In the past few years, tremendous progress has been made in

ORIGINAL ARTICLE Clinical and Testing Protocols for the Analysis of Epidermal Growth Factor Receptor Mutations in East Asian Patients with Non-small Cell Lung Cancer A Combined Clinical-Molecular Pathological Approach Manuel Salto-Tellez, MD,* Ming-Sound Tsao, MD, Jin-Yuan Shih, MD, PhD, Sumitra Thongprasert, MD, Shun Lu, MD, PhD, Gee-Chen Chang, MD, PhD, Joseph Siu-Kie Au, MD,# Teh-Ying Chou, MD, PhD,** Jong-Seok Lee, MD, Yuan-Kai Shi, MD, PhD, Ahmad Radzi, MD, Jin-Hyoung Kang, MD, PhD, Sang-We Kim, MD, PhD, Soo-Yong Tan, MD, PhD,## and James Chih-Hsin Yang, MD, PhD*** Background: Several randomized phase III studies in advanced stage non-small cell lung cancer (NSCLC) confirmed the superior response rate and progression-free survival of using epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor as first-line therapy compared with chemotherapy in patients with activating *Department of Pathology, National University Health System, Cancer Science Institute of Singapore, National University of Singapore, Singapore; Department of Pathology, Princess Margaret Hospital and University of Toronto, Toronto, Canada; Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan; Department of Medicine, Chiang Mai University, Chiang Mai, Thailand; Shanghai Lung Tumor Clinical Medical Center, Chest Hospital Affiliated to Shanghai Jiaotong University, Shanghai, China; Division of Chest Medicine, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan; #Department of Clinical Oncology, Queen Elizabeth Hospital, Hong Kong, China; **Institute of Clinical Medicine, School of Medicine, National Yang-Ming University and Department of Pathology and Laboratory Medicine, Taipei Veterans General Hospital, Taipei, Taiwan; Department of Internal Medicine, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Korea; Department of Medical Oncology, Cancer Institution/Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China; Department of Oncology, KPJ Johor Specialist Hospital, Johor, Malaysia; Division of Medical Oncology, Department of Internal Medicine, Seoul St. Mary s Hospital, The Catholic University of Korea, Seoul, Korea; Department of Oncology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea; ##Department of Pathology, Singapore General Hospital, Singapore, Singapore; and ***Graduate Institute of Oncology and Cancer Research Center, National Taiwan University, Taipei, Taiwan. Disclosure: M Salto-Tellez, MS Tsao, JY Shih, S Thongprasert, S Lu, TY Chiou, JS Lee, YK Shi, A Radzi, JH Kang, SW Kim, SY Tan, JC Yang received an honorarium for their advisory role from Astrazeneca. MS Tsao and JC Yang have served as advisors to Roche and OSI. M Salto-Tellez has served as advisor to Ventana-Roche, and is currently a consultant for Almac Diagnostics. Address for correspondence: James Chih-Hsin Yang, MD, PhD, Department of Oncology, National Taiwan University Hospital, National Taiwan University, 7, Chung-Shan South Road, Taipei, Taiwan. E-mail: chihyang@ntu.edu.tw Copyright 2011 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/11/0610-1663 EGFR mutations. Despite the need for EGFR mutation tests to guide first-line therapy in East Asian NSCLC, there are no current standard clinical and testing protocols. Methods: A consensus meeting was held involving expert oncologists, pulmonologists, and pathologists to discuss the current status and variations in EGFR mutation testing of NSCLC across Asia and to recommend a standard clinical and laboratory testing approach for future use. Results: Currently, EGFR mutation tests are only routinely performed in some East Asian countries and medical centers. The consensus recommendation was to perform the test in all newly diagnosed patients with advanced stage nonsquamous lung cancer and some squamous patients with clinical features associated with higher prevalence of EGFR mutations. To increase the sensitivity and specificity of the EGFR mutation tests, tissue acquisition and pretest sample evaluation are important steps in addition to standardization of the EGFR mutation test methodology. Conclusion: A standardized EGFR mutation testing protocol is an essential step toward realization of personalized medicine in East Asian NSCLC treatment. Key Words: NSCLC, EGFR, Tyrosine kinase inhibitor. (J Thorac Oncol. 2011;6: 1663 1669) In the past few years, tremendous progress has been made in the understanding of the molecular pathology of non-small cell lung cancer (NSCLC) and the development of molecular targeted treatment against this fatal disease. This has led to epidermal growth factor receptor (EGFR) mutations being demonstrated as the most robust predictive biomarker for response to EGFR-targeted therapies. In addition to standard clinical examination, pathology and imaging studies for the diagnosis of patients with NSCLC, EGFR mutation testing should be considered as a requirement for the complete diagnosis of NSCLC. Single-arm studies of first-line EGFR tyrosine kinase inhibitors (TKI) gefitinib or erlotinib monotherapy in NSCLC Journal of Thoracic Oncology Volume 6, Number 10, October 2011 1663

Salto-Tellez et al. Journal of Thoracic Oncology Volume 6, Number 10, October 2011 demonstrated high response rates in patients whose tumor harbors activating EGFR mutations. 1 3 In a large phase III IRESSA Pan-Asia Study (IPASS) involving advanced stage non/light smoker East Asian patients with lung adenocarcinoma randomized to treatment with EGFR-TKI gefitinib or chemotherapy, significant differences in objective response rates (ORRs) and progression-free survival (PFS) were reported in favor of patients treated with gefitinib. Further exploratory biomarker analysis showed that patients with EGFR-activating mutations achieved superior outcomes in PFS and ORR when first treated with EGFR-TKI compared with chemotherapy, whereas patients without EGFR mutation experienced better PFS when combination chemotherapy was given first. 4 This is despite the results of final analysis revealing no difference in OS between the EGFR mutation positive or negative patients after 2 or more years of followup, probably due to the extensive crossing-over of subsequent treatments. 5 Despite no difference in the final OS outcome, initial use of gefitinib in EGFR mutation positive patients did result in better quality of life, longer PFS, and higher ORR than chemotherapy-treated patients. 6 The results demonstrate that clinical selection alone was not sufficient for the best management of patients with NSCLC, and EGFR mutation test should be recommended to patients with advanced NSCLC during the time of initial diagnosis. Several subsequent clinical trials randomizing patients with EGFR mutation-positive NSCLC to chemotherapy or EGFR-TKIs such as the Northeast Japan, 7 West Japan Thoracic Oncology Group Trial (WJTOG), 8 and erlotinib versus gemcitabine/carboplatin in chemonaive patients with stage IIIB/IV NSCLC with EGFR exon 19 or 21 mutation (ML20981) (OPTIMAL) 9 studies have confirmed the superiority of first-line EGFR-TKI to combination chemotherapy in terms of PFS and ORR. Several other phase II to phase III studies suggested similar predictive outcomes for patients receiving EGFR-TKI as first-line or subsequent lines of treatment. 1 3,10 16 Importantly, these studies have used different EGFR mutation testing methodologies, and there has been limited details regarding the tumor sampling and the pretesting sample evaluation procedures used from literature. Although EGFR mutation testing is becoming a standard practice for the treatment of NSCLC, variances for testing in clinical practice remain across East Asia. This clinical and testing protocol collate the expert opinions and recommendations for EGFR mutation testing from a panel of medical oncologists, pulmonologists, and molecular pathologists across East Asia, providing a reference for further discussion and improvement for management of patients with NSCLC in the molecular targeted age. PATIENTS AND METHODS An expert meeting was held with a panel of medical oncologists, pulmonologists, and molecular pathologists from Taiwan, Hong Kong, China, South Korea, Thailand, Malaysia, Singapore, and Canada. The objective was to discuss the current status and variances in EGFR mutation testing of NSCLC across Asia and to provide recommendations to standardize the clinical and testing protocols across all Asian clinical centers and hospitals. The discussions examined current versus ideal EGFR mutation testing practices, current local practices, and the barriers to routine EGFR mutation testing across Asia. The discussions led to the participants gaining a subtle understanding on the situation of EGFR mutation testing across these countries, and from there, a standard approach was established and outlined for EGFR mutation testing in Asia. RESULTS AND DISCUSSIONS Status of EGFR Mutation Testing across Asia In most East Asian medical centers, hospitals, or clinics, EGFR mutation test are not routinely performed in patients with NSCLC. A simple survey on the panel of experts estimated that the proportion of nonsquamous patients tested for EGFR mutations in their practice across East Asia, which may vary from 30 to 80%. The rates were closely associated with whether they were referred to medical centers, public/private hospitals or clinics, size of the medical centers or hospitals, and facilities available and clinical expertise of the medical centers or hospitals. In addition, EGFR mutation test is not yet available for most of Asian medical facilities. This is in contrast to some research or academic medical centers in Japan, Taiwan, Korea, Singapore, Thailand, China, and Hong Kong, which routinely (or plan to routinely) perform the test. As many patients with advanced NSCLC presenting to local hospitals in these or other Asian countries are currently excluded from the test, with the shift in treatment paradigm from cytotoxic chemotherapies to EGFR-targeted therapy on the basis of the mutation test results, more patients with NSCLC need to be screened, and EGFR testing is expected to increase. Potential Barriers to EGFR Mutation Testing across East Asia At present, the cost for EGFR mutation tests in most countries is funded by the patients or research funds and can vary from US$150 to several hundred US$. Currently, the most popular method is direct sequencing; however, many centers are investigating the use of more sensitive methods such as Scorpion-amplification refractory mutation system method and other polymerase chain reaction (PCR)-based methods. As multiple studies have confirmed the efficacy of first-line gefitinib in improving the PFS of patients with NSCLC with EGFR mutations, there is potential for the national insurance systems to extend coverage for the test and first-line treatment with EGFR-TKI as reimbursable. Besides cost, other factors that should be considered in setting up routine EGFR mutation testing include the number of preliminary screenings and sampling times required for patients, availability of optimal specimen for testing, challenges in specimen acquisition, clinical protocol at the medical center or hospital, turn-around time (TAT), availability of experienced clinical pathologists for the required tests, number of laboratories capable of performing the test in each country, communication/coordination efforts between the clinician and pathologist, and the predefined criteria for the type of 1664 Copyright 2011 by the International Association for the Study of Lung Cancer

Journal of Thoracic Oncology Volume 6, Number 10, October 2011 Protocols for the Analysis of EGFR patients (first-line only or include second- and third-line patients) who should be tested. TAT: From Clinical Consultation to the Management of Treatment If there is a short order-to-report time, first-line treatment with EGFR-TKI can be initiated once EGFR test results are released. According to a simple survey on the panel of experts, the time of patient consultation to the release of test results may be as quick as 5 days or require as long as 14 days, and treatment with EGFR TKI can be recommended only within 3 days after the results are released. Most patients can wait and tolerate a 1 to 2-week order-to-report time. Patients without EGFR mutations may not benefit from gefitinib treatment at all and should be treated with chemotherapy. 8 Patients should be discouraged from taking EGFR-TKI if the result for mutation is not available based on the IPASS results that demonstrated even in patients who are enriched clinically for EGFR mutations, as much as 40% have no mutations. Nevertheless, in a terminally ill patients who are not fit for chemotherapy, a trial of EGFR-TKI may be justified, provided the family members and the patients have been informed regarding the possible worsening of symptoms and disease, based on the IPASS findings. Several factors may influence the duration of the orderto-report time. In general, histology and EGFR tests that are performed within the same pathology laboratory compared with those performed in separate laboratories would have shorter order-to-report times. The communication and coordination efforts between clinicians and pathologists in hospitals lacking proper clinical protocols may also contribute to an increase in the order-to-report time. Most small to medium sized medical centers or hospitals are not set up to perform the test in-house and would have to send the tests to accredited clinical molecular pathology laboratories for testing. This situation could lead to longer order-to-report times. Technical Challenges and Shortcomings of Mutation Testing Methodology The most widely used mutation testing methodology is direct sequencing of PCR products from amplification of EGFR exons 18 to 21. This method can detect all types of mutations, but it is time consuming and its sensitivity can be limited by contamination of nonmalignant cells in the samples. Other types of confirmatory tests if required after the first or second direct sequencing tests are also time consuming. Most tests are conducted using archival formalin-fixed and paraffin-embedded materials, which may include diagnostic bronchoscopic, core or excisional biopsies or previous tumor resection specimens. Materials obtained by fine needle aspiration may present difficulties, especially if they have been obtained from small lung lesions. In some circumstances, tumor tissue is not available or suitable for EGFR mutation analysis, and this may apply to a large proportion of patients with lung cancer. A sensitive and accurate method to detect EGFR mutation from plasma, serum or circulating tumor cells DNA can greatly improve the feasibility of personalized medicine for lung cancer. If the tissue cannot be obtained, the patient s plasma is an alternative choice for DNA extraction. 17 19 Nevertheless, the sensitivity and applicability of plasma, serum circulating tumor cells testing is yet to be established. Malignant pleural effusion is possible source to collect malignant cells for molecular studies. Nevertheless, microdissection is difficult to perform on cytology specimens, which often contain substantial proportions of nontumor cells. The technique of forming a cell pellet, followed by formalin fixation and paraffin embedding, allows the availability of material for conventional bright-field microscopy examination of the presence and percentage of malignant cells and, as such, a very good alternative for DNA extraction and molecular analysis. 20 22 There should be standard internal quality control (QC) procedures implemented and adopted within medical institutions or pathology laboratories to ensure the quality of biopsy sampled is sufficient and met quality requirements before EGFR mutation testing. Other Predictive Biomarkers for EGFR TKI Therapy Several markers including EGFR mutations, increases in EGFR gene copy number, single-nucleotide polymorphisms of the EGFR gene, EGFR protein expression, and KRAS mutations have been evaluated as predictive markers to select patients with NSCLC for increased treatment efficacy to EGFR-TKI. 23 For EGFR mutation testing, many methodologies are available. Methodologies such as immunohistochemistry, fluorescence in situ hybridization, and mutational analysis have all been used in research. 24 Many of these testing methodologies have not been standardized in a clinical trial setting. 24 Current Clinical Screening and Management Protocols Although a consensus has not been established in recommending the type of patients with NSCLC who should be tested, most participants agreed that Asian patients with nonsquamous histology, particularly adenocarcinoma histology should be routinely tested for EGFR mutations. For patients with NSCLC of Asian origin with squamous histology where the EGFR mutation rates are only between 3 and 5%, clinicians are recommended to consider EGFR mutation testing but with an option of excluding male and heavy smokers. Nevertheless, EGFR mutation test may be useful for Asian male and heavy smokers when they have poor performance status or comorbidities. In terms of issues with the specificity of EGFR testing methodologies, it can often be resolved and validated using repeated sequencing or more stringent laboratory protocols. It is recommended that for EGFR wild-type patients, EGFR-TKI should be reserved for second or further lines of treatment after patients exhaust chemotherapy options. Rebiopsy of tumor tissue after disease progression for further evaluation of new mutation such as EGFR T790M or other activated pathways such as hepatocyte growth factor cmet pathway, should be consider. Use of clinical information and histology alone to guide first-line EGFR treatment for patients with NSCLC should be discouraged, unless an EGFR mutation test cannot be performed. Copyright 2011 by the International Association for the Study of Lung Cancer 1665

Salto-Tellez et al. Journal of Thoracic Oncology Volume 6, Number 10, October 2011 EGFR Molecular Diagnostic Testing Practical Laboratory Considerations This section of the article aims to provide a framework to understand some practical aspects related to EGFR mutation testing. EGFR Mutation Testing Model The operational organization of diagnostic testing of solid tumors may be such that morphology-based diagnostics and molecular diagnostics are run in independent laboratories, representing different departmental and management structures (focused approach) or, alternatively, both aspects may be run under a single laboratory umbrella that coordinates both the morphologic and the genetic analysis of the tumors (integrated approach). In practical terms, the integrated approaches to pathology diagnostics provide significant advantages that have been emphasized recently 25,26 including the following: a. An optimal QA of the tissues, including adequate fixation, processing, embedding, and storage. This optimal treatment of diagnostic tissues should lead to best possible histomorphology, immunohistochemistry, and DNA preservation. b. A combined interpretative analysis, as the histological diagnosis may help in interpreting the clinical significance of the molecular diagnostic results. c. The organization of a diagnostic flow within the department to improve TAT (see later). EGFR Mutation Testing Methodology The EGFR gene is large but the region with clinical relevance (the catalytic kinase domain) includes only four relatively small exons 18 to 21, which are analyzable by four individual PCR reactions, within the constraints of small amplicon sizes inherent to formalin-fixed and paraffin-embedded materials. The many different approaches to the analysis of EGFR status in lung cancer have been well reviewed elsewhere. 27,28 Although mutation testing may be performed using different methods including Sanger direct sequencing or other more sensitive methods, 28 regardless of the method used, it is important to make sure that the validation, verification, and quality assurance (QA)/QC of the test must happen in an accredited laboratory environment. In view of the relevance of EGFR testing in the Asian clinical environment, an Asian program for EGFR QA/QC, which could hold a repository of external QCs for general use, is recommended. Figure 1 shows the careful steps for a successful Sanger sequencing analysis of EGFR mutations. We hereby stress the degree of necessary redundancy in some of the steps, which represents a conditio sine qua non to ensure accurate analyses at all times. All laboratories are encouraged to participate in an external QA program in EGFR mutational analysis, perform validation studies to ensure their assay is sufficiently sensitive in particular for the detection of point mutations, and report their mutation pickup rates and compare the data with other laboratories in the Asian context. FIGURE 1. Steps for the Sanger sequencing analysis of epidermal growth factor receptor (EGFR) mutations. EGFR Mutation Testing Materials The starting materials for EGFR mutation analysis are usually the materials still available in the diagnostic samples. Thus, EGFR analysis can be performed in materials from (a) bronchoscopic biopsies from central neoplasms; (b) core biopsies from peripheral neoplasms, (c) the surgical resection of the primary tumor, (d) fine needle aspiration or pleural effusion samples, and (e) biopsy of metastatic lesions. The cytology cell block technique from malignant pleural exudates may represent an alternative to samples where the original diagnostic sample has not yield enough materials for therapeutic testing. As mutation testing will become more common or routine, the acquisition of tumor materials especially during biopsy should take into account the following important considerations: a. The biopsy sampling of lung cancer should take into account the need to perform EGFR therapeutic testing in addition to diagnostic needs (see later). b. EGFR preanalytical protocols for best DNA preservation are essential for successful testing. The type of fixative and the length of fixation, the age of the paraffin block, and the conditions for storage are clear determinants of the DNA quality. Optimal fixation and embedding as recommended by the College of American Pathologists 29 or the Royal College of Pathologists of Great Britain and Ireland are adequate to ensure appropriate DNA quality. This issue also highlights the importance of embedding strong internal controls in the protocol (from DNA extraction to PCR reaction) to avoid the presence of false negative results, a matter of 1666 Copyright 2011 by the International Association for the Study of Lung Cancer

Journal of Thoracic Oncology Volume 6, Number 10, October 2011 Protocols for the Analysis of EGFR importance to take into account when designing QA reviews. c. It is essential to review the hematoxylin and eosin stain of the material to be analyzed, to ensure the nature of the sample, its adequacy for analysis and, crucially, that the ratio of tumor versus normal is sufficient for the reported sensitivity of the test, as the calculations of the individual laboratory. In most occasions, manual microdissection may be necessary to ensure this. d. The samples that normally have more material (i.e., resection specimens) are not necessarily the ones that have a higher success rate. This may be related to adequacy of fixation. e. Most cytology samples may be adequate for analyzing EGFR mutations, 30 provided that the laboratory test has been validated as well for that purpose. f. The importance of preanalytic steps can be illustrated by these figures from the laboratory of one of the meeting participants: when analyzing samples from their own hospital (where fixation, embedding, and other preanalytical variables are carefully looked after) the unsatisfactory rate is below 3% whereas, when considering the broad spectrum or referred cases from all over Asia, the unsatisfactory rate increases close to 13% (unpublished data). EGFR Mutation Testing TAT This aspect of testing may be determinant, as on occasions the test may take as long as it would require to empirically observe if the patient will respond to treatment by first-line EGFR TKI (2 3 weeks). 31 Much of the time gap between when the test is requested and the process indicated in Figure 2 is initiated may be related to internal issues between laboratories within the same institutions. One option FIGURE 2. Overall process of epidermal growth factor receptor (EGFR) mutation testing turn-around time (TAT) from request for testing, diagnostic testing to recommendations for first-line EGFR tyrosine kinase inhibitor (TKI) therapy. to overcome this is by instituting reflex testing for EGFR mutation. The overall process is depicted in Figure 2. When this protocol is applied, both the morphologic and genetic characterization will become available to the oncologist simultaneously, thus streamlining the whole therapeutic decision process. EGFR Mutation Testing Reporting Classically, a complete molecular diagnostic report should include the following components: type of molecular test, results, diagnostic comment (where applicable), clinical significance of the test (in general), materials submitted, clinical background, methods, references, and signatures. In combined morphology molecular report: gross description, microscopic description, and histo/cytopathological diagnosis should also be included. Whenever possible, and if the technology applied to the mutation detection allows it, it is important to report the results in the right clinical value of the detected mutation. Some examples of how this can be done include the following: Y A mutation XXXX is identified in exon XXXX. This is one of the commonest mutations reported conferring sensitivity to erlotinib or gefitinib. Y A mutation XXXX is identified in exon XXXX. This is an uncommon mutation which, nevertheless, has been reported as conferring sensitivity to erlotinib or gefitinib (ideally, a reference should be incorporated). Y A mutation XXXX is identified in exon XXXX. This is an uncommon mutation; to our knowledge, the clinical significance of it is unknown. Y A mutation XXXX is identified in exon 20. This mutation is reported to confer resistance to erlotinib or gefitinib. Y A mutation XXXX is identified in exon 20. This is an uncommon mutation; to our knowledge, the clinical significance of it is unknown. EGFR Mutation Testing Alternative Testing Methods In view of the fact that the majority of laboratories in this study review use Sanger sequencing as the technique of choice for EGFR mutation detection, our considerations have been centered in this method, considered by many as the gold standard. Other approaches have been reported, and the results are equally successful. Methods such as the denaturing high performance liquid chromatography, 30 the peptic nucleic acid-locked nucleic acid clamp, 32 the limited copy number-high resolution melting (LCN-HRM) approach 33 or the amplification refractory mutation system, and DxS EGFR mutation detection kit 4 have shown a good performance with reported increased sensitivity when compared with Sanger sequencing. Nevertheless, aspects such as reagent cost, availability of the technology, or the calculated sensitivity of the direct sequencing-based home-brew test are strong variables to choose a testing modality. The latter is particularly important: although it is true that alternative techniques are more sensitive than direct sequencing when working on DNA dilutions from tumor cell lines, the usefulness of some of these techniques remains to be demonstrated on paraffin-fixed Copyright 2011 by the International Association for the Study of Lung Cancer 1667

Salto-Tellez et al. Journal of Thoracic Oncology Volume 6, Number 10, October 2011 tumor biopsies. The use of immunohistochemistry by using mutation-specific antibodies 34 can serve as a useful prescreening tool, although confirmation by other PCR-based methods is still desirable. EGFR Mutation Testing Single Biomarker versus Multiple Biomarkers It is the impression of the pathology community that we are increasingly asked to provide more information from samples that are getting smaller. This small sample revolution may have to be revisited in the context of predictive pathology and will clearly affect the testing of EGFR and other mutations. In the future, it is likely that we will be testing for different biomarkers of the same pathway, as the KRAS/ BRAF paradigm in metastatic colorectal cancer, 35 different biomarkers that are mutually exclusive leading to different therapeutic options, such as EGFR or KRAS mutation analyses and EML4-ALK rearrangements in lung adenocarcinoma, 36 or perform multiple biomarker tests for several biologically distinct therapeutic potentials. The latter is exemplified by the Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination trial, 37 in which additional to their original diagnostic sampling, patients tumors are characterized for a large panel of biomarkers to guide multiple therapeutic trials. The need for more generous sampling at the time of diagnosis, or the introduction of multiplex biomarker assays, will require a significant change of mindset in those who are at the first line of the biopsy taking, such as radiologists or pulmonologists (Figure 3). Current Recommendations in Other Parts of the World The issues of EGFR mutation testing confronted by pathologists and oncologists are similar worldwide. Nevertheless, the recommendations may differ due to different proportions of patients with EGFR mutation in a given population and different practicing environment. A European FIGURE 3. Diagnostic sampling process in the combined morpho-molecular diagnostic approach. workshop established consensus of EGFR mutation testing in NSCLC among European lung cancer experts. 38 It is recommended in European centers that EGFR mutation tests should be ordered by treating physicians, whereas in Asian centers, the EGFR mutation test results can preferably be performed immediately with pathological diagnosis as part of the molecular pathology diagnosis report. In the National Comprehensive Cancer Network guideline, EGFR testing is a category 1 recommendation for adenocarcinoma, large cell carcinoma, and NSCLC not otherwise specified and not recommended for squamous cell carcinoma. Nevertheless, the EGFR mutation testing can be considered in Asian patients with squamous cell carcinoma, although not routinely performed. CONCLUSION All committee members agreed and were confident that the expert opinions recommended in this article would improve clinical screening of EGFR mutations in East Asian patients with NSCLC and lead to more rapid and effective treatment of Asian patients with NSCLC with EGFR-directed therapies. The protocol will facilitate multiple biomarkers testing in the future. ACKNOWLEDGMENTS The advisory board for Asian pathologists and clinicians was supported by Astrazeneca. The authors thank EMD Asia for their editorial help of this publication. REFERENCES 1. Sequist LV, Martins RG, Spigel D, et al. 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