etc., were indicated to be associated with primary resistance for EGFR-TKIs treatment [11–15]. EGFR T790M mutation, MET amplification and PIK3CA mutation, etc., were reported to be responsible for secondary resistance, of which the patients are EGFR-TKIs sensitive initially but require drug resistance after a few months [16–18]. However, none of these alterations can explain the majority of the cases with primary or secondary resistance. Tumor heterogeneity is now commonly believed to be the cause of response variation to EGFR-TKI treatment. Theoretically, patients who have higher proportion of cancer cells carrying heterozygous activating mutations would gain more benefit from EGFR-TKIs. Through quantifying the EGFR mutations using quantitative real-time PCR (qRT-PCR) and Sanger sequencing, Zhou et al. showed that the patients whose tumors had a high abundance of EGFR mutations benefited more from EGFR-TKIs than those with a low abundance of EGFR mutations [19]. Although PCR-based techniques could reveal the EGFR mutant abundance at some extent, the accuracy of the result largely depends on the purity of cancer cells. Ideally, non-tumor cells should be all excluded or to a maximum extent. However, it is not practical in clinical work, especially not for biopsies. Increased EGFR gene copy number has been variably reported in 10–44% of lung adenocarcinoma and at higher frequency, 52–75%, in the patients with EGFR mutations [20–26]. In addition, it has been well demonstrated that the EGFR amplification usually affects the mutant allele but not the wild-type locus [24,27,28]. Given all above evidence, we hypothesize that EGFR amplification might be the fundamental cause for EGFR mutant abundance and direct evaluation of EGFR gene copy number could predict the extend of EGFR-TKIs response. In the present study, we applied Dual-color Silver in-situ Hybridization (DISH) assay on 86 NSCLC patients harboring EGFR-sensitive mutations and evaluated the association of EGFR amplification with efficacy of EGFR-TKIs treatment.

2. Materials and methods 2.1. Patient selection, data collection and tissue microarray (TMA) construction We first recruited 92 consecutive patients who carried TKIs sensitive EGFR mutations and had received anti-EGFR therapy for metastatic lung ADC at the Cancer Hospital, Chinese Academy of Medical Sciences (CICAMS), Beijing, China. In the 92 patients, 6 were not eligible because of a lack of archival tumor tissue. Eightysix patients had formalin-fixed paraffin-embedded (FFPE) tumor tissue blocks obtained at the time of the primary diagnosis by biopsy (n = 25, 29%) or surgical resection (n = 61, 71%). The sampling sites were the primary tumor (n = 84, 98%), or metastatic sites (n = 2, 2%). Tissue microarray blocks for surgical resection specimens were built as described previously [29]. Briefly, for each case, cancer tissues were duplicated with a diameter of 1 mm on a slide. Before sample acquisition, the HE-stained FFPE slide of each case was observed under a microscope and the locations of typically characteristic morphology of cancer tissues were circled. Samples were taken from the circled location in the paraffin block using the Minicore 3 Tissue Arrayer (Alphelys, Plaisir, France). For each block, two 1 mm cores were punched from the circled regions in the donor block and arrayed on the recipient block to ensure the representation of the samples, and avoid missing information due to a loss of tissue cores. As for biopsies, whole tissue section was applied for EGFR expression and gene copy number analysis. The medical records of the patients were then reviewed to obtain demographic, clinical, and pathologic information. The results of EGFR mutations in exons 18–21 were also collected.

Imaging data were independently reviewed by authors to evaluate their treatment responses according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. PFS was calculated from the date of initiating TKI treatment to a radiologic or clinical observation of disease progression. Disease progression in

Concurrence of EGFR amplification and sensitizing mutations indicate a better survival benefit from EGFR-TKI therapy in lung adenocarcinoma patients.

Tumor heterogeneity, which causes different EGFR mutation abundance, is believed to be responsible for varied progression-free survival (PFS) in lung ...
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