Published OnlineFirst March 10, 2020; DOI: 10.1158/1078-0432.CCR-19-3972 CLINICAL CANCER RESEARCH | PRECISION MEDICINE AND IMAGING Integrative Omics Analysis Reveals Soluble Cadherin-3 as a Survival Predictor and an Early Monitoring Marker of EGFR Tyrosine Kinase Inhibitor Therapy in Lung Cancer Ting-Feng Hsiao1, Chih-Liang Wang2,3, Yi-Cheng Wu4, Hsiang-Pu Feng1, Yen-Chuan Chiu1, Hao-Yu Lin1, Ko-Jiunn Liu5,6, Gee-Chen Chang7,8, Kun-Yi Chien1,9,10, Jau-Song Yu9,10,11,12, and Chia-Jung Yu1,3,9,10 ABSTRACT ◥ Purpose: EGFR tyrosine kinase inhibitors (EGFR-TKI) (OS) were assessed to evaluate the prognostic values of the benefit patients with advanced lung adenocarcinoma (ADC) potential biomarkers. harboring activating EGFR mutations. We aimed to iden- Results: Fifteen proteins were identified as potential biomarkers tify biomarkers to monitor and predict the progression of of EGFR-TKI resistance. Cadherin-3 (CDH3) was overexpressed in patients receiving EGFR-TKIs via a comprehensive omic ADC tissues compared with normal tissues. CDH3 knockdown analysis. enhanced EGFR-TKI sensitivity in ADC cells. The PE level of Experimental Design: We applied quantitative proteomics soluble CDH3 (sCDH3) was increased in patients with resistance. to generate the TKI resistance–associated pleural effusion (PE) The altered sCDH3 serum level reflected the efficacy of EGFR-TKI proteome from patients with ADC with or without EGFR-TKI after 1 month of treatment (n ¼ 43). Baseline sCDH3 was signif- resistance. Candidates were selected from integrated genomic icantly associated with PFS and OS in patients with ADC after and proteomic datasets. The PE (n ¼ 33) and serum (n ¼ 329) EGFR-TKI therapy (n ¼ 76). Moreover, sCDH3 was positively levels of potential biomarkers were validated with ELISAs. associated with tumor stage in non–small cell lung cancer (n ¼ 272). Western blotting was applied to detect protein expression in Conclusions: We provide useful marker candidates for drug tissues, PEs, and a cell line. Gene knockdown, TKI treatment, resistance studies. sCDH3 is a survival predictor and real-time and proliferation assays were used to determine EGFR-TKI indicator of treatment efficacy in patients with ADC treated with sensitivity. Progression-free survival (PFS) and overall survival EGFR-TKIs. Introduction lung cancers, and adenocarcinoma (ADC) is the most common subtype of NSCLC (2). EGFR mutations specifically target patients Lung cancer is the leading cause of cancer-related death world- with lung ADC (60%) in East Asian countries, including Tai- wide (1). Non–small cell lung cancer (NSCLC) represents 80% of all wan (3, 4). EGFR-tyrosine kinase inhibitors (EGFR-TKI), such as the first-generation TKIs erlotinib and gefitinib and the second-generation TKI afatinib, have been determined to benefit patients with ADC with 1Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung EGFR-activating mutations (5). These response evaluations of targeted University, Taoyuan, Taiwan. 2School of Medicine, College of Medicine, Chang therapy are based on the RECIST guidelines (6), and an EGFR Gung University, Taoyuan, Taiwan. 3Division of Pulmonary Oncology and Inter- mutation is recognized as a good predictor for the initial efficacy of ventional Bronchoscopy, Department of Thoracic Medicine, Chang Gung Memo- EGFR-TKI treatments, in which the response rates and disease control 4 rial Hospital, Linkou, Taoyuan, Taiwan. Department of Thoracic Surgery, Chang rates are 60%–70% and 85%–95%, respectively. These TKIs are Gung Memorial Hospital, Linkou, Taoyuan, Taiwan. 5National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan. 6School of associated with a median progression-free survival (PFS) time of – – Medical Laboratory Science and Biotechnology, Taipei Medical University, 9 14 months compared with 5 7 months for platinum-based Taipei, Taiwan. 7Division of Chest Medicine, Department of Internal Medicine, chemotherapy (7). Unfortunately, patients treated with first/second- Taichung Veterans General Hospital, Taichung, Taiwan. 8Faculty of Medicine, generation EGFR-TKIs eventually develop acquired resistance to these 9 School of Medicine, National Yang-Ming University, Taipei, Taiwan. Department agents after a median of 10–16 months that most commonly (50%– of Cell and Molecular Biology, College of Medicine, Chang Gung University, 60% of cases) results from a secondary mutation at position 790 in Taoyuan, Taiwan. 10Molecular Medicine Research Center, Chang Gung Univer- sity, Taoyuan, Taiwan. 11Liver Research Center, Chang Gung Memorial Hospital, EGFR (T790M; refs. 8, 9). Osimertinib, a third-generation irreversible – Linkou, Taoyuan, Taiwan. 12Research Center for Food and Cosmetic Safety, EGFR-TKI that selectively inhibits both EGFR-TKI sensitizing and College of Human Ecology, Chang Gung University of Science and Technology, EGFR T790M–resistance mutations (10), obtained FDA-accelerated Taoyuan, Taiwan. approval in November 2015. Recent studies have also shown that Note: Supplementary data for this article are available at Clinical Cancer osimertinib improves both the median PFS (18.9 vs. 10.2 months) and Research Online (http://clincancerres.aacrjournals.org/). overall survival (OS; 38.6 vs. 31.8 months) compared with first- € T.-F. Hsiao and C.-L. Wang contributed equally to this article. generation EGFR-TKIs in patients with treatment-na ve, EGFR muta- tion–positive (exon 19 deletion or L858R) advanced NSCLC (11, 12), Corresponding Author: Chia-Jung Yu, Chang Gung University, No. 259 Wenhua fi 1st Rd., Guishan Dist., Taoyuan 33302, Taiwan. Phone: 886-3211-8800, ext. 3424; suggesting that osimertinib will be a new standard of care in rst-line Fax: 886-3211-8042; E-mail: [email protected] EGFR-mutated NSCLC. T790M can be evaluated via either a tissue rebiopsy or circulating cell-free tumor DNA in the plasma, and its Clin Cancer Res 2020;XX:XX–XX evaluation is currently mandatory after progression to first/second- doi: 10.1158/1078-0432.CCR-19-3972 generation EGFR-TKIs (13). However, neither cancer progression nor Ó2020 American Association for Cancer Research. an improvement in the patient's OS is correlated with EGFR AACRJournals.org | OF1 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst March 10, 2020; DOI: 10.1158/1078-0432.CCR-19-3972 Hsiao et al. Hospital (Linkou, Taoyuan, Taiwan; approval number: 99-3738B and Translational Relevance 99-3671B). This study was conducted in accordance with the Decla- Mutated EGFR is a predictor of the initial treatment efficacy of ration of Helsinki and Good Clinical Practice. Written informed EGFR–tyrosine kinase inhibitors (TKI) in advanced lung adeno- consent was received from all patients before collection. For the carcinoma (ADC). Unfortunately, neither EGFR mutations nor discovery phase, malignant pleural effusion (MPE) from 23 patients molecular markers can accurately monitor cancer progression or with advanced lung ADC with EGFR mutations who received EGFR- predict patient survival. EGFR-TKI susceptibility associated bio- TKI–targeted therapy with or without following chemotherapy regi- markers to benefit patients with lung ADC are in high demand. mens were included. PE from 10 patients with tuberculosis (TB) Pleural effusion (PE) has been recognized as a promising source for represents benign pulmonary disease. These 33 patients' PEs were biomarker discovery. By integrating PE proteomic and tissue divided into four groups (Fig. 1A; Supplementary Table S2): MPE genomic analyses, we herein identified soluble cadherin-3 (sCDH3) obtained prior to treatment (before treatment, BT group) from 11 as a marker for monitoring first/second-generation EGFR-TKI patients who achieved partial response (PR) for at least 6 months, MPE resistance in real time. Cadherin-3 (CDH3) knockdown enhances obtained on the day of failure from 8 patients who received EGFR-TKI EGFR-TKI sensitivity in ADC cells. Importantly, the baseline treatment followed by chemotherapy [progressive disease; PD (TþC)], serum sCDH3 level is associated with progression-free survival in MPE obtained on the day of failure from 4 patients who received patients with ADC who received EGFR-TKI therapy and with EGFR-TKI treatment [PD (T)], and PE from 10 patients with TB. For overall survival in patients with non–small cell lung cancer the verification phase, these 33 PEs used in the discovery phase were (NSCLC). Our results establish a biomarker dataset for EGFR- included in ELISA. To determine the sCDH3 level via ELISA, serum TKI resistance and prognosis. This study significantly advances the samples were obtained from 272 treatment-na€ve NSCLC patients development of a liquid biopsy to benefit patients with NSCLC and (baseline) and 57 healthy controls (Table 1). Specifically, 23 MPE and provides new insights into EGFR-TKI resistance in lung ADC. 76 serum samples from patients with ADC with mutated EGFR and who received first/second-generation EGFR-TKIs were collected between 2009 and 2016. Among these 272 patients with NSCLC, serum samples collected 1 month (29 Æ 8 days) after EGFR-TKI mutations. This suggests that even in patients with the same EGFR treatment from 43 patients with ADC (28 PR and 15 non-PR) with mutation status, the event of acquiring resistance and the timing of EGFR mutations were included to examine alterations in sCDH3 levels resistance are heterogeneous and unpredictable. To improve the upon EGFR-TKI treatment. To detect