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INTERNATIONAL ASSOCIATION FOR THE STUDY OF LUNG CANCER

IASLC ATLAS OF EGFR TESTING IN LUNG CANCER

Conquering Thoracic Cancers Worldwide

EDITED BY TONY S. MOK, MDDAVID P. CARBONE, MD, PHD

FRED R. HIRSCH, MD, PHD

International Association for the Study of Lung Cancer, Aurora, CO, U.S.A.

Editors:Tony S. Mok, MD David P. Carbone, MD, PhD Fred R. Hirsch, MD, PhD

An IASLC publication published by Editorial Rx Press, North Fort Myers, FL, U.S.A.

Cover and interior design by Amy Boches, Biographics

IASLC Office:IASLC, 13100 East Colfax Ave., Unit 10, Aurora, Colorado 80011, U.S.A.www.iaslc.org

First Editorial Rx Press Printing October 201710 9 8 7 6 5 4 3 2 1

ISBN: 978-1-947768-00-0

Copyright © 2017 International Association for the Study of Lung CancerAll rights reserved

Cover images:Top image: Courtesy of Dara Aisner, MD.

Bottom left image: Reprinted by permission from Macmillan Publishers Ltd: Modern Pathology; 2012; 25:347–369, © 2012.

Bottom right image: Reprinted from Cancer Sci. 2016; 107: 1179–1186 courtesy of Yoshihisa Kobayashi, MD, Tetsuya Mitsudomi, MD, PhD, under a Creative Commons License CC-BY-NC-ND 4.0 https://creativecommons.org/licenses/by-nc-nd/4.0/).

Without limiting the rights under copyright reserved above, no part of thispublication may be reproduced, stored in or introduced into a retrieval system,or transmitted in any form, or by any means without prior written permission.

While the information in this book is believed to be true and accurate as ofthe publication date, neither the IASLC nor the editors nor the publisher canaccept any legal responsibility for any errors or omissions that may be made.The publisher makes no warranty, express or implied, with response to thematerial contained therein.

AN INTERNATIONAL ASSOCIATION FOR THE STUDY OF LUNG CANCER PUBLICATION

Editorial Rx Press

North Fort Myers, FL


EDITED BY TONY S. MOK, MD DAVID P. CARBONE, MD, PHD FRED R. HIRSCH, MD, PHD

IASLC acknowledges the generous funding and support provided by AstraZeneca for the IASLC Atlas of EGFR Testing in Lung Cancer.

The coeditors and contributors also acknowledge the assistance of Jacinta Wiens, MS, PhD, Scientific Affairs Project Manager, IASLC, for coordinating the project; the editorial assistance of Joy Curzio and Lori Alexander, MTPW, ELS, MWC; the graphic arts talents of Amy Boches, Biographics; and, the publishing support of Deb Whippen, Editor and Publisher, Editorial Rx Press, for the publication of this text.

Acknowledgments

Contents

Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Manufacturers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11

1 Therapeutic Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13

2 EGFR Testing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19

3 Sample Acquisition, Processing, and General Diagnostic Procedures. . . . . . . . . . . . . . . . . . . . . . . . . .27

4 EGFR Gene Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33

5 Types of Assays for EGFR Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .43

6 Reporting, Interpretations, and Quality Assurance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53

7 Access to Testing Guidelines and Algorithms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .59

8 Summary and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .65

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69

6 IASLC ATLAS OF EGFR TESTING IN LUNG CANCER

Contributors

EditorsTony S. Mok, MDProfessorDepartment of Clinical OncologyChinese University of Hong Kong Prince of Wales HospitalHong Kong, China

David P. Carbone, MD, PhDProfessor and DirectorJames Thoracic Center The Ohio State University Comprehensive Cancer CenterOhio, U.S.A.

Fred R. Hirsch, MD, PhDProfessor of Medicine and PathologyPia and Fred R. Hirsch Endowed ChairUniversity of Colorado Cancer CenterCEO, International Association for the Study of Lung Cancer (IASLC)Colorado, U.S.A.

Contributing AuthorsMyung-Ju Ahn, MD, PhDProfessorDivision of Hematology-OncologyDepartment of MedicineSamsung Medical CenterSungkyunkwan University School of MedicineSeoul, South Korea

Dara L. Aisner, MD, PhDAssociate ProfessorDepartment of PathologySchool of MedicineUniversity of Colorado Anschutz Medical CampusColorado, U.S.A.

Sanja Dacic, MD, PhDProfessorDepartment of PathologyUniversity of Pittsburgh Medical CenterPennsylvania, U.S.A.

Leora Horn, MD, MScAssociate Professor of MedicineDirector, Thoracic Oncology ProgramAssistant Vice Chairman for Faculty Development

Department of MedicineVanderbilt University Medical CenterTennessee, U.S.A.

Peter B. Illei, MDAssistant Professor of PathologyAssistant Professor of OncologyDepartment of PathologyJohns Hopkins University School of MedicineMaryland, U.S.A.

Yuichi Ishikawa, MD, PhDChief, Division of PathologyDirector, Clinicopathology CenterVice Director, Cancer InstituteKeio UniversityThe Cancer Institute Hospital of JFCRTokyo, Japan

Pasi A. Jänne MD, PhDDirector, Lowe Center for Thoracic OncologyDirector, Belfer Center for Applied Cancer ScienceSenior Physician Professor of MedicineDana-Farber Cancer InstituteHarvard Medical SchoolMassachusetts, U.S.A.

Keith M. Kerr, FRCPathProfessorDepartment of PathologyAberdeen University Medical School,Aberdeen Royal InfirmaryAberdeen, Scotland, United Kingdom

Philip Mack, PhDProfessorDirector of Molecular PharmacologyDepartment of Internal MedicineDivision of Hematology and OncologyUC Davis Comprehensive Cancer CenterCalifornia, U.S.A.

Alberto M. Marchevsky, MDDirector Pulmonary and Mediastinal PathologyCedars-Sinai Medical CenterClinical Professor of PathologyDavid Geffen UCLA School of MedicineCalifornia, U.S.A.

7CONTRIBUTORS

Geoffrey R. Oxnard, MDAssistant Professor of MedicineDana-Farber Cancer InstituteHarvard Medical SchoolMassachusetts, U.S.A.

Keunchil Park, MD, PhDProfessorDivision of Hematology-OncologyDepartment of MedicineSamsung Medical CenterSungkyunkwan University School of MedicineSeoul, South Korea

Luis Paz-Ares, MD Associate ProfessorChair, Medical Oncology DepartmentUniversity ComplutenseHospital Doce De OctubreMadrid, Spain

Solange Peters, MD, PhDCHUV Medical Oncology Service ChiefChair of Thoracic OncologyAssociate ProfessorOncology DepartmentCentre Hospilalier Universitaire VaudoisLausanne, Switzerland

Daniel SW Tan, MBBS, MRCP, PhDSenior Consultant, Division of Medical OncologyPrincipal Investigator, Cancer Therapeutics Research LaboratoryNational Cancer Centre SingaporeSingapore

Erik Thunnissen, MD, PhDConsultant PathologistVU University Medical centerAmsterdam, The Netherlands

Ming Sound Tsao, MD, FRCPCPathologist, Senior Scientist, and ProfessorM. Qasim Choksi Chair in Lung CancerTranslational ResearchPrincess Margaret Cancer Centre, University Health NetworkDepartment of Laboratory Medicine and PathobiologyUniversity of TorontoToronto, Canada

Walter Weder, MDProfessor and ChairDepartment of Thoracic SurgeryUniversity Hospital ZurichZurich, Switzerland

Jacinta Wiens, PhDScientific Affairs Project ManagerInternational Association for the Study ofLung CancerColorado, U.S.A.

Ignacio I. Wistuba, MDProfessor and ChairAnderson Clinical Faculty Chair for Cancer Treatment and ResearchDepartment of Translational Molecular PathologyThe University of Texas MD Anderson Cancer CenterTexas, U.S.A.

James Chih-Hsin Yang, MD, PhDDistinguished Professor, DirectorGraduate Institute of OncologyCollege of Medicine, National Taiwan UniversityDirector, Department of OncologyNational Taiwan University HospitalTaipei City, Taiwan

Yasushi Yatabe, MD, PhDChiefDepartment of Pathology and MolecularDiagnosticsAichi Cancer CenterNagoya, Japan

Rex Yung, MDPulmonologistGreater Baltimore Medical CenterMaryland, U.S.A.


Abbreviations

The following abbreviations are used in the text without being expanded.

ALK: Anaplastic lymphoma kinase

BRAF: B-raf proto-oncogene

cfDNA: Cell-free DNA

ctDNA: Circulating-tumor DNA

EDTA: Ethylenediaminetetraacetic acid

EGFR: Epidermal growth factor receptor

FISH: Fluorescent in situ hybridization

FDA: Food and Drug Administration

H&E: Hematoxylin and eosin

HER2: Human epidermal growth factor receptor 2

HGF: Hepatocyte growth factor

IHC: Immunohistochemistry

KRAS: Kirsten rate sarcoma viral oncogene homolog

MAPK: Mitogen-activated protein kinase

MET: MET proto-oncogene

NSCLC: Non-small cell lung cancer

PCR: Polymerase chain reaction

PD-L1: Programmed cell death ligand-1

P13KCA: Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α

PTEN: Phosphatase and tensin homolog

RET: RET proto-oncogene

ROS1: c-ros oncogene 1

RTK: Receptor tyrosine kinase

SCLC: Small cell lung cancer

STAT: Signal transducer and activator of transcription

TTF1: Thyroid transcription factor-1

9MANUFACTURERS

Manufacturers

The following manufacturers and their EGFR testing-related products are noted in this Atlas. The location given for each manufacturer is not the only location; most manufacturers have offices worldwide.

Agena BioscienceSan Diego, California, USAMassARRAY systems, OncoCarta Panel v1.0, Lung Carta Panel

Applied BiosystemsFoster City, California, USASNapShot Multiplex Kit

HologicMarlborough, Massachusetts, USACyoLyt Solution

Illumina, Inc.San Diego, California, USAMiSeq Sequencing System, HiSeq Sequencing System

Qiagen N.V.Venlo, the Netherlandstherascreen EGFR RCQ PCR Kit

Roche Molecular Systems, Inc.Pleasanton, California, USAcobas EGFR Mutation Test v2

Streck Omaha, Nebraska, USACell-Free DNA BCT

Thermo Fisher ScientificWaltham, Masschusetts, USAOncomine Dx Target Test, Ion Personal Genome Machine (PGM) (Ion Torrent)

Introduction By Tony S. Mok, David P. Carbone, and Fred R. Hirsch

The discovery of somatic driver mutations in EGFR over a decade ago set the stage for science-based precision medicine in the management of advanced NSCLC. Since then, research has achieved many milestones that have transformed the clinical management of this disease. Starting with the IPASS study, we demonstrated that selection of patients by the presence of EGFR mutations rather than clinical/pathologic characteristics for treat-ment with first-line EGFR TKIs resulted in improved outcomes with less toxicity compared to chemotherapy. We now have more potent second-generation EGFR TKIs that may be superior to the first-generation ones, and have also found that EGFR TKIs may potentially be beneficially combined with other agents, such as bevacizumab. Identification of EGFR exon 20 T790M mutations as a common resistance mechanism, and selectively targeted by the third-generation EGFR TKI osimertinib, defined standard therapy for patients with acquired resistance to EGFR TKIs. All of these developments have helped to establish this new molecular treatment paradigm, making precision medicine a reality for many thousands of patients with lung cancer. Testing for EGFR mutations was the key to this success. From the time when EGFR testing became available at academic centers to general acceptance of EGFR testing as an essential aspect of lung cancer management globally, intense research efforts have been dedicated to the advancement in technology, standardization, validation, and interpretation of this analysis. Now that we are blessed with multiple efficient platforms, we need consensus on the establishment and optimization of EGFR testing. The IASLC Atlas of EGFR Testing in Lung Cancer is a useful guidebook for this purpose. We aim to provide the pathologists with “know-how” information on EGFR testing, and to provide the clinicians with the “know-why” information on how to interpret the results. From the retrieval and handling of tumor sample to the different available assays, and from interpretation of results to reporting and quality assurance, the Atlas is a comprehensive yet user friendly compendium for the general oncology readership. We have also summarized the relevant clinical data supporting the application of EGFR testing in patients with either treatment-naïve or EGFR TKI-resistant disease. It is the vision of IASLC to disseminate these


important educational messages around the world, and I hope you will share our vision to improve the quality of lung cancer care, and thus improve and prolong the lives of our lung cancer patients.

Therapeutic PerspectivesBy Tony S. Mok, Solange Peters, Daniel SW Tan, Luiz Paz-Ares, and Keunchil Park 1Non-small cell lung cancer (NSCLC) is the leading cause of death around the globe, resulting in more than 1 million deaths worldwide annually (GLOBOCAN 2015). The 5-year survival rate for patients with stage IV disease is less than 5% (Siegel 2017). The identification of specific somatic aberrations has led to a personalized therapeutic approach, resulting in substantially improved outcomes in NSCLC. In Europe and North America, 10% to 20% of patients with NSCLC possess an activating EGFR gene mutation (Moran 2009). Incidence of this driver alteration is more prevalent in East Asia, affecting 50% to 60% of patients with NSCLC. Regardless of geographic region, prevalence is higher among never-smokers, women, and the adenocarcinoma subtype (Shi 2014).

EGFR Mutation Incidence, Associated Treatment OutcomesThe EGFR gene is located on the short arm of chromosome 7 (7p11.2) and encodes a 170-kDa type I transmembrane growth factor receptor with tyrosine kinase activity. EGFR belongs to the HER/ErbB family of receptor tyrosine kinases. Intracellular signaling is mediated mainly through the RAS/RAF/MEK/MAPK pathway, the PI3K/PTEN/AKT pathway, and the STAT pathway. Downstream EGFR signaling ultimately leads to increased proliferation, angiogenesis, metastasis, and decreased apoptosis. Gain-of-function or activating muta-tions of EGFR were first identified in 2004, leading to constitutive tyrosine kinase activity (Lynch 2004). Given the magnitude of the benefit of targeted therapy associated with this disease, management of EGFR-mutated NSCLC is a major issue worldwide, particularly in regions with high prevalence. An EGFR mutation status should be systematically analyzed—with sequencing as the criterion standard—for patients who have advanced NSCLC with nonsqua-mous histology (Novello 2016). Activating (and sensitizing) EGFR mutations are predictive for response to EGFR tyrosine kinase inhibitors (TKIs), including first-generation gefitinib and erlotinib, second-generation afatinib and dacomitinib, and the third-generation drug osimertinib. In patients with advanced NSCLC, both first- and second-generation EGFR TKIs are standard first-line treatment options. Such treatments result in improved response and


progression-free survival rates, better tolerability, and superior quality of life compared with platinum-based chemotherapy in the first-line setting, as demonstrated in several randomized trials (Mok 2009; Maemondo 2010; Mitsudomi 2010; Rosell 2012; Sequist 2013; Lee 2014; Wu 2014; Wu 2015; Zhou 2015). Exon 19 LREA frame-deletion mutations and the single-point substitution mutation L858R in exon 21 are the most frequent in NSCLC and are considered to be so-called clas-sic mutations, accounting for 85% to 90% of all EGFR mutations (Mok 2009; da Cunha Santos 2011). Most of the large randomized trials comparing EGFR TKIs with chemotherapy enrolled patients with classic mutations; only four trials included patients with uncommon EGFR mutations. Uncommon mutations comprise approximately 10% of EGFR mutations and are defined as all mutations excluding exon 19 deletion and L858R. The objective response rate to TKIs for patients with classic mutations is approximately 60%. Between 50% and 60% of classic EGFR mutations are exon 19 LREA frame deletions (Lee 2015). Patients with this specific activating mutation have consistently had better out-comes with EGFR TKIs than patients with L858R single-point substitutions (Jackman 2006; Jackman 2009; Riely 2006; Lee 2015). A recent meta-analysis evaluating seven trials (1,649 patients) found that EGFR TKIs significantly prolonged progression-free survival (HR: 0.37; 95% CI: 0.32–0.42) in all subgroups compared with chemotherapy. TKI treatment benefit was 50% greater (HR: 0.24; 95% CI: 0.20–0.29) for patients with exon 19 deletions than for those with exon 21 L858R substitution (HR: 0.48; 95% CI: 0.39–0.58; P = 0.001). Patients with exon 19 deletions had a greater overall survival than those with exon 21 L858R substitution after gefitinib or elotinib therapy. Interestingly, exon 21 L858R substitutions, rather than exon 19 deletions, have been associated with longer overall survival for TKI treatment-naive patients (Shigematsu 2005). In the meta-analysis by Lee et al., chemotherapy resulted in significantly greater progression-free survival for patients with exon 21 L858R substitutions than for patients harboring exon 19 deletions (median progression-free survival, 6.1 vs. 5.1 months; P = 0.003). Within this context, the findings of a pooled analysis focusing on the two randomized trials comparing afatinib and chemotherapy—LUX-Lung 3 and LUX Lung-6 trials—suggested this TKI could improve overall survival compared with chemotherapy among patients with exon 19 deletions but not for patients with L858R substituted disease (Yang 2015). The cause of this difference in response to EGFR TKIs, or possibly chemotherapy, by EGFR mutation subtype is not known. However, in the randomized trial in which afatinib was compared with gefitinib (LUX-Lung 7), the overall survival did not differ significantly in the two treatment arms among patients with EGFR exon 19 deletion (Paz-Ares 2017).

EGFR TKIs Despite the first encouraging report in 1948 by Karnofsky et al. that advanced lung cancer could respond to cytotoxic chemotherapy, progress in controlling advanced or metastatic lung cancer has been slower than expected (Kennedy 1998). Until the early 2000s, the standard of care for patients with advanced NSCLC was a third-generation platinum-based chemotherapy doublet, irrespective of histopathology, for patients with good performance statuses and best supportive care for patients with poor performance statuses. At the dawn of the new millennium, the thoracic oncology community experienced a temporary

15THERAPEUTIC PERSPECTIVES

disappointment following an observation that survival outcomes of modern chemotherapy regimens had reached a therapeutic plateau (Schiller 2002). Fortunately, that was merely the beginning of a completely new treatment paradigm. The initial studies (IDEAL-1 and -2) of gefitinib, the first EGFR TKI, evaluated the agent as second- and third-line treatment for pretreated patients with advanced NSCLC. A once-daily dose of 250 or 500 mg showed meaningful antitumor activity (overall response rate, 9% to 19%; overall survival, 6 to 8 months) and provided symptom relief with mild toxici-ties (Fukuoka 2003; Kris 2003). In two large randomized phase III trials (INTACT-1 and -2), chemotherapy-naive patients with unresectable stage III and IV NSCLC were randomly assigned to receive gefitinib (250 or 500 mg daily) or placebo in combination with platinum-doublet chemotherapy. There was no added benefit for survival, time to progression, or response rate compared with standard chemotherapy alone (Giaccone 2004; Herbst 2004). Based on the early results of these trials, gefitinib received accelerated approval in 2003 by the US FDA as monotherapy treatment for patients with locally advanced or metastatic NSCLC after failure of both platinum- and docetaxel-based chemotherapies. Erlotinib, another reversible EGFR TKI, was approved by the FDA in 2004 as a single-agent treatment for patients with locally advanced or metastatic NSCLC who had disease progression after other treatments, including at least one prior chemotherapy regimen. Approval was based on the early results of a randomized placebo-controlled double-blind trial that demonstrated improved overall survival for patients treated with erlotinib (6.7 months) compared with placebo (4.7 months; HR: 0.70; p < 0.001) (Shepherd 2005).

Response Prediction of EGFR TKIsIn the IDEAL-1 study, the response rate was greater for Japanese patients than for non-Japanese patients (27.5% vs. 10.4%; odds ratio = 3.27; P = 0.0023), which suggested that potential ethnic and/or genetic factors may be involved in gefitinib response (Fukuoka 2003). Investigators soon discovered that a mutation in the EGFR kinase domain is strongly associated with response to gefitinib (Lynch 2004; Paez 2004), as discussed previously. The introduction of EGFR TKIs in the management of advanced EGFR-mutated NSCLC has opened the door to precision medicine and paved the way for studies of other solid tumors, such as ALK-rearranged and ROS-1-rearranged NSCLC. Several randomized phase III trials comparing EGFR TKIs with the standard platinum-based chemotherapy doublet in chemotherapy-naive patients with EGFR-mutated NSCLC have been conducted following encouraging results from trials on EGFR TKIs for patients with advanced NSCLC whose disease did not respond cytotoxic chemotherapies. EGFR TKIs demonstrated superior outcomes compared with standard chemotherapy in terms of objective response rate (60% to 80% vs. 25% to 35%, respectively) and progression-free survival (10 to 14 months vs. 5 to 6 months, respectively) with better tolerability (Haaland 2014). These results formed the basis of approval of first-generation EGFR TKIs, such as gefitinib and erlotinib, and second-generation agents, such as afatinib and this drug class is the current standard of care for the first-line management of EGFR-mutated NSCLC. One strategy to further improve first-line management of EGFR-mutated NSCLC includes the addition of an antiangiogenic agent to an EGFR TKI. A phase II Japanese trial demon-strated superior progression-free survival with erlotinib and bevacizumab (16.0 months)


compared with erlotinib alone (9.7 months) (Seto 2014). Confirmatory trials are ongoing in Japan. As was previously described, afatinib was approved for first-line treatment of advanced EGFR-mutated NSCLC, and a prospective head-to-head randomized phase IIb compara-tive study in the first-line setting demonstrated better progression-free survival, time to treatment failure, and overall response rate compared with gefitinib, which suggests that a second-generation EGFR TKI is not interchangeable with a first-generation agent (Park 2016). A prospective randomized phase III trial of dacomitinib also demonstrated superior progression-free survival compared with the first-generation EGFR TKIs (median 14.7 vs 9.2 months) (Mok 2017).

EGFR TKI Resistance Almost all patients who had a response to first- or second-generation EGFR TKIs eventually had resistance to these drugs. The most common mechanism of resistance, accounting for approximately 50% of all cases, is the acquired T790M mutation in exon 20 (Kobayashi 2005; Yun 2008; Yu 2013). Upon occurrence of T790M mutation at exon 20 of the EGFR gene, the quinazoline-based first-generation EGFR TKI can no longer bind to the ATP binding pocket at the receptor, thus losing efficacy on inhibition of downstream signalling. The biology and epidemiology of the T790M resistance mutation has been well studied. The incidence of this mutation is similar among ethnicities and among different EGFR TKIs. Preclinical studies have shown that the more potent EGFR inhibition with second-generation EGFR TKIs, such as afatinib, may overcome the T790M mutation-associated acquired resistance, but severe toxicities associated with inhibition of normal EGFR receptor at this dose level would be prohibitive for clinical use. Afatinib was given to patients with EGFR-mutated NSCLC that was nonresponsive to prior treatment with gefitinib and/or erlotinib in the LUX-Lung 1 and LUX-Lung 4 trials (Miller 2012, Katakami 2013). The overall response rate was 7% to 8%, with a median progression-free survival of 3 to 4 months, suggesting that second-generation EGFR TKIs cannot overcome T790M-associated acquired resistance. Other mechanisms of resistance are relatively heterogeneous, and these may include HER2 and/or MET amplifi-cation, PIK3CA and/or BRAF mutation, and small cell lung cancer transformation (Stewart 2015). The incidence of each of these mechanisms of resistance is much lower than that of the T790M mutation, and only rarely do these mutations or amplifications occur concur-rently with T790M mutation. Third-generation EGFR TKIs were designed to target the T790M mutation while sparing wild-type EGFR, as first reported by Zhou et al (Zhou 2009). These authors studied three closely related pyrimidines—namely WZ3146, WZ4002, and WZ8040—and found WZ4002 to be most potent against EGFR T790M. In an in vivo study of WZ4002, tumor regressions were significant compared with vehicle alone in both EGFR L858R/T790M and deletion E746_A750/T790M-containing murine models. However, the compound was never devel-oped into a commercial drug. The first and only commercially available third-generation EGFR TKI is osimertinib. The FDA granted regular approval on March 30, 2017, to osimertinib for the treatment of patients with metastatic EGFR T790M mutation-positive NSCLC, as detected by an FDA-approved test, and disease progression during or after EGFR TKI therapy. Molecular structure is

17THERAPEUTIC PERSPECTIVES

distinct from WZ4002 but the background concept of a pyrimidine-based structure and the idea of the electrophilic functionality residing in different regions of the chemical structure were similar (Cross 2014). The authors of a preclinical study reported the IC

50 of osimertinib

against cell line with L858R/T790M at 1 nM against L858R/T790M, which is approximately 200 times greater potency than wild-type EGFR. Jänne et al. reported results from the first phase I/II multicenter study (253 patients). Five dose cohorts (20, 40, 80, 160, and 240 mg daily), were studied in patients with EGFR mutation-positive disease who had prior benefit from first- or second-generation EGFR TKIs but subsequently experienced dis-ease progression. Both patients with or without T790M resistance were included. Focusing on the T790M-positive subgroup, the overall response rate was 61% and progression-free survival was 9.6 months (Jänne 2015). The finding was supported by a single-arm phase II study in which a daily dose of 80 mg of osimertinib was evaluated in 210 patients with T790M-positive disease (Yang 2017). The response rate was 64% (95% CI: 57%-71%) and the median progression-free survival was 8.6 months. Toxicities were well tolerated and included diarrhea (47%), rash (40%), nausea (22%), and decreased appetite (21%). Grade 3 or higher toxicities were uncommon. AURA 3 is the first randomized phase III study to compare osimertinib with standard platinum-based doublet chemotherapy in patients with T790M-positive lung cancer (Mok 2017). To be eligible, patients must have had treatment with an EGFR TKI that was unsuc-cessful according to RECIST criteria and proven with the presence of T790M mutation confirmed by repeat biopsy. Tumor response rates were 71% and 31% for the osimertinib and chemotherapy groups, respectively. Progression-free survival was significantly longer for the osimertinib group (median, 10.1 vs. 4.4 months; HR: 0.30; 95% CI: 0.23–0.41, P = 0.0001) The overall toxicity profile of osimertinib is also more favorable than chemotherapy. The study has defined osimertinib to be the standard of care for T790M-positive acquired resistance; therefore, patients who experience treatment failure of a first-line EGFR TKI should be tested for the presence of a T790M mutation. However, management of T790M-negative resistance continues to be controversial. Chemotherapy is the current standard, and ongoing research is investigating the role of immunotherapy in this population. Although third-generation EGFR TKIs have demonstrated impressive efficacy, develop-ment of acquired resistance is inevitable. Little is known about the mechanisms of acquired resistance to these agents, and active investigation is ongoing.

EGFR TestingBy Ming Sound Tsao, Ignacio I. Wistuba, and Yasushi Yatabe 2In 2013, the College of American Pathologists (CAP), International Association for the Study of Lung Cancer (IASLC), and the Association for Molecular Pathology (AMP) published the molecular testing guideline in lung cancer, which included information about testing of EGFR gene mutations (Lindeman 2013). The guideline, which was subsequently endorsed by ASCO (Leighl 2014), has served as an international standard for EGFR mutation testing. The guideline update, authored by experts from each of the three organizations, is expected to be published by the end of 2017. For this update, a systematic review was conducted of all applicable data since the initial publication. The update includes recommendations on a range of additional molecular tests in lung cancer that are used either routinely or to identify patients who might benefit from novel therapies in clinical trials. Several updated recom-mendations are related to EGFR testing (Box 1). The recommendations offered in the 2013 guideline largely have been reaffirmed, except for the following points (Lindeman 2017). • Any cytology sample with adequate cellularity and preservation may be tested. The develop-

ment of sensitive molecular testing techniques allows cytology specimens to be used in clinical practice, and the findings of the systematic review demonstrated excellent perfor-mance of smear preparations, if the tumor cells were cytologically confirmed in samples (Roy-Chowdhuri 2016, Rekhtman 2016, Malapelle 2017a). In patients with advanced lung cancer, which represents two-thirds of the lung cancer population, cytology specimens often are the only samples available for molecular testing to guide treatment decisions.

• Analytic methods must be able to detect mutations in a sample with 20% or more malignant cell content. In the 2013 guideline, Sanger sequencing was still one of the recommended testing methods, but evidence for the clinical utility of more sensitive PCR-based tech-niques has been established in subsequent studies. Along with the widespread availability of these sensitive techniques, Sanger sequencing is not considered suitable for clinical use, particularly for biopsy specimen testing.


• IHC is not appropriate for EGFR mutation testing. The 2013 guideline indicated that EGFR IHC testing does not have a role in clinical practice, but IHC tests with mutation-specific EGFR antibodies are allowed when the sample is extremely limited. The findings of sub-sequent studies have shown that these antibodies have poor sensitivity for many exon 19 deletions, insensitivity for less-common mutations (eg, codon 719 mutations), and false-positive results with exon 20 insertions (Kitamura 2010). With the availability of very sensitive detection techniques and circulating cfDNA, EGFR mutation-specific IHC was considered to have no role in routine clinical practice.

In its endorsement of the CAP/IASCL/AMP guideline, ASCO suggested routine molecular testing of early-stage disease, similar to the testing paradigm for patients with breast cancer (Leighl 2014). The updated guideline also addresses this point, encouraging such testing but at the discretion of the individual institution and local oncology team. The updated guideline is more focused than the original version on testing of other genes, including ROS1, BRAF, RET, HER2, RAS, and MET. Furthermore, testing for second-ary mutations that cause therapeutic resistance, multigene panel testing, and liquid biopsy application also are described. The updated guideline also includes the following recom-mendations related to EGFR testing.

T790M. The results of the AURA trials have demonstrated improved outcome using osimertinib after the development of resistance to first- and second-generation EGFR tyrosine kinase inhibitors (TKIs), and the findings of a systematic review support the increased therapeutic benefit (Janne 2015, Goss 2016, Mok 2017, Yang 2017, Lindemann 2017). (See Chapter 1.) The updated guideline recommends EGFR T790M testing to select patient candidates for treatment with a third-generation EGFR TKI. The results of some studies have shown that a small proportion of T790-mutated tumor cells that existed in a bulk tumor-cell population caused the resistance (Inukai 2006, Engleman 2006, Chmielecki 2011, Su 2012, Chen 2016). In clinical samples, the number of T790M-mutated tumor cells is almost always less than the number of cells that bear only the sensitizing EGFR mutations (Engelman 2006, Chmielecki 2011). Therefore, the sensitiv-ity to detect the EGFR T790M resistance mutation was set to detect as low as 5% of mutant allele. A commercial real-time PCR allele-specific assay, droplet digital PCR, and next-generation sequencing are currently the preferred methods to meet the requirement.

Liquid biopsy. There is much enthusiasm regarding the development of noninvasive methods for EGFR mutation testing. For example, so-called liquid biopsy, in which blood is used as the test sample, has been evaluated in several studies (Oxnard 2014, Qiu 2015, Mok 2015). (See Chapter 3.) A systematic review of this new diagnostic modality demonstrated insufficient evidence for use as a molecular test that involves circulating tumor cells. However, testing of cfDNA provided useful information as an alterna-tive molecular test when tissue is limited and/or insufficient for regular tissue-based molecular testing. Because of the high specificity (95.6%; 95% CI: 83.3%–99.0%) but only intermediate sensitivity (66.4%; 95% CI: 62.7–69.9%) of cfDNA testing, a negative result does not represent tumor negativity for EGFR mutation (Lindemann 2017).

21EGFR TESTING

Patient Selection for EGFR Mutation Testing1.1a: Recommendation: EGFR molecular testing should be used to select patients for EGFR- targeted

tyrosine kinase inhibitor (TKI) therapy; patients with lung adenocarcinoma should not be excluded from testing based on clinical characteristics.

1.2: Recommendation: In the setting of lung cancer resection specimens, EGFR testing is recommended for adenocarcinomas and mixed lung cancers with an adenocarcinoma component, regardless of his-tologic grade. In the setting of full excised lung cancer specimens, EGFR testing is not recommended in lung cancers that lack any adenocarcinoma component, such as pure squamous cell carcinomas and pure small cell carcinomas.

1.3: Recommendation: In the setting of more limited lung cancer specimens (i.e., biopsies or cytology) where an adenocarcinoma component cannot be completely excluded, EGFR testing may be per-formed in cases showing squamous or small cell histology but clinical criteria (e.g., young age and lack of smoking history) may be useful in selecting a subset of these samples for testing.

1.4: Recommendation: To determine EGFR status for initial treatment selection, primary tumors or meta-static lesions are equally suitable for testing.

1.5: Expert consensus opinion: In patients with multiple, apparently separate, primary lung adenocarci-nomas, each tumor may be tested, but testing of multiple different areas within a single tumor is not necessary.

Specimen Testing for EGFR Mutation 2.1a: Recommendation: EGFR mutation testing should be ordered at the time of diagnosis for patients

presenting with advanced-stage disease—stage IV according to the 7th edition Tumor Node Metastasis (TNM) staging system—that meets the therapeutic requirements, or at time of recurrence or progres-sion in patients who originally presented with lower-stage disease but were not previously tested.

2.2a: Expert consensus opinion: EGFR testing of tumors at diagnosis from patients presenting with stage I, II, or III disease is encouraged, but the decision to do so should be made locally by each laboratory in collaboration with the oncology team.

2.3: Recommendation: Tissue should be prioritized for EGFR, ALK, and ROS1 testing.

Turnaround Times for Test Results3.1: Expert consensus opinion: EGFR results should be available within 2 weeks (10 working days) of

receiving the specimen in the testing laboratory. 3.2: Expert consensus opinion: Laboratories with average turnaround times beyond 2 weeks must make a

more rapid test available—either in-house or through a reference laboratory—in instances of clinical urgency.

3.3: Expert consensus opinion: Laboratory departments should establish processes to ensure that speci-mens that have final histopathologic diagnosis are sent to outside molecular pathology laboratories within 3 working days of receiving requests and to intramural molecular pathology laboratories within 24 hours.

Specimen Processing 4.1: Expert consensus opinion: Pathologists should use formalin-fixed, paraffin-embedded specimens or

fresh, frozen, or alcohol-fixed specimens for polymerase chain reaction (PCR)-based EGFR mutation tests. Other tissue treatments (e.g., acidic or heavy-metal fixatives, or decalcifying solutions) should be avoided in specimens destined for EGFR testing.

4.2: Expert consensus opinion: Cytologic samples are also suitable for EGFR testing, with cell block, cell pellets and smears.

Box 1. Recommendations for EGFR Testing in the Updated College of American Pathologists (CAP), International Association for the Study of Lung Cancer (IASLC), and the Association for Molecular Pathology (AMP) Molecular Testing Guideline

continued on next page


Specimen Requirements 5.1: Expert consensus opinion: Pathologists should determine the adequacy of specimens for EGFR test-

ing by assessing cancer cell content and DNA quantity and quality. 5.2: Expert consensus opinion: Each laboratory should establish the minimum proportion and number

of cancer cells needed for mutation detection during validation. 5.3: Expert consensus opinion: A pathologist should assess the tumor content of each specimen and

either perform or guide a trained technologist to perform microdissection for tumor cell enrichment, when needed.

Performing EGFR Testing6.1: Recommendation: Laboratories may use any validated EGFR testing method with sufficient

performance characteristics. 6.2: Expert consensus opinion: Laboratories should use EGFR test methods that are able to detect

mutations in specimens with at least 20 % cancer cell content.6.3: Expert consensus opinion: Clinical EGFR mutation testing should be able to detect all individual muta-

tions that have been reported with a frequency of at least 1% of EGFR-mutated lung adenocarcinomas. 6.4: Recommendation: Immunohistochemistry (IHC) for total EGFR IHC and EGFR-mutation specific IHC

is not recommended for selection of EGFR TKI therapy.6.5: Recommendation: EGFR copy number analysis (i.e., fluorescence in situ hybridization [FISH] or chro-

mogenic in situ hybridization [CISH]) is not recommended for selection of EGFR TKI therapy.

New RecommendationsKey Question IV: What testing is indicated for patients with targetable mutations who have relapsed on targeted therapy? IVA. EGFR T790M

Strong Recommendation: In patients with lung adenocarcinoma who harbor sensitizing EGFR muta-tions and whose disease has progressed after treatment with an EGFR-targeted TKI, physicians must use EGFR T790M mutational testing when selecting patients for third-generation EGFR-targeted therapy. Recommendation: Laboratories testing for EGFR T790M mutation in patients with secondary clinical resistance to EGFR-targeted TKIs should deploy assays capable of detecting EGFR T790M mutations in as few as 5% of mutant alleles.

Key Question V: What is the role of testing for circulating, cell-free DNA (cfDNA), for patients with lung cancer Va. No Recommendation: There is currently insufficient evidence to support the use of cfDNA molecular

methods for the diagnosis of primary lung adenocarcinoma. Vb. Recommendation: In some clinical settings in which tissue is limited and/or insufficient for molecular

testing, physicians may use a cfDNA assay to identify EGFR mutations. Vc. Expert Consensus Opinion: Physicians may use cfDNA methods to identify EGFR T790M mutations

in patients with lung adenocarcinoma with disease progression or secondary clinical resistance to EGFR-targeted tyrosine kinase inhibitors; testing of the tumor sample is recommended if the plasma result is negative.

Vd. No Recommendation: There is currently insufficient evidence to support the use of circulating tumor cell (CTC) molecular analysis for the diagnosis of primary lung adenocarcinoma, the identification of EGFR or other mutations, or the identification of EGFR T790M mutations at the time of EGFR TKI resistance.

Patient Candidates for Testing In early studies, the incidence of EGFR kinase domain mutations was more prevalent in the East Asian population, never-smokers, women, and the adenocarcinoma subtype, as sum-marized in Tables 6 and 9 in the 2013 guideline (Lindeman 2013). The guideline includes recommendations for patient selection for EGFR testing (Box 1).

23EGFR TESTING

Although the number of studies on squamous cell carcinoma testing is limited, the updated CAP/IASLC/AMP guideline addresses the molecular tests for lung cancers that do not have an adenocarcinoma component. The guideline states that molecular testing is rec-ommended for tumors with a histology other than adenocarcinoma when clinical features indicate a higher probability of the presence of an oncogenic driver. Major clinical features include young age and the absence of tobacco exposure. Ample data demonstrate associa-tion of EGFR, ALK, and ROS1 alterations with no or minimal tobacco exposure. Although it is generally recognized that EGFR mutations rarely occur in patients with squamous cell carcinoma, the authors of some studies (especially from East Asian countries) have reported the presence of EGFR mutations in 3% to 13% of patients with this type of lung cancer. However, the prevalence of nonsmokers is higher among these patients than among patients without EGFR mutations (Hata 2013, Dearden 2013). The findings suggest that light or absent tobacco exposure is a sufficient rationale to prompt testing, regardless of sample types or nonadenocarcinoma morphology. However, the response rates and survival benefit associated with EGFR TKI therapies among these patients appear to be lower than among patients with adenocarcinoma (Hata 2013, Xu J 2016, Liu 2017, Joshi 2017). In terms of patient age, the findings of the systematic review for the 2013 CAP/IASLC/AMP guideline showed that the mean age of patients with EGFR-mutated tumors was signifi-cantly less than that for patients without EGFR-mutated tumors (Lindeman 2013). However, prevalence of younger age is more frequently reported for patients with ALK and ROS1 alterations. The updated guideline recommends testing for all patients younger than 50 years who have nonadenocarcinoma histology. It is important to emphasize that in the current practice of lung cancer pathology, routine performance of histologic markers (TTF-1/mucin and P40/P63) on poorly differentiated NSCLC specimens is mandatory, both in the context of more accurate histologic classi-fication of the tumors and especially in the context of the CAP/IASLC/AMP guideline (Loo 2010, Mukhopadhyay 2011, Travis 2011, Warth 2012, Rekhtman 2012, Rekhtman 2013).

Timing of EGFR Mutation Testing Primary DiagnosisKnowledge of EGFR mutation status is crucial to therapeutic decision-making for patients with advanced nonsquamous cell NSCLC given that EGFR TKIs are used as first-line treat-ment of patients with EGFR-sensitizing mutations. Such knowledge is also essential when treating patients with recurrence after surgical resection of earlier-stage disease. This prin-ciple was articulated in the 2013 CAP/IASLC/AMP guideline and is reaffirmed in the update. Because lung cancer is at an advanced stage at the time of diagnosis in two-thirds of patients, only small biopsy or cytology specimens are available for testing in most patients. Except for bone specimens that have been fixed in acidic solutions (eg, Bouin or bone-decalcify-ing solutions) or heavy-metal fixatives (eg, Zenker’s, B5, or B-Plus fixative, or acid zinc formalin), all types of small biopsy or cytology specimens are suitable for mutation testing; this has been confirmed in reports from various laboratories around the world (Table 1) (Shiau 2014, Shi 2014, Vigliar 2015, Yatabe 2015). The main issue in the timing of EGFR testing is whether testing should be ordered by the treating physician (known as bespoke testing) or by the pathologists responsible


for the primary diagnosis (reflex testing). Bespoke testing remains the cus-tomary practice in many parts of the world because it may avoid the cost of testing in patients with early-stage disease. In contrast, bespoke testing may result in a substan-tial delay in the initiation of first-line treatment for patients with advanced dis-ease, potentially negatively affecting the outcome. Lim et al compared bespoke and reflex EGFR testing and found the median time from consultation to treatment decision was sig-nificantly shorter (0 vs. 22 days, P = 0.0008) for reflex testing, as was the time to treatment start (16 vs. 29 days, P = 0.004) (Lim 2015). Similarly, Cheema et al. compared 74 patients who had reflex EGFR testing with 232 patients who had routine bespoke testing (Cheema 2016). Reflex testing was associated with a shorter median time from first medical oncology visit to treatment (26 vs. 36 days, P = 0.071) and time to optimal first-line systemic therapy (24 vs. 36 days, P = 0.036), as well as greater quality of biomarker testing, as indicated by the percentage of unsuccessful tests (4% vs. 14%, P = 0.039). A substantial pitfall in reflex testing is the common lack of knowledge of disease stage at the time of initial biopsy examination by the pathologist. It is expected that 30% to 40% of patients have early-stage disease and may be treated by surgical resection. Awareness of EGFR mutation status is not crucial for primary treatment because there is no adjuvant role for EGFR TKIs; therefore, EGFR testing is not required. The 2013 CAP/IASLC/AMP guideline stated that “EGFR testing of tumors at diagnosis from patients presenting with stage I, II, or III disease is encouraged but the decision to do so should be made locally by each laboratory, in collaboration with its oncology team.” (Lindeman 2013). Analogous to the use of adenocarcinoma histology to limit EGFR testing, whether testing should be done in patients with early-stage disease is largely an economic issue because these patients with EGFR mutations may be cured by surgery alone and do not require EGFR TKI therapy. However, metastatic recurrence is expected to develop in 40% to 60% of patients with early-stage disease, which requires treatment similar to that for advanced disease. Overall,

Table 1. Published Data on Sample Types Used in EGFR Testing Around the World

Sample Available for Testing (%)

Study Source of Data Surgical Biopsy Cytology

Yatabe 2015 China 46.2 45.8 8

Hong Kong 12.6 32.5 52.9

Indonesia 0 2 98

Japan 25.2 24.8 50

South Korea 23.9 70.4 5.7

Philippines 34.7 32.3 33.0

Singapore 21.0 69.8 9.2

Taiwan 16.7 74.8 8.5

Thailand 26.0 70.4 3.6

Vietnam 33.3(annotated as biopsy)

66.7

Shiau 2014 Ontario, Canada 39.1 38.5 22.4

Sholl 2015 United States, multi-institutional triala

47.2 35.8 17.0

Shi 2014 Multiple countries in Asiab (PIONEER trial)

88.6 (annotated as biopsy)

11.4

Hlinkova 2013

Eastern Europe, single institution

0.5 13.6 85.9

aLung Cancer Mutation Consortium.bPIONEER trial.

25EGFR TESTING

15% of patients with nonsquamous NSCLC may not need testing at all. Reflex testing on tumors of all stages may streamline pathology workload, mitigate turnaround time for test-ing, minimize testing failure rates when testing small samples, and fast-track repeat biopsy when it is required.

Disease Progression and MonitoringEGFR TKIs such as gefitinib, erlotinib, and afatinib are the standard of treatment for patients with EGFR-mutated tumors because of the high response rates (55% to 78%) and substantially longer progression-free survival rates for these patients (Mok 2011). However, resistance and relapse develop within a short time in most patients, caused by the T790M mutation in exon 20 of the EGFR kinase domain (approximately 50% of cases), amplification of the MET oncogene, or mutations of the PI3KCA gene. Other changes, including SCLC transformation, also have been also described in approximately 4% of cases. (PMID: 21862980) Osimertinib is the first FDA-approved TKI for patients with EGFR T790M-mutated NSCLC who had disease progression during treatment with an EGFR TKI (Goss 2016, Mok 2017). These data high-light the importance of repeat biopsy to identify the underlying mechanisms of resistance to EGFR TKIs, particularly the actionable EGFR T790M mutation, MET amplification, and SCLC transformation. A new EGFR acquired-resistance mutation (C797S) has been noted during treatment with osimertinib (Yu HA 2015); no specific effective treatment, however, has been developed for this new mutation. The isolation of cfDNA in blood samples from patients with lung cancer has provided an alternative source of tumor material that can be used as a biopsy surrogate for EGFR T790M mutation testing (Bordi 2015, Oxnard 2016, Sacher 2016). cfDNA genotyping testing approaches allow serial assessments of repeat blood samples from patients with lung cancer, which may be especially important when monitoring for acquisition of second-site EGFR mutation in patients with NSCLC who have disease progression during treatment with an EGFR TKI (Oxnard 2016). Eligibility for osimertinib therapy is determined by the presence of an EGFR T790M mutation. Currently, the companion diagnostic for this drug is approved by the FDA for both tissue biopsy and plasma cfDNA testing. Therefore, liquid biopsy provides an attractive alternative to tissue biopsy and could help widen the selection of eligible patients with the acquired T790M mutation. The results of a retrospective analysis showed that, for patients with tumors that were positive for T790M mutations on plasma testing, outcomes with osimertinib were equivalent to those for patients who had positive results on a tissue-based assay (Oxnard 2016). These findings suggest that, with the availability of validated plasma T790M assays, a tumor biopsy for T790M genotyping may be avoided in some patients. However, because of the 30% rate of false-negative results for plasma genotyping reported in the study, a tumor biopsy is required to determine the presence of a T790M mutation when the results of a plasma assay are negative.

Conclusion The 2013 CAP/IASLC/AMP guideline has served as an international guideline for molecu-lar testing in lung cancer. The anticipated update of this guideline generally reaffirms the recommendations made in the original guideline, with a few additional recommendations


based on the findings of systemic reviews conducted since the initial publication. These new recommendations address such topics as testing of cytology samples, testing methods, testing by IHC, testing for T790M acquired-resistance mutations, and testing of cfDNA in blood. Although EGFR testing is recommended mainly for patients with adenocarcinoma or for patients in whom adenocarcinoma cannot be ruled out, testing is also recommended for tumors with nonadenocarcinoma histologies when clinical features indicate a higher probability for the presence of an oncogenic driver.

Sample Acquisition, Processing, and General Diagnostic ProceduresBy Philip Mack, Rex Yung, Walter Weder, and Dara L. Aisner 3The standard principles for lung cancer diagnosis apply when acquiring sufficient tissue for definitive diagnosis and characterization of lung cancer at the highest stage and with minimal risk to the patient. Adequate characterization is defined as the ability to distinguish cell type by IHC because the diagnosis of NSCLC-not otherwise specified is unsatisfactory in the era of cell type-specific cytotoxic therapy, limiting direction to perform follow-up testing for targeted therapy-associated actionable biomarkers. Surgical biopsies, including excision biopsy of cutaneously accessible metastatic lymph nodes, mediastinoscopy, or video-assisted thoracoscopic surgery (VATS) will provide the greatest amount of tissue for biomarker analysis. The majority of lung cancer cases may continue to be diagnosed at later stages (ie, multistation IIB, IIIA/B, and IV) until advances in early detection occur and screening is routinely established; less-invasive procedures that combine diagnosis and staging are preferable. The only noninvasive approach to tissue acquisition and examination involves spontane-ously expectorated or induced sputum. The general diagnostic yield from sputum is in the range of 5% to 20%, depending on the location of tumor, technician familiarity with process-ing techniques, and cytologic diagnosis of lung cancer from sputum. Studies on EGFR and other biomarker testing in sputum are limited. The only controlled paired study comparing sputum testing with EGFR-proved primary tumor demonstrated sensitivity of 30% to 50% (Hubers 2013). As with the other minimally invasive techniques discussed further in this chapter, harvesting of sufficient tissue amounts for multiple biomarker analyses is an even greater challenge with sputum. Pleural effusion with positive results of cytologic testing will provide a stage IVa diagno-sis; however, sensitivity of pleural cytology for pleural metastases is between 40% and 70%. In addition, previous reports of EGFR testing on pleural samples showed that results were dependent on the number of viable cells recovered in a cell block (Kimura 2006). Testing by next-generation sequencing (Buttitta 2013) or cfDNA (Park 2017), which is explained in greater detail in this chapter, may be more promising. Pleural extension and metastases is most effectively diagnosed by VATS or medical thoracoscopy, the latter of which allows


for simultaneous treatment of surgical and chemical pleurodesis by insertion of a long-term drainage catheter, which also improves lung re-expansion. Bronchoalveolar lavage is the least invasive of the bronchoscopy approaches and requires the least specialized training to perform. This technique, however, also provides the lowest diagnostic yield for lung cancer (Xin 2016), although incorporation of next-gener-ation sequencing and cfDNA testing can also improve detection rates for EGFR mutations (Buttitta 2012; Park 2017). Nevertheless, a bronchoscopy performed specifically for cancer diagnosis should be planned with procedures that maximize tissue acquisition for predic-tive biomarker testing. Comprehensive sampling of multiple accessible targets should be considered (provided it does not compromise patient safety) because it is often unclear whether enlarged lymph nodes are malignant or only reactive (Zhang 2012; Hyo 2016). Aside from transbronchial brushings, endobronchial and transbronchial forceps biopsies, and cryobiopsies, the most versatile biopsy technique may be transbronchial needle aspiration (TBNA) because it can be used to sample lymph nodes, as well as endobronchial lesions and parenchymal masses. Although conventional TBNA-acquired tissue can be tested for EGFR mutations (Horiike 2007), the advent of the endobronchial ultrasound (EBUS)-adapted bron-choscope (Nakajima 2007) is a definite improvement because this tool provides real-time image guidance, allowing for accurate sampling of lymph nodes (Navani 2012). However, mediastinoscopy remains the criterion standard when these less-invasive methods fail to provide an accurate diagnosis. There are different methods of processing tissue collected using EBUS-TBNA (outside of typical sampling of lymph nodes and other masses) to enhance diagnostic yield for cancer-cell typing and for molecular testing (Yung 2012; van der Heijden 2014). These methods should be standardized for a given institution. Given the improvements in biomarker testing regarding use of smaller quantities of tissue, the choice for sample acquisition should be customized for the local institution according to the available experience and expertise of the radiologists, interventional pulmonologists, gastroenterologists, and surgeons. Ideally, cases are discussed at a multidisciplinary tumor board, and decisions regarding biomarker testing should be predetermined for the patient if biomarker reflex testing is not a part of the pathologist’s standard operating procedure.

Quality, Amount, and Location of a Tissue Biopsy Numerous factors regarding the quantity and quality of tumor tissue for molecular analysis should be considered. Key among these factors is the quantity of tumor available, either as formalin-fixed paraffin embedded (FFPE) tissue or, in some cases, preserved in the format of cytopathology materials (eg, smears and touch preps). As most assay approaches to evaluating EGFR mutations involve PCR-based strategies, testing can be performed on relatively small quantities of tissue, even on exceptionally scant samples. Standard minimally invasive approaches that are often performed in an interventional radiology setting, such as transbronchial biopsy, EBUS-fine-needle aspiration, and core biopsies, may yield sufficient tissue for molecular analysis. However, balancing the needs for adequate primary diagnosis and for subsequent molecular and ancillary studies requires careful consideration of tissue management. Specialized strategies can be used to optimize tissue handling and manage-ment; however, these strategies are often time- and labor-intensive (Aisner 2016). Measures

29SAMPLE ACQUISITION, PROCESSING, AND GENERAL DIAGNOSTIC PROCEDURES

to ensure preservation of tissue from initial diagnostic procedures should be maximized to reduce the need for repeat biopsy. When considering the quantity of tissue collected, practicing pathologists should know the minimum quantity needed by the laboratory performing the molecular biomarker test-ing, including for EGFR mutations, to best identify and preserve samples that are adequate for subsequent analysis. However, when it is uncertain whether the size of a sample is suf-ficient, the sample should be referred to the molecular laboratory for evaluation because testing may sometimes be possible on samples that are initially perceived to be insufficient. The possibility of testing also largely depends on the capacity of the testing laboratory to individually tailor the tumor enrichment and extraction in difficult cases. Body fluids are often used for molecular testing. For example, pleural fluid is often a sample type used for EGFR mutation analysis. The ability to use a pleural fluid sample is often highly dependent on a combination of three factors: the tumor cellularity of the speci-men (pleural fluids often contain a high proportion of nontumor cells); the ability of the laboratory to enrich for tumor, which can be exceptionally challenging or impossible with pleural fluid samples; and the analytic sensitivity of the platform used for molecular testing. For example, a pleural fluid with a high degree of nontumor cells in which enrichment is not feasible would not be appropriately tested with a low analytic-sensitivity methodology, such as Sanger sequencing. A common question posed to a molecular laboratory is “What is the absolute minimum number of tumor cells needed for testing?” Few data are available regarding a minimum number of cells, and the answer is likely to depend on variables at each testing laboratory. In addition, the number of cells visible on a single section stained with H&E may not be representative of what remains in a FFPE block. Furthermore, the ability to test exception-ally scant specimens may be largely dependent on the quality of the nucleic acids extracted from the specimen, a feature that cannot be determined by histopathologic examination. The quality of nucleic acids derived from solid tumor samples depends on postprocedural specimen handling. Key factors relate to duration of formalin exposure, histopathologic processing parameters, exposure to other agents (specifically decalcifying agents or acids), and elapsed time in paraffin. Although optimal formalin exposure time has not been estab-lished for lung cancer samples, this time has been determined in other clinical settings, most notably breast cancer. Exposure of fewer than 6 hours and more than 72 hours has been associated with increased error rates in ancillary testing in this setting. On the basis of this information, it is suggested that lung cancer samples not be subjected to more than 72 hours of formalin exposure, including histopathologic processing time. When possible, formalin exposure should be limited to fewer than 48 hours. Other factors that can be mitigated include use of only neutral buffered formalin (because unbuffered formalin will render samples unsuitable for molecular testing), avoidance of decalcification treatments when bone metastases are sampled (Aisner 2016), and avoidance of other acid-based solutions, such as Bouin’s fixative. Slides that are cut from a paraffin block and left for extended peri-ods of time may undergo oxidation, resulting in reduced-quality nucleic acids. To the extent possible, tissue should be preserved in paraffin blocks rather than on archived unstained slides.


The quality of nucleic acids derived from non-FFPE cytopathology preparations are often superior to FFPE-derived specimens, owing to lack of exposure to formalin. Ethanol- and methanol-based fixatives used in most cytopathology specimens are generally less damag-ing to nucleic acids; however, there are insufficient data regarding additional variables that may affect the nucleic acid quality, such as coverslip mounting media or method of coverslip removal.

Best Practices for Sample CollectionLimited data exist at this time to suggest evidence-based best practices for sample collec-tion. For tissue samples intended for FFPE, immediate deposition into formalin can reduce postprocedural degradation that may occur if a sample is kept in saline or other media or remains dry until receipt in the pathology laboratory. Numerous studies have demonstrated that rapid onsite evaluation can improve diagnostic yield, as well as procurement of addi-tional tissue for molecular and ancillary studies when fine-needle aspiration is performed. Cytopathologists who are trained in the use of additional tissue for molecular and ancillary studies can also effectively triage samples into the appropriate preparations (eg, smears, spins, and cell blocks) to maximize the likelihood of successful testing. In 2016, the College of American Pathologists convened a multidisciplinary guideline group to address the outstanding questions related to specimen acquisition and handling. These highly anticipated guidelines are expected to be of significant assistance in identify-ing best practices in this rapidly evolving field.

Circulating Tumor DNA from Plasma In the absence of sufficient tumor tissue for molecular analysis, examination of ctDNA isolated from patient plasma has been shown to be a viable alternative for most patients with stage IV disease. Tumors often shed DNA into peripheral circulation, and the extent to which this occurs appears to be based on a variety of factors, including stage, disease burden, metastatic dissemination, tumor aggressiveness, proliferation, and apoptotic rate. DNA released into circulation in this manner is referred to as cfDNA or ctDNA. Although these terms are often used interchangeably, they have distinct meanings: cfDNA refers to the totality of mutant and wild-type DNA shed into body fluids from both normal and tumor cells, and ctDNA refers to the specific subset that is tumor in origin. Identification of tumor-specific DNA abnormalities in cfDNA, such as EGFR-activating mutations, requires assays that are both highly sensitive (able to identify very small propor-tions of mutant alleles in a large pool of wild-type alleles) and highly specific (not prone to false-positive findings). The need for an assay with a specificity approaching 100% stems from the observation that, in most cases, ctDNA mutations are detected at extremely low mutant allele frequencies (MAFs). The MAF is defined as the percentage of mutant alleles at a specific locus; thus, a MAF of 1% indicates a ratio of one mutant sequence to 99 wild-type sequences. Large-scale analysis of patients with NSCLC adenocarcinoma has shown that the vast majority with mutations that were detectable in cfDNA also had MAFs of less than 1% (Mack 2016). When using a technique that requires such high levels of sensitivity, any assay that risks generation of false-positive results, even at very low frequencies, may result in misdiagnosis. (See Chapter 5 for a discussion of assays appropriate for detection of EGFR

31SAMPLE ACQUISITION, PROCESSING, AND GENERAL DIAGNOSTIC PROCEDURES

mutations in cfDNA.) Regardless of the assay used, great care must be taken to maximize detection of EGFR mutations during the sample-acquisition stage.

Plasma Sample Preparation and StorageDue to the highly sensitive assays required for mutation detection in cfDNA, all precautions must be taken to avoid contamination. Processing of plasma for ctDNA analysis should be conducted by trained personnel in a dedicated blood laboratory. It is essential to avoid any risk of cross-contamination with mutation-positive samples (such as assay controls), and processing should take place in facilities that are isolated from PCR- amplification prod-ucts. However, perhaps the biggest threat to ctDNA analysis is the release of genomic DNA from white blood cells during sample preparation. Inadvertent lysis of white blood cells can significantly increase the total amount of cfDNA in the tube, diluting the MAF. Thus, plasma is preferable to serum. When standard blood tubes, such as EDTA tubes are used, best practice includes rapid processing and a double-spin procedure. It is recommended that samples be processed within 4 hours of being collected (El Messaoudi 2013); if immediate processing is not possible, tubes should be kept at 4oC. Centrifugation of blood at 1,000 to 2,000 g for 10 to 20 minutes separates the plasma from the cell fractions. However, a second centrifugation step is recommended because this initial process fails to remove all cells and cellular debris (Normanno 2017). This step is typically accomplished by transferring the plasma supernatant from the first spin into a 15-mL conical tube and centrifuging a second time at a minimum of 3,000 g for another 10 minutes. If possible, a refrigerated centrifuge should be used. The plasma supernatant from the second spin should be carefully pipetted into prelabelled freezer vials and stored at -80oC in aliquots to avoid freeze–thaw cycles. Alternatively, the use of a blood-collection tube that contains stabilizers that prevent both nuclease-mediated DNA degradation and lysis of nucleated cells can be used. A commonly used example is the Cell-Free DNA BCT® (Streck). Although stabilizer tubes are expensive, their advantage is the ability to be stored at room temperature for days before processing without compromising the recovery of cfDNA. If an outside service for ctDNA analysis is being used, it is essential to strictly follow the service’s plasma collection procedures, as they are developed and optimized for its analytical approaches.

Other ConsiderationsThe timing of the blood collection relative to patient treatment is a crucial but often unad-dressed factor for maximizing ctDNA yield. Patients who are undergoing treatment will often have limited or nondetectable levels of ctDNA. For example, Mok et al. demonstrated that after two cycles of chemotherapy (with or without sequential erlotinib), detection of EGFR mutations in cfDNA was significantly ablated, particularly for patients who received an effective EGFR tyrosine kinase inhibitor (Mok 2015). The dynamic drop in the pres-ence of mutant-positive alleles is likely a product of reduced tumor burden; however, even cytostatic responses to therapy may have a dramatic effect in the shedding of DNA because apoptosis typically occurs in proliferating cells. Thus, blood for cfDNA analysis should be collected before treatment is initiated or at the time of disease progression. In patients with disease progression during treatment with an EGFR tyrosine kinase inhibitor, ctDNA analysis can be instrumental in identifying emergent resistance mechanisms, particularly


in cases where secondary tissue biopsies are unfeasible or uninformative. For instance, detection of the EGFR T790M gatekeeper mutation in plasma can guide use of osimertinib, as discussed in Chapter 1. However, the absence of mutation positivity in plasma should not be construed as a definitive negative result. Small, indolent tumors may not shed suf-ficient DNA into peripheral circulation for detection. In treatment-naïve tumors, detection rates for a known EGFR-activating mutation in tissue range from 60% to 85%. Detection rates are lower still for patients with disease in the initial stages of progression (Qiu 2015). If a well-validated approach is used, positive detection of an actionable mutation in plasma ctDNA can be used to guide clinical decision making; however, a lack of detection should be taken as inconclusive—it cannot be determined whether the tumor is truly negative or is simply not shedding sufficient DNA for detection in plasma.

EGFR Gene Mutations By James Chih-Hsin Yang, Pasi A. Jänne, Myung-Ju Ahn, and Leora Horn 4Sensitizing EGFR gene mutations are the most common actionable driver mutations found in patients with NSCLC, occurring in approximately 10% to 15% of white patients and as many as 50% of Asian patients (Hirsch 2009, Shi 2015, Skov 2015). These mutations occur within EGFR exons 18–21, which encode a portion of the EGFR kinase domain, and are a prime example of the complexity of the disease at the molecular level. Mutations involving exons 18, 19, and 21 are considered predictive of sensitivity to EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib, erlotinib, afatinib, and icotinib; mutations in exon 20, however, are typically resistant to therapy, with the exception of S768I mutations and A763_Y764insFQEA variants (Figure 1) (Asahina 2006, Sasaki 2007, Ladanyi 2008, Wu 2008, Arcila 2013, Beau-Faller 2014, Baek 2015, Chiu 2015, Cheng 2015, Yang 2015a, Naidoo 2015). In addition, some uncommon mutations may be associated with primary drug sensitivity and resistance, whereas other rare EGFR mutations are of less clear clinical significance (Dahabreh 2010).

Figure 1. Location of hotspots for EGFR gene mutations in chromosome 7p11.2, with a focus on exons 18-21 and corresponding percentages of responsiveness to EGFR tyrosine kinase inhibitors. Reprinted by per-mission from Macmillan Publishers Ltd: Modern Pathology; 2012; 25:347–369, © 2012.


Common MutationsThe two most common EGFR gene mutations are L858R and exon 19 deletion. The L858R mutation within exon 21 results in an amino acid substitution from a leucine (L) to an arginine (R) at position 858 in EGFR. L858R mutations are seen in approximately 43% of EGFR mutation-positive NSCLC (Mitsudomi 2010). L858R occurring as a single mutation is associated with sensitivity to treatment with EGFR TKIs. In contrast, L858R mutations occurring in combination with either A871E, L747S, de novo G719S, de novo T790M, L861P, or R776G mutations are associated with disease progression (Table 1) (Yeh 2013). EGFR exon 19 deletions are in-frame deletions occurring within exon 19 and are found in approximately 48% of EGFR-mutated lung tumors (Mitsudomi 2010). As many as 53 different exon 19 dele-tions have been described—E746_A750 being most common, accounting for approximately 60% of exon 19 deletions. The majority of these deletions are associated with response to EGFR TKIs; however, deletion 19 unspecified in combination with either exon 20 insertion unspecified or V769M were noted to be associated with disease progression (Yeh 2013). Biochemical analyses of the deletion 19 and L858R mutations showed that exon 19 deletions appeared to be more sensitive than the L858R substitution to erlotinib inhibition (Carey 2006). In a meta-analysis of seven randomized trials (1,649 patients) comparing first-line treatment with first- and second-generation EGFR TKIs (950 patients) and chemotherapy (699 patients), the authors examined the effects of mutation subtype and clinical charac-teristics on progression-free survival rates (Lee 2015). Across all EGFR mutation subtypes, treatment with an EGFR TKI was associated with a 63% reduction in the risk of disease progression or death compared with chemotherapy (HR: 0.37; 95% CI: 0.32–0.42; P < 0.001). The vast majority of patients harbored common EGFR mutations: 872 patients had exon 19 deletions, and 686 patients had exon 21 L858R substitutions. Subgroup analyses dem-onstrated that patients with exon 19 deletions had a 50% greater progression-free survival benefit when treated with an EGFR TKI than patients with exon 21 L858R substitutions (P < 0.001 for interaction). Another mutation, EGFR exon 20 insertion, is seen in 4% to 9% of EGFR mutation-positive NSCLC (Naidoo 2015). This mutation is most often between amino acids 767 and 774 of exon 20 and confers primary resistance to EGFR TKIs (Sasaki 2007, Wu 2008, Arcila 2013, Yasuda 2013, Oxnard 2013, Beau-Faller 2014, Yang 2015a). Structural and molecular analyses have shown that, although this mutation may activate EGFR, it does so without increasing recep-tor affinity for EGFR TKIs (Yasuda 2013). One exception may be the A763_Y764insFQEA variant, which appears to be sensitive to EGFR TKIs (Yasuda 2013, Naidoo 2015).

Secondary MutationsIn most patients treated with an EGFR TKI, resistance develops within approximately 10 to 12 months. The T790M mutation is a second-site mutation occurring within exon 20 of EGFR that has been detected in approximately 50% to 60% of patients in whom acquired resistance to EGFR TKIs develops (Table 2) (Kobayashi 2005, Pao 2005). This so-called gatekeeper mutation, which involves a threonine-to-methionine substitution in exon 20, increases the affinity of mutant EGFR for ATP, thereby competitively inhibiting the bind-ing ability of reversible EGFR TKIs (Yun 2008). Osimertinib was recently approved for patients with T790M mutation resistance to a first- or second-generation EGFR TKI, such

35EGFR GENE MUTATIONS

Table 1. EGFR Gene Mutations Associated with Disease Progression+

Mutation

Number of Instances Number of Unique

StudyResponses to Therapy

Progressive Diseasea Patientsb Studiesc

A763V 1 1 1 1 Chou 2005

A859T 2 2 2 2 Cappuzzo 2006, Taron 2005

Del19 A767-V769 1 1 1 1 Chou 2005

Del19 unspecified + ins20 unspecified

1 1 1 1 Wu JY 2011

Del19 unspecified + V769M 1 1 1 1 Wu JY 2011

E709G + G719C 1 1 1 1 Wu 2011

E711K 1 1 1 1 Pallis 2007

G719A + S768I 2 2 2 2 Wu SG 2008, Wu JY 2008

G721D 1 1 1 1 Ichihara 2007

G729R 1 1 1 1 Pallis 2007

I744M 1 1 1 1 Hsieh 2006

I759T 1 1 1 1 Takahashi 2010

Ins20 769-770insGVV 1 1 1 1 Ichihara 2007

Ins20 A767-V769dupASV 1 1 1 1 Wu JY 2008

Ins20 D770-N771insD 1 1 1 1 Wu JY 2008

Ins20 P772-H773insYNP + H773Y

1 1 1 1 Wu JY 2008

Ins20 S768-D770dupSVD 4 3 4 1 Wu JY 2008

Ins20 SVD768-770 3 2 3 1 Yang CH 2008

Ins20 unspecified 3 3 3 1 Cappuzzo 2007

K806E 1 1 1 1 Wu JY 2008

K860E 1 1 1 1 Pallis 2007

L703F 1 1 1 1 Pallis 2007

L747P 2 2 2 1 Kimura 2007

L777G 1 1 1 1 Ichihara 2007

L838P + E868G 1 1 1 1 Hsieh 2006

L858R + A871E 1 1 1 1 Wu JY 2011

L858R + G719S 3 2 3 1 Hata 2010

L858R + L747S (de novo) 1 1 1 1 Wu JY 2011

L858R + L861P 1 1 1 1 Pallis 2007

L858R + T790M (de novo) 6 5 6 5 Wu JY 2008, Ichihara 2007, Yang CH 2008, Jackman 2009, Tokumo 2006

L858R + R776G 1 1 1 1 Wu JY 2008

L861Q + G719S 1 1 1 1 Chou 2005 continued on next page


N826Y 1 1 1 1 Wu JY 2011

N842S 1 1 1 1 Wu JY 2011

S768I + V769L 1 1 1 1 Asahina 2006

S784F 1 1 1 1 Wu JY 2011

T847A + G863S 1 1 1 1 Pallis 2007

T847I 1 1 1 1 Wu JY 2011

T790M (de novo) 2 2 2 2 Jackman 2009, Donovan 2009

V774M 1 1 1 1 Wu JY 2011

V802I 1 1 1 1 Jackman 2009

V852I 1 1 1 1 Cappuzzo 2005 aDisease progression is defined by the Response Evaluation Criteria in Solid Tumors (RECIST) criteria (7), following treatment with an EGFR tyrosine kinase inhibitor (TKI, gefitinib or erlotinib therapy) as best response.

bThe number of unique patients does not match the number of responses to therapy because one patient may have been treat-ed with EGFR TKIs in different lines of treatment and, therefore, may have multiple associated instances of response to therapy.cThe number of unique studies refers to the number of different studies that encompass all of the patients with a particular mutation in the DNA Inventory to Refine and Enhance Cancer Treatment (DIRECT) database.

Table 2. EGFR Gene Mutations in Patients with Acquired Resistance to EGFR Tyrosine Kinase Inhibitors (TKIs)

Initial EGFR Status

Acquired Resistance Mutationa

Number of Unique

StudyPatients Studiesb

Del19 E746-A750 T790M 14 5 Costa 2008, Chen 2009, Balak 2006, Bean 2007, Engelman 2007

Del19 E746-T751insA T790M 1 1 Balak 2006

Del19 E746-T751insV T790M 1 1 Bean 2008

Del19 L747-A750insP T790M 1 1 Chen 2009

Del19 L747-E749;A750P T790M 3 2 Engelman 2007, Bean 2008

Del19 L747-P753insQ T790M 1 1 Ruppert 2009

Del19 L747-P753insS T790M 2 2 Balak 2008, Engelman 2007

Del19 L747-S752 T790M 2 2 Chen 2009, Balak 2006

Del19 L747-T751 T790M 1 1 Bean 2008

Del19 L747-T751;K754E T790M 3 2 Engelman 2007, Bean 2008

Del19 unspecified T790M 3 1 Onitsuka 2010

L858R D761Y 1 1 Balak 2006

L858R L747S 1 1 Costa 2008

L858R T854A 1 1 Bean 2008

L858R T790M 21 6 Chen 2009, Balak 2006, Engelman 2007, Bean 2008, Onitsuka 2010, Inukai 2006

Wild type T790M 4 1 Chen 2009 aResistance mutations were acquired after treatment with an EGFR TKI.bThe number of unique studies refers to the number of different studies that encompass all of the patients with a particular mutation in the DNA Inventory to Refine and Enhance Cancer Treatment (DIRECT) database.


as gefitinb, erlotinb, or afatinib (Janne 2015). Germline EGFR T790M mutations occur in approximately 1% of white patients with NSCLC; cancers in such patients often also contain a second activating EGFR mutation. In addition, patients with germline T790M mutations may have familial cancer syndromes (Bell 2005). Studies have found that patients carrying a germline T790M mutation have a high lifetime risk of the development of lung cancer; this risk is as high as 31% among never-smoking genetic carriers (Gazdar 2014, Bell 2005). In addition, T790M mutations may be detected de novo in conjunction with an EGFR sensitizing mutation and are associated with decreased sensitivity to EGFR TKIs (Wu 2011). In a meta-analysis of three randomized controlled trials and 15 observational studies, pretreatment T790M mutations were more likely to be present with L858R mutations than with exon 19 deletions (Chen 2016). This association may provide an explanation for better progression-free survival for patients with exon 19 deletions than for patients with L858R mutations (Jackman 2006, Riely 2006, Yang 2015b). T790M is most often seen in the cis position with an L858R mutation or exon 19 deletion; however, it can occur in the trans position as well (Kobayashi 2005). Among patients with resistance to osimertinib, a third mutation, C797S, was identified along with the activating mutation and T790M (Thress 2015). Preclinical studies have shown that, when C797S and T790M mutations were present in the trans posi-tion, cells were resistant to third-generation TKIs but sensitive to combined treatment with first- and third-generation inhibitors. When these mutations were in cis conformation, no TKIs were able to suppress EGFR activity (Niederst 2015).

Uncommon EGFR MutationsStudies in which patients with uncommon EGFR mutations have been analyzed as a single group have often shown that the response to EGFR TKIs is lower among this group than among patients with either of the common mutations alone (L858R and exon 19 deletions) (Watanabe 2014, Bek 2015, Chiu 2015, Arrieta 2015,). However, when analyses are performed on individual mutations or on smaller selective subsets, substantial clinical heterogeneity clearly exists. The most frequently detected exon 18 mutation is G719X, followed by the E709X mutation (Beau-Faller 2014, Cheng 2015). Mutations in G719A, G719C, and G719S, among others, are seen in approximately 2% to 3% of patients with EGFR-positive NSCLC. These mutations are substitution at position 719 in EGFR; substitution from a glycine (G) to an alanine (A), cysteine (C), or serine (S) are associated with responses to an EGFR TKI but are less sensitive than the common mutations in vitro (Yeh 2013, Yun 2007, Kancha 2009). A retrospective analysis of patients pretreated with an EGFR TKI who received afatinib as part of the Afatinib Compassionate-Use Program following disease progression during one prior regimen of an EGFR TKI and chemotherapy included 10 patients with G719X mutations, and the median time to treatment failure was 2.6 months (Heigener 2015). In contrast, among patients with E709X mutations, the median time to treatment failure was 12.2 months with afatinib therapy. In a prospective pool analysis of 18 patients who had not been treated with an EGFR TKI and had G719X mutation and received afatinib, the response rate was 78%, with a progression-free survival of 13.8 months and an overall survival of 26.9 months (Yang 2015b). The L861Q mutation is seen in 2% of patients with EGFR mutations. This mutation occurs in exon 21 because of a substitution at position 861 in EGFR, from a leucine (L) to a glutamine


(Q), and is associated with disease control. However, patients with these mutations are less likely to have a response to therapy than patients with the common mutations deletion 19 and L858R (Yeh 2013). A post-hoc analysis of 838 patients from LUX-Lung 2, LUX-Lung 3, and LUX-Lung 6 showed uncommon EGFR mutations in 100 (12%) of the patients; 16 of these mutations were L861Q substitutions (Yang 2015a). The overall response rate associated with afatinib for patients with L861Q mutations was 56.3%, with a median progression-free and overall survival rate of 8.2 months and 17.1 months, respectively. In an analysis of next-generation sequencing data, rare EGFR mutations were identified in approximately 20% of samples (Costa 2016), a higher proportion than has been estimated (Mitsudomi 2010, Krawczyk 2015). Retrospective data from the DNA Inventory to Refine and Enhance Cancer Treatment (DIRECT) database provides a comprehensive review of known and reported EGFR mutations, including 188 unique EGFR mutations occurring in 207 different combinations, as well as of responses to targeted therapy (Yeh 2013). The information in this electronic catalog of EGFR mutations in NSCLC paired with clinical outcome was collected with use of a retrospective PubMed medical-subject heading search to identify patient-level, mutation-specific, drug-response data from different studies of EGFR-mutant NSCLCs (Yeh 2013). Electronic queries of DIRECT will result in a customized report for clinical use at the bedside. These data are continuously updated for public use. A growing number of genomic profiling services that include pertinent clinical relevance data are becoming available for community use.

Molecular Mechanisms EGFR proteins belong to the ErbB family, which consists of ErbB1 (EGFR), ErbB2 (HER2), ErbB3 (HER3), and ErbB4 (HER4). ErbB family proteins are membrane proteins that bind to extracellular ligands and form homodimer or heterodimer with each other (Figure 2) (Blume-Jensen 2001). Dimerization of ErbB proteins plus ligand binding results in phosphorylation of tyrosine kinase in the intracellular domain, leading to activation of tyrosine kinase and subsequently activating the downstream pathways (Ferguson 2008). Dimerization of ErbB proteins are usually pre-formed before ligand binding. However, ErbB dimerization is not sufficient for activation of ErbB downstream pathways. Activation occurs only after ErbB dimerization and ligand binding (Tao 2008). The results of studies suggest that tetramers and clusters of ErbB proteins are frequently seen with ligand binding and ErbB activation (Clayton 2005). Of note, there is no known ligand for HER2 and no intracellular active kinase domain for HER3. Phosphorylation of different EGFR tyrosine kinases through dimerization of EGFR and ATP binding to the kinase pocket leads to diverse downstream pathway activation. There are two important activated pathways: one leads to activation of RAS, RAF, MEK, and ERK and eventually results in cellular proliferation, and the second pathway activates PI3K and AKT and prevents apoptosis of cells (Figure 2). ErbB family downstream signals are con-trolled through binding to different adaptor proteins and different binding sites of ErbB family proteins, and protein is recruited to phosphorylation sites of intracellular domain (Figure 3). EGFR is the most important member of the ErbB family. EGFR is present in most epithelial, mucosal, and glandular cells, and its primary function is to maintain the structure


Figure 2. ERBB signaling pathways. Binding of a family of specific ligands to extracellular domain of ERBB leads to formation of homo- and heterodimers. In this case, HER2 is a preferred dimerization partner and heterodimers containing HER2 mediate a stronger signal than homodimers. Dimerization consequently stimulates intrinsic tyrosine kinase activity of the recep-tors and triggers autophosphorylation of specific tyrosine residues within the cytoplasmic domain. These phosphorylated tyrosines serve as specific binding sites for several signal transducers that initiate multiple signaling pathways including mitogen-activated protein kinase (MAPK), phosphatidyl inositol 3 kinase (PI3K)-AKT and signal transducer and activator of transcription protein (STAT) 3 and 5 pathways. These eventually result in cell proliferation, migration and metastasis, evasion from apoptosis, or angiogenesis, all of which are associated with cancer. P85 and p110 is a regulatory and catalytic subunit of phosphatidyl inositol 3 kinase (PI3K), respectively. STAT, SRC and mTOR are also activated by ERBB sinaling. AR, amphiregulin; BTC, betacellulin; EPR, epirefulin; ERK, extracellular signal-regulated kinase; HB-EGFR, heparin binding EGF; MEK, MAP an ERK kinase; mTOR, mammmalian target of rapamycin; NRG, neuregulin; TGF, transforming growth factor. Reprinted with permission from Cancer Sci. 2007;98:1817-1824.

1818 doi: 10.1111/j.1349-7006.2007.00607.x© 2007 Japanese Cancer Association

small deletions encompassing five amino acids from codons 746through 750 (ELREA) or missense mutations resulting inleucine to arginine at codon 858 (L858R). There are over 20variant types of deletion, for example, larger deletion, deletion

plus point mutation, deletion plus insertion, etc. Approximately3% of the mutations occur at codon 719 resulting in thesubstitution of glycine to cysteine, alanine or serine (G719X).Also, approximately 3% are in-frame insertion mutations inexon 20.(11) These four types of mutations seldom occursimultaneously. There are many rare point mutations, some ofwhich occur with L858R.

Biological consequences of the EGFR mutations. Exon 19 dele-tional mutation and L858R result in increased and sustainedphosphorylation of EGFR and other HER family proteins withoutligand stimulation. Sordella et al. reported that mutant EGFRselectively activate AKT and signal transducer and activator oftranscription protein (STAT) signaling pathways that promotecell survival but no effect on the mitogen-activated protein kinase(MAPK) pathway that induces proliferation.(14)

Two groups of researchers have recently developed transgenicmice that express either exon 19 deletion mutant or the L858Rmutant in type II pneumocytes under the control of doxycyclin.(15,16)

Expression of either EGFR mutant leads to the developmentof adenocarcinoma similar to human bronchioloalveolar cellcarcinoma and withdrawal of doxycyclin to reduce expressionof transgene or erlotinib treatment resulting in tumor regression.These experiments showed that persistent EGFR signaling is

Fig. 2. Incidence of epidermal growth factor receptor gene (EGFR)mutations by ethnicity, gender, smoking history and histology. Datawere compiled from the published reports (n = 2880).(11)

Fig. 1. ERBB signaling pathways. Binding of a family of specific ligands to extracellular domain of ERBB leads to formation of homo- andheterodimers. In this case, HER2 is a preferred dimerization partner and heterodimers containing HER2 mediate a stronger signal thanhomodimers. Dimerization consequently stimulates intrinsic tyrosine kinase activity of the receptors and triggers autophosphorylation of specifictyrosine residues within the cytoplasmic domain. These phosphorylated tyrosines serve as specific binding sites for several signal transducers thatinitiate multiple signaling pathways including mitogen-activated protein kinase (MAPK), phosphatidyl inositol 3 kinase (PI3K)-AKT and signaltransducer and activator of transcription protein (STAT) 3 and 5 pathways. These eventually result in cell proliferation, migration and metastasis,evasion from apoptosis, or angiogenesis, all of which are associated with cancer. P85 and p110 is a regulatory and catalytic subunit of phosphatidylinositol 3 kinase (PI3K), respectively. STAT, SRC and mTOR are also activated by ERBB sinaling. AR, amphiregulin; BTC, betacellulin; EPR, epirefulin;ERK, extracellular signal-regulated kinase; HB-EGFR, heparin binding EGF; MEK, MAP an ERK kinase; mTOR, mammmalian target of rapamycin;NRG, neuregulin; TGF, transforming growth factor.

Figure 3. Protein recruited to phosphorylation sites of ErbB intracellular sites. (Adapted from Bradshaw R, Dennis EA (eds). Functioning of Transmembrane Receptors in Signaling Mechanisms. Academic Press, 2011. p. 104. Used with permission.)

EGFR

HER4

HER3

HER2

PI3K Grb7

Chk1Cb1

Shc/Grb2Stat5

Src

OLCg Gab1PTP2c/Shp1


of these cells by replacing apoptotic cells in the surface with newly divided and differenti-ated cells from the stem cells. The number of EGFR molecules per cell range from 20,000 in physiologic conditions to more than several million per cell in pathologic conditions. Overexpression of EGFR has been reported in more than 65% of patients with NSCLC (Franklin 2002). Gene amplification shown by fluorescence in situ hybridization is the main mechanism of EGFR overexpression. EGFR mutation in the kinase domain (exons 18-21) was discovered after the successful administration of EGFR TKIs to patients with lung cancer (Paez 2004, Lynch 2004). Most of the patients with NSCLC who had a response to EGFR TKIs had one of these mutations in their tumors. The mutations found in patients with NSCLC are always in frame, which suggests that these mutations are functional. The fact that inhibition of the activating EGFR mutation by an EGFR TKI resulted in dramatic tumor response in most patients suggests that these activating EGFR mutations play a dominant role in main-taining the survival of these cancer cells. Among patients with EGFR-positive NSCLC, 80% also had EGFR gene amplification. Patients who had only EGFR gene amplification but no EGFR mutations did not have a response to gefitinib and had short progression-free survival times, suggesting that the presence of EGFR mutation but not EGFR gene amplification is the mechanism for maintaining the addicted EGFR pathways in patients with NSCLC who had a response to EGFR TKIs (Fukuoka 2011). Mutation of EGFR resulted in alteration of affinity of EGFR tyrosine kinase to ATP and EGFR TKIs (Yun 2008). This is due to the three-dimensional structural alterations in the EGFR tyrosine kinase domain (Figure 4). Resistance mutations, such as T790M, may accu-mulate in cancer cells through survival advantage by selective pressure after exposure of EGFR mutant cells to gefitinib, erlotinib, or afatinib (Ercan 2010). Mutant-specific EGFR TKIs, such as osimertinib, were designed to inhibit both activating and T790M EGFR muta-tions (Finlay 2014). However, a novel mutation, C797S, can emerge in response to prolonged exposure to osimertinib (Figure 5) (Thress 2015). Several resistance mechanisms to EGFR TKIs have been suggested (Table 3).

Figure 4. Three-dimensional structural alterations in the EGFR tyrosine kinase domain. Structural modeling of CO-1686 binding to EGFRT790M. The EGFRT790M kinase is shown in a ribbon represen-tation (green) with the bound CO-1686 in orange. The aminopy-rimidine binds to the hinge residue Met793 through hydrogen bonding (yellow dashed lines). The C5-CF3 substitution points to the gatekeeper residue Met790. Both C2 and C4 substitutions adapt a U-shaped binding mode. The piperazine ring is facing an open space in the solvent exposure area. The meta-acrylamide points to Cys797 and forms the covalent bond. Reprinted with permission from Cancer Discov. 2013; 3:1404-1415.

M790

L792

P794 M793

C797

on August 12, 2017. © 2013 American Association for Cancer Research.cancerdiscovery.aacrjournals.org Downloaded from

Published OnlineFirst September 24, 2013; DOI: 10.1158/2159-8290.CD-13-0314


Table 3. Possible Resistance Mechanisms to EGFR Tyrosine Kinase Inhibitors (TKIs)

EGFR TKI

First- and Second-Generation

Third-Generation: Osimertinib

Third-Generation: Rociletinib

Third-Generation:WZ4002

Primary resistance

EGFR modification Exon 20 insertion

Alternative pathway activation

BIM deletion

Acquired resistance

EGFR modification T790MAmplification

C797SG769S/RL792F/HT790M loss

C797SL718QL844VAmplification

C797SL718QL844V

Alternative pathway activation

Bypass MET amplification HGF overexpression AXL overexpression HER2 amplificationDownstream PTEN loss PI3K mutation BRAF V600E

Bypass MET amplification HER2 amplificationDownstream BRAF V600E NRAS mutation

Bypass IGF1R activationDownstream ERK1 and ERK2 activation

Histologic SCLC transformation(RB loss)Epithelial-mesenchy-mal transition

SCLC transformation SCLC transformation Epithelial-mesenchy-mal transition

Other Drug efflux (ABCG2)RTK internalization

(Cortot 2014, Piotrowska 2015, Ercan 2015, Tricker 2015, Eberlein 2015, Wlater 2013, Oxnard 2015, Park 2016, Ou 2016, Ham 2016, Ou 2017, Chen 2017).

Figure 5. A 3-dimensional structure of the C797S allele in the EGFR gene. The structure of a typical tyrosine-specific kinase domain of EGFR is shown in the middle panel, along with an ATP analog, 5’-adenylyl-imidodiphosphate (AMP-PNP). The P-loop and the activation loop are in light blue and light green, respectively. Leucine 858 (L858), which is frequently mutated in lung cancer, is highlighted in pink. Similarly labeled is threonine 790 (purple), which is replaced by a methionine in some advanced lung tumors and confers resistance to first-generation tyrosine kinase inhibitors (TKIs). The squared area is magnified in the side panels and shows the structure of the T790M mutant in a complex with either a first-generation TKI, erlotinib (right), or with a second-generation TKI, afatinib (left). The latter structure shows a replacement of cysteine 797 to second- and third-generation EGFR TKIs. The chemical structures of both erlotinib and afatinib are shown. Reprinted with permission from Semin Cell Dev Biol. 2016;50:164-176.

Types of Assays for EGFR TestingBy Daniel SW Tan, Erik Thunnissen, Geoffrey R. Oxnard, and Sanja Dacic 5Molecular Diagnostics on Tissue or Cytologic Samples Traditional PlatformsEGFR mutation assays have evolved over the past decade from a single-gene test (eg, Sanger DNA sequencing or pyrosequencing) to multiplex hotspot mutation tests (eg, PCR-based SNapShot Multiplex Kit [Applied Biosystems] and MassARRAY SNP [Agena Bioscience]) to next-generation sequencing (NGS). There is no consensus about the best EGFR mutation assay that should be used in clinical practice, but an ideal assay should be sensitive and specific enough to comprehensively cover all clinically relevant targets with use of limited samples, while being cost-effective and efficient. All of the EGFR mutation assays can be performed on fresh, frozen, formalin-fixed paraffin-embedded (FFPE), or alcohol-fixed tissue samples, including surgical resection specimens or various cytologic preparations (samples from fine-needle aspiration or smears). Fresh or frozen samples typically provide better nucleic acid quality, but the lack of correlation with histology is a major drawback. In contrast, FFPE samples allow much better assessment of the quality and quantity of the tumor cells in the sample; however, the DNA is fragmented in FFPE material, requiring adjustment of the assays to smaller base-pair fragments (usually less than 150 to 200 bp). In pulmonary pathology, samples are frequently small and also contain non-neoplastic cells (eg, stromal components). Depending on the analytic threshold of the test or platform used, enrichment of tumor content of the sample by microdissection or macrodissection of a tumor area designated by a pathologist is usually necessary. In this way, nontumor areas are left out for DNA extraction, resulting in less total DNA, but a higher fraction of tumor DNA, which should be higher than the threshold of the platform (see later).

Nontargeted AssaysSanger sequencing, or direct DNA sequencing. Bidirectional sequence determination by the Sanger method with fluorescence-tagged dideoxyterminators was a method of choice in the first clinical trials with erlotinib and gefitinib and were once the criterion standard for EGFR testing. However, this method can precisely detect mutant sequences when they


constitute 25% of the total DNA (analytic threshold), which corresponds to a tumor content of 50% for a heterozygous mutation with no polysomy or amplification (Lindeman 2013). Because of the high tumor content required, many samples were deemed inadequate for testing in clinical practice. Assay sensitivity remained an issue despite the application of a mutant-enriching strategy, such as locked nucleic acid or peptide nucleic acid clamps, or coamplification at lower denaturation temperature (COLD)-PCR. The main advantage of Sanger sequencing is the identification of all known and previously unknown mutations in the studied region. The technique requires a small amount of input DNA (5 to 10 ng), but because of a low sensitivity, it is no longer a method of choice for detection of EGFR mutations.

Pyrosequencing. Pyrosequencing (also known as sequencing by synthesis) is a quick and sensitive method that involves the use of chemiluminescent detection of pyrophos-phate released by DNA polymerase after each nucleotide addition during the synthesis of a DNA strand. This method is limited by the length of the template DNA strand that can be sequenced, which is substantially shorter than for Sanger chain termination sequenc-ing. The method is also prone to more reading errors through homopolymer sequences. However, it is more sensitive than Sanger sequencing and can detect mutations in samples with a proportion of tumor cells as low as 5%.

Targeted Assays Targeted EGFR assays are much more sensitive than direct sequencing assays and require 5% to 10% of the starting tumor DNA. Targeted EGFR assays allow rapid multiplex genotyping of specific known hotspot mutations within a single assay, but they are unable to identify novel mutations, more specifically, mutations outside the amplified regions. Following are brief summaries of the commercially available EGFR targeted assays. Cobas EGFR Mutation Test (Roche Molecular Systems, Inc.) is the allele-specific real-time PCR assay for selection of patients who may be candidates for treatment with erlotinib. There are two versions of this US FDA-approved companion diagnostic test. The first ver-sion was approved in 2013 and included detection of EGFR exon 19 deletion and exon 21 L858R substitution mutation in tumor tissue specimens. The second version was approved in 2015 and identifies 42 clinically relevant mutations in exons 18-21, including the T790M mutation. This second version is also approved for plasma specimens. Therascreen EGFR RCQ PCR Kit (Qiagen N.V.) is another allele-specific real-time PCR assay. This test is also an FDA-approved companion diagnostic test and was used in the LUX–Lung 3 trial, the results of which led to FDA approval of afatinib as first-line treat-ment of patients with advanced NSCLC harboring EGFR mutations (Sequist 2013). This assay allows detection of 29 mutations in the EGFR gene with use of Scorpion primers and an amplification-refractory mutation system (ARMS). The MassARRAY system (Agena Bioscience) involves the use of PCR amplification and allele-specific single-base primer extension. The primer extension products are analyzed according to the principles of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. Time of flight is proportional to the mass/charge, which is translated into specific genotype calls (Brevet 2011, Sherwood 2014, Yu 2014). An advantage

45TYPES OF ASSAYS FOR EGFR TESTING

of this system is its ability to identify any mutant base at the given position in one assay with-out the need for a separate assay for each potential mutation. Multiplex customized somatic mutation panels, including OncoCarta Panel v1.0 and LungCarta panel (Agena Bioscience), can detect EGFR mutations in addition to other oncogenes (eg, KRAS, BRAF) (Fleitas 2016, Quinn 2015, Okamoto 2014). The SNaPshot Multiplex Kit (Applied Biosystems) is a primer extension-based method that uses fluorescent-labelled probes, and the detection method is capillary electrophoresis. The SNaPshot panel tests smaller panels of genes (up to 14) and more than 50 hotspot muta-tions. The assay rapidly identifies single-nucleotide polymorphism at the specific sites of the PCR-generated template. It may require less than 5% of the mutant DNA. The SNaPshot assay is able to detect mutations, and sizing assays performed in parallel can detect deletions and insertions. The main disadvantage of this test is the limited number of reactions that can be multiplexed (fewer than 10). (Su 2011, Sequist 2011).

Relevance of IHC to EGFR TestingIHC is the most common testing method available in pathology laboratories, but major shortcomings include interlaboratory variability in assay performance and interobserver variability in assay interpretation. Overexpression of EGFR protein has been reported in as many as 70% of NSCLCs, with overexpression depending highly on the type of antibody clone, antigen retrieval method, and tissue processing. Although EGFR mutant-specific antibodies are now available, IHC with these antibodies must be distinguished from other types of EGFR IHC, including total EGFR and phosphorylated EGFR. EGFR mutation-specific antibodies recognize the three-dimensional conformation change of the protein due to a specific mutation, which translates into another combina-tion of amino acids, but do not bind to the wild-type EGFR protein. Different antibodies have been developed to identify a 15bp deletion in exon 19 and an L858R point mutation in exon 21. The sensitivity and positive-predictive value for EGFR L858R-mutant antibody are 95% as high as 100%, respectively, depending on the cutoff value (Brevet 2010). The EGFR exon 19 mutant-specific antibody has a lower sensitivity in detecting exon 19 dele-tions other than the 15bp deletion (Yu 2009, Brevet 2010, Simonetti 2010, Kitamura 2010, Angulo 2012). Among cytologic preparations, staining has been reported to be more intense in formalin-fixed cell blocks and samples fixed with CytoLyt Solution (Hologic) or alcohol, including Papanicolaou-destained samples; the least intense staining has been found in air-dried slides (Bellevicine 2015). The CAP/IASLC/AMP guideline for molecular testing in lung cancer allows for the use of EGFR mutation-specific antibodies after careful validation against a valid molecular assay in a setting with limited material (Lindeman 2013). However, the routine use of IHC in selecting anti-EGFR treatment for patients with lung cancer is not recommended in the updated guideline (Lindeman 2017). IHC with antibodies against wild-type (total) EGFR has no relevance for prediction of treatment with an EGFR TKI, as the results of this method correlate poorly or not at all with the presence of EGFR mutations (Pinter 2008, Li 2008, Sholl 2010). In contrast, there appears to be some predictive value of protein expression and the use of necitumumab, a second-generation human monoclonal antibody that, when combined with chemotherapy, has shown promise in the treatment of squamous cell lung cancer (Thatcher 2015). Of


interest, although necitumumab received FDA approval in the United States as first-line treatment (in combination with cisplatin and gemcitabine), regardless of EGFR expres-sion status, the European Medicines Agency approved necitumumab only for patients with advanced squamous NSCLC that expresses wild-type EGFR protein by IHC. Experience with IHC for phosphorylated EGFR is limited, and concerns remain regarding the stability of phosphorylation status in routinely processed pathologic specimens. Use of such IHC assays for selection of patients for treatment with EGFR TKIs is premature at this point (Tsakiridis 2008, Wang 2012).

Next-Generation Sequencing (NGS)NGS, or massive parallel sequencing technology, enables high-throughput detection of multiple genetic alterations in both constitutional and cancer genomes. It provides clear advantages over traditional sequencing techniques, such as Sanger sequencing, by allowing for the sequencing of large regions of the genome with higher sensitivity. Effective imple-mentation of NGS requires good-quality DNA, preferably from a tumor-rich sample; careful amplification and library construction; and careful interpretation of postanalytic data. NGS can be performed on FFPE and freshly collected tissue specimens and on fine-needle aspi-ration samples and small biopsy specimens. A number of preanalytic and analytic factors, particularly with FFPE tissue, can result in errors in the detection of somatic variants by NGS (Loudig 2011, Spencer 2013, Jennings 2017). It is well known that formalin reacts with DNA and proteins to form covalent crosslinks and other chemical changes, resulting in errors in low-coverage NGS datasets or assays designed to detect variants at low variant allele frequencies (Auerbach 1977, Karlsen 1994). A guideline for the validation of NGS-based oncology panels addressed troubleshooting of these potential sources of errors from a laboratory standpoint (Jennings 2017). NGS can be used for whole-genome sequencing, exome sequencing, transcriptome sequencing (mRNA sequencing), and targeted sequencing of multigene panels. According to surveys conducted by AMP, the American College of Medical Genetics and Genomics, the American Society of Clinical Oncology, and CAP to determine the use of NGS in clinical practice, a minority of laboratories perform exome (12%) or genome (5%) analysis on tumor tissue (Li 2017). Whole-genome sequencing produces a large amount of data on single-nucleotide vari-ants, insertions and deletions (indels), structural arrangements, and copy number variations that require complex bioinformatics analysis and support. It is expensive to implement this technique as a standard clinical assay and, given its high sensitivity, the significance of incidentally discovered novel variants is frequently of uncertain clinical significance. In contrast, targeted NGS is more affordable, efficient, and suitable for clinical use. The design of a targeted panel largely depends on the purpose of the panel: clinical testing for available approved therapeutic targets or testing in the context of clinical trials. Therefore, targeted NGS panels can range from hotspot panels of a limited number of codons to large panels that include the entire coding regions of hundreds of genes. Targeted NGS panels can be designed to detect single-nucleotide variants/point mutations, gene fusions, small indels, copy number variations, and structural variants. However, the results of the aforementioned survey showed that all laboratories can detect


single-nucleotide variants; 95% could identify small indels by their testing methods, whereas copy number variations and gene fusions were assessed by 35% and 37%, respectively (Li 2017). The two most commonly used strategies for target enrichment include the hybrid capture approach and PCR-based amplification (Mamanova 2010). Amplification-based NGS is susceptible to issues associated with PCR primer design, such as allele dropout resulting in lower-than-expected coverage for the amplicon and potential for incorrect assessment of variant allele frequencies (Jennings 2017). Amplification-based library preparation works less effectively for genes with high guanine-cytosine content, and the sequence quality diminishes at the ends of amplicons, leading to potential miscalling of variants (Jennings 2017). In contrast, hybrid capture-based assays are sensitive to sample base composition (eg, adenine-thymine and guanine-cytosine-rich samples) that may result in interpretation errors (Mamanova 2010). The most commonly used NGS platforms involve different chemistries for sequencing, including ion semiconductor-based sequencing and sequencing by synthesis. In numerous studies, performance concordance between commercially available platforms has been high with these two different sequencing approaches (Loman 2012, Quail 2012, Boland 2013, Misyura 2016, Malapelle 2017a). Despite many similarities in these platforms, there are also differences, such as DNA input requirements, cost of reagents, turnaround time, and depth of coverage (Table 1) (Dacic 2014, Jennings 2017).

Table 1. Most Commonly Used Next-Generation Sequencing (NGS) Platforms in Clinical Laboratories

NGS Platform

Parameter Ion PGM MiSeq HiSeq

Method of sequencing Semiconductor sequencing technology, detecting pH change

Sequencing by synthesis, detecting fluorescent signal

Sequencing by synthesis

Clinical application Targeted sequencing for genes or gene panels

Targeted sequencing for genes or gene panels

Whole genome, exome, transcriptome, targeted sequencing for large gene panels

Time/run 4-5 hours 24 hours 3-10 days

Length of reads(base pairs)

100-200 bp 150-200 bp 150-300 bp

Minimal DNA input 10 ng 50-250 ng 250 ng-1 ug

Advantages Low amount of DNA allows use of NGS on small biopsies and FNA samples; fastest sequencing time

Fast; high accuracy Allows performance of broadest number of NGS applications; discovery tool

Disadvantages Errors in homopolymer regions

Cost of reagents; higher amount of starting DNA

Cost of reagents; higher amount of starting DNA; complex bioinformatic analysis

Ion Personal Genome Machine (PGM) (Ion Torrent) is a product of Thermo Fisher Scientific, and MiSeq and HiSeq Sequencing Systems are products of Illumina, Inc.


Depth of coverage or sequencing is defined as the number of aligned reads that con-tain a given nucleotide position (Jennings 2017). Bioinformatics tools used for analysis are dependent on depth of coverage for sensitive and specific detection of sequence vari-ants. The sequencing platform and the complexity of the targeted region (eg, pseudogenes, guanine-cytosine content) are the most common factors that may influence the required depth of coverage (Loman 2012, Sims 2014). High depth of coverage is necessary for tumor samples because of tumor clonal heterogeneity, tissue heterogeneity (admixed stromal, inflammatory, and normal cells), and tumor viability. According to the 2017 guideline for NGS-based oncology panels, an average coverage of at least 1,000 times may be required to identify heterogenous variants in tissue specimens with low tumor cellularity (Jennings 2017). The required depth of coverage can be estimated on the basis of the required lower limit of detection, the quality of the reads, and the acceptable rate of false-positive and false-negative results (Jennings 2017). The implementation of NGS technology in a clinical laboratory is complex and requires substantial expertise in clinical, technical, and bioinformatic aspects of sequencing. Laboratories in the United States should strictly apply all quality measures for NGS test development, validation, and quality assurance under the guidance of the Clinical Laboratory Improvement Amendments (CLIA) and CAP regulations (Jennings 2017). Similarly, a European expert opinion was provided for integration of NGS in clinical diagnostic molecu-lar pathology laboratories for analysis of solid tumors (Deans 2017). In June 2017, the FDA approved Oncomine Dx Target Test (Thermo Fisher Scientific), a 23-gene panel, as the first NGS-based companion diagnostic that can be used to select patients for three FDA-approved therapies for NSCLC: gefitinib (EGFR), crizotinib (ALK and ROS1), and the combination of dabrafenib and trametinib (BRAF) in NSCLC.

Diagnostic Testing on Nontraditional and Other SpecimensPlasma-based EGFR Testing One emerging alternative to genotyping of tumor specimens is genotyping of cfDNA, which is present in blood and other body fluids (Wan 2017, Oxnard 2017). This free DNA is released through cell death or apoptosis and is primarily made up of germline (leukocyte) DNA. In people with cancer, a portion of this DNA may originate from tumor cells and can be detected with appropriate assays, creating an opportunity to detect EGFR mutations in cfDNA, thus reducing the need for an invasive biopsy. However, important technical differences exist between genotyping of biopsy-derived tumor DNA and plasma cfDNA. First, with genotyping of a tissue biopsy, pathology review is performed to confirm the adequacy of the specimen, and genotyping is not performed if the tumor content is too low. Such an adequacy assessment is not routinely performed for plasma genotyping, and the level of tumor DNA in plasma is often low, such that highly sensitive technologies are needed for detection; in some cases, no tumor DNA is present. Second, biopsy-derived DNA has usually been formalin fixed, which can result in low-level assay artifact; this means that some assays cannot confidently call low-level signals below an allelic fraction of 5% to 10%. Plasma cfDNA can be collected fresh and frozen without a fixative, potentially reducing DNA artifact and allowing accurate detection of mutations below an allelic fraction of 5%. Third, genomic analysis of a tumor biopsy provides data on


a single metastatic site. Genomic analysis of cfDNA has the potential to instead offer insight across multiple metastatic sites, which could be valuable in settings of increased genomic heterogeneity, such as in patients with treatment resistance. Techniques for genotyping of plasma cfDNA have been under development for decades, but only recently have they begun to demonstrate the diagnostic characteristics needed for routine clinical use (Sacher 2017). A number of plasma genotyping assays are now associated with excellent specificity (greater than 95%) and moderately high sensitivity (70% to 80%) for detection of driver EGFR mutations (Table 2) (Douillard 2014, Marchetti 2015, Sacher 2016, Oxnard 2016). The imperfect sensitivity is believed to be related to the fact that not all cancers shed tumor DNA in the circulation; sensitivity is improved with greater meta-static extent, such as metastatic involvement of bone or liver. The high positive-predictive value of plasma EGFR genotyping has led to the FDA approval of one well-validated assay (cobas, Thermo Fisher Scientific) for the detection of EGFR mutations in patients with advanced NSCLC when tumor genotyping is not possible, with the stipulation that a nega-tive result should prompt standard tumor genotyping because of the imperfect sensitivity of the assay.

Table 2. Plasma Droplet Digital Polymerase Chain Reaction Assay Sensitivity, Specificity, and Positive Predictive Value

Assay

Sensitivity Analysis Specificity Analysis

Positive Predictive Value, % (95% CI)

No. No.

Sensitivity, % (95% CI)

True Positivea

False Negativeb

Specificity, % (95% CI)

True Negativec

False Positived

EGFR exon 19 del

Newly diagnosed

86 (57-98) 12 2 100 (96-100) 101 0 100 (74-100)

Acquired resistance

81 (64-92) 29 7 100 (85-100) 23 0 100 (88-100)

Overall 82 (69-91) 41 9 100 (97-100) 124 0 100 (91-100)

EGFR L858R

Newly diagnosed

69 (39-91) 9 4 100 (96-100) 102 0 100 (66-100)

Acquired resistance

78 (52-94) 14 4 100 (91-100) 41 0 100 (77-100)

Overall 74 (55-88) 23 8 100 (97-100) 143 0 100 (85-100)

EGFR T790M

Acquired resistance

77 (60-90) 27 8 63 (38-84) 12 7 79 (62-91)

KRAS G12X

Newly diagnosed 64 (43-82)

16 9 100 (94-100) 62 0 100 (79-100)

aTrue positive indicates positive test result in both tissue and plasma.bFalse negative indicates positive test result in tissue and negative result in plasma.cTrue negative indicates negative test result in both tissue and plasma.dFalse positive indicates negative test result in tissue and positive result in plasma.Reprinted with permission from JAMA Oncol. 2016;2:1014-1022.


Plasma genotyping of EGFR is also compelling in the setting of acquired resistance, where a high response rate and durable progression-free survival have been reported for patients with EGFR T790M-positive resistance who were treated with osimertinib (Mok 2017). In this setting, plasma genotyping has a sensitivity of 50% to 70% for EGFR T790M mutations, slightly lower than the sensitivity for driver EGFR mutations because resistance mutations are inherently a subclonal genomic event (Sacher 2016, Oxnard 2016, Karlovich 2016, Mok 2017, Jenkins 2017). Of interest, the authors of several studies have described a specificity of approximately 70% to 80% for EGFR T790M testing in this setting, which means that T790M is detected in plasma even though T790M is not shown in a biopsy sample taken after resistance (Sacher 2016, Jenkins 2017). This discordance is believed to be related to the heterogeneity of resistance across different sites of disease, and the find-ings of small series suggest less favorable outcomes for osimertinib (Oxnard 2017). Overall, in a cohort of 308 patients in the AURA trial for whom tumor or plasma genotyping was available, patients with positive results for T790M in tumor or plasma both had similarly favorable outcomes after treatment with osimertinib (Oxnard 2017). Because of these find-ings, plasma genotyping is increasingly being adopted as a screening test to determine the presence of EGFR T790M mutation in patients with acquired resistance before performing an invasive biopsy. Supporting this approach are studies showing a high response rate among smaller cohorts of patients who were treated with osimertinib on the basis of detection of EGFR T790M in plasma (Remon 2017). Plasma genotyping may have an additional role as a tool for understanding acquired resistance to osimertinib, as loss of the T790M muta-tion and acquired C797S mutations can both be detected by plasma cfDNA (Thress 2015b, Chabon 2016). One important question is whether there are clinical differences in the performance of different plasma genotyping assays. This question is challenging to answer, as few stud-ies have rigorously compared different assays on paired clinical specimens. One post hoc study of plasma from the AURA trial found a slightly higher sensitivity with digital PCR than with allele-specific PCR (Thress 2015a). One limitation of allele-specific PCR is that it is less quantitative than digital PCR approaches, although it has been reported that a semi-quantitative index can allow accurate quantification of EGFR mutations in cfDNA (Marchetti 2015). Quantitative plasma genotyping is attractive because it can allow easy monitoring of mutational burden within plasma, which may offer insight into treatment outcome (Marchetti 2015, Sacher 2016, Mok 2017). More comprehensive genomic assays such as plasma NGS are also attractive because they have the potential to detect a wide range of resistance mechanisms, although the cost and turnaround time may be greater than those associated with more focused PCR assays (Paweletz 2016, Thompson 2016, Chabon 2016).

EGFR Testing on Other FluidsThere has been general enthusiasm to examine the role of other noninvasive approaches for EGFR testing, including the use of urine and saliva samples. Both of these approaches have the benefit of permitting repeated sampling without the need for venesection. One of the largest studies to date in which DNA testing was done on urine samples is from the TIGER-X phase I trial of rociletinib; the sensitivity of EGFR mutation detection was 72% for T790M, 75% for L858R, and 67% for exon 19 deletions (Reckamp 2016). In this study,


both urine and plasma samples were tested on the same high-depth NGS platform of up to 200,000 times, with comparable sensitivity and specificity. Testing on cerebrospinal fluid has been attempted with various assays, including Sanger sequencing and ARMS-PCR (Shingyoigi 2011, Yang 2014). Although the results of studies have shown feasibility in the context of tumors with and without positive cytologic results on cerebrospinal fluid, it remains uncertain if the discordance with paired primary tumor biopsy samples are mainly technical or related to heterogeneity of mutations at different metastatic sites.

Reporting, Interpretations, and Quality AssuranceBy Dara L. Aisner, Peter B. Illei, Yuichi Ishikawa, and Alberto M. Marchevsky 6Reports of EGFR testing include a summary of technical elements and interpretive elements. Multiple technical elements should be presented in an easy-to-read and transparent manner, regardless of the testing methodology. These elements include discrete results (ie, whether specific mutations are present) and the type of alteration, such as point mutation, insertion, and deletion, using appropriate Human Genome Variation Society nomenclature (Box 1 and Table 1) (den Dunnen 2000, den Dunnen 2016). Some assays detect mutations but do not distinguish specific molecular alterations. For example, some assays detect the presence of an exon 19 deletion without identifying the specific altered base sequence. Similarly, some assays detect the presence of a mutation at C719 but do not distinguish between the individual point mutations at this location. In these situations, it should be clearly stated in the report that the assay does not distinguish the specific mutation sequence. The testing methodology should always be included in EGFR test-ing reports, and the allelic/analytic sensitivity of the assay should be provided. Inherent to performing any assay is an understanding of its limitations, which (to the extent

Box 1. Mutation Nomenclature

In 2016, the Human Genome Variation Society (HGVS) published an updated version of its recommendation for describing sequence variants (den Dunnen 2016). The HGVS nomenclature was first recommended in 2000 and was widely adopted worldwide (den Dunnen 2000). Changes to the nomenclature are currently administered through a Sequence Variant Description Working Group supervised by three international organization: HGVS, the Human Variome Project (HVP) and the Human Genome Organization (HUGO). Version numbers are assigned to the nomenclature if significant changes are made. The most recent update is version 15.11, and the most important changes were removal of inconsistencies and the strengthening of definitions to enable automatic data entry (den Dunnen 2016).

Reports of EGFR gene sequence alterations and/or variations should include the location (exon) of the sequence and a description of the alteration using HGVS nomenclature. In general, the first letter (in small caps) should indicate the type of sequences (nucleotide or amino acid), followed by the position numbering in relation to the first nucleotide or protein of the reference sequence as shown in Table 1. Inclusion of both c. and p. nomenclature is strongly encour-aged in testing reports.


they are known) should be elaborated on in the report. For example, common real-time PCR assays for EGFR mutations do not detect many of the known EGFR exon 20 insertions; known limitations such as these should be clearly stated. EGFR testing reports should also include clinical interpretations based on current infor-mation. Data about EGFR variations are far from static and this area of research is constantly evolving, providing a shifting basis for an understanding of the likely clinical effects of spe-cific alterations. The most commonly reported alterations within EGFR—L858R and exon 19 deletions—are widely accepted as conferring sensitivity to EGFR targeted therapy and should be reported as such. However, as the frequency with which an individual mutation is reported decreases, the certainty with which its clinical significance can be delineated also decreases. For example, some mutations are identified cumulatively in approximately 5% to 10% of patients with EGFR-positive NSCLC (eg, G719X, S768I, and L861Q) and are thought to confer sensitivity to targeted therapy either alone or in combination. It is incumbent on the personnel reporting the results to remain up-to-date with the most recent developments in clinical science. Similarly, EGFR exon 20 insertions were once thought to universally portend resistance to EGFR tyrosine kinase inhibitor (TKI) therapy; however, additional studies have dem-onstrated that a single rare EGFR exon 20 variant (A763_Y764insFQEA) actually confers sensitivity to targeted therapy (Yasuda 2013). This fact also underscores the importance of adapting clinical interpretations to current knowledge and, in some cases, suggests the need for additional testing. For example, if a test identifies the presence of an EGFR exon 20 insertion but does not specify the actual sequence, additional testing should be done to further characterize the alteration at the sequence level once the alteration is identified. Another recent development in the clinical interpretation of EGFR variants relates to the T790M alteration when it is identified before exposure to EGFR TKI therapy. The presence of this alteration in the germline has been associated with familial predisposition to lung cancer (Bell 2005); therefore, identification of this variant prior to administration of EGFR TKI therapy should prompt a recommendation for genetic counseling.

Table 1. Definition of Prefixes for Reporting Sequence Variations

Sequence type Prefix Position

Coding DNA c. First nucleotide of the translation start codon of the coding reference DNA sequence (most commonly used)

Protein p. First nucleotide of genomic reference DNA sequence(provides information most recognizable)

Genomic DNA g. First nucleotide of genomic reference DNA sequence

Noncoding DNA n. First nucleotide of the noncoding reference DNA sequence

Mitochondrial DNA m. First nucleotide of mitochondrial reference DNA sequence

RNA r. First nucleotide of the translation start codon of the RNA reference sequence or first nucleotide of the noncoding RNA reference sequence

Protein p. First nucleotide of genomic reference DNA sequence

Modified with permission from den Dunnen JT, Dalgleish R, Maglott DR, et al. HGVS recommendations for the description of sequence variants: 2016 update. Hum Mutat. 2016;37(6):564-569.

55REPORTING, INTERPRETATIONS, AND QUALITY ASSURANCE

Of note, several population polymorphisms are seen in the EGFR coding region. According to the guidelines developed by the Association for Molecular Pathology (AMP), American Society for Clinical Oncology (ASCO), and the College of American Pathologists (CAP) regarding the reporting of somatic alterations, alterations that can be reliably classified as Tier IV (variants of known insignificance) should not be included in clinical reports (Li 2017).

Reporting of EGFR Mutation Status in Patients Who Have Received TKI TherapyInformation about whether a patient has received TKI therapy should be given to the labora-tory before EGFR testing is done. This information is important particularly if a different assay with higher allelic/analytic sensitivity is used to examine the presence of T790M. Specifically, assays that are not designed to sensitively detect T790M may underdetect this alteration, which can lead to changes in therapy selection. Furthermore, if testing conclu-sively demonstrates the absence of T790M after treatment, this absence deserves specific mention in the report. This interpretation also can be aided, and to some extent modified, by the knowledge of the pre-existing alteration that conferred eligibility for TKI because it can serve as a marker of tumor enrichment in the tested sample. For example, a sample in which the original alteration is seen at a level close to the assay limit of detection is likely to underdetect a T790M mutation because the tumor enrichment is unlikely to be sufficient to identify a subclonal alteration. In addition, evolving molecular alterations are continually being identified in the setting of disease progression during targeted therapy. For example, C797S has been identified as a mechanism of resistance to third-generation EGFR TKIs; however, most commercially available real-time PCR assays for EGFR mutation assessment do not include this alteration. Thus, the need to adapt assays to ongoing knowledge is crucial.

Reporting of EGFR Mutation Status Using Next-Generation Sequencing PlatformsThe rapid adoption of methodologies based on next-generation sequencing (NGS) adds new concerns and opportunities for the clinical interpretation of results. As noted in the AMP/ASCO/CAP guideline for clinical reporting of variants, a four-tier system has been recommended for classification of variants, with only the first three tiers being included in clinical reports (Li 2017). Tier I includes variants of strong clinical significance, which in the case of EGFR includes all known sensitizing and resistance mutations with appro-priate interpretation. Tier II includes variants of potential clinical significance, Tier III is for variants of unknown clinical significance, and Tier IV comprises variants of known insignificance. However, distinguishing between Tiers II and III for rare EGFR variants may be challenging, and the vast majority of so-called novel EGFR variants are likely to be classified in Tier III. Researching of individual variants is needed for this classification, and usually requires examination of the databases referenced in the published guideline docu-ment, such as the Catalogue of Somatic Mutations in Cancer (http://cancer.sanger.ac.uk/cosmic) and the cBioPortal for Cancer Genomics (Cerami 2012, Gao 2013). Among other helpful websites is a publicly available database of EGFR structure (http://bcc.ee.cityu.edu.hk/data/EGFR.html). In addition, the editors of the Journal of Thoracic Oncology specifically seek case reports on rare EGFR variants and the resulting response to targeted therapy to expand the knowledge base.


Interpretation of EGFR mutation status should be correlated with the evaluation of the tissue and extent of tumor enrichment. Specifically, a strongly worded disclaimer should be included when the results of EGFR testing is negative and tumor enrichment is not certainly to a sufficient degree to meet the analytic requirements of the assay. Such a disclaimer should include a statement about the possibility of false-negative findings attributable to the ability to enrich tumor content, as well as information about the potential for repeat testing on an alternate sample if clinically indicated.

Assay Validation and Quality ManagementAssay ValidationAssay analytic validation refers to the laboratory process of ensuring that numerous param-eters of an individual assay are well defined and understood. Analytic validation plays a central role in ensuring the ongoing reliability and reproducibility of an assay, as well as in understanding the limitations of the assay that may lead to reduced confidence in an individual result. Full analytic validation of an assay comprises evaluation of multiple param-eters, which can be time- and labor-intensive. The list of individual assay parameters to be evaluated is extensive, but includes elements such as reproducibility among technologists, reproducibility between days, and allelic sensitivity (also referred to as analytic sensitivity). For NGS-based assays, additional validation requirements are centered on bioinformatic processes and types of alterations (eg, single-nucleotide variants, insertions, deletions, and structural variants). The burden for analytic validation is largely dependent on the regulatory status of the assay used. In the United States, an assay that has been approved by the US FDA (typically referred to as a companion diagnostic assay) may be used in a laboratory with a lower level of rigor, referred to as verification. In this circumstance, the assay validation param-eters have been pre-established, and the laboratory is responsible for ensuring that the assay performs as expected. For assays that are not approved by the FDA or when modifying a FDA-approved assay, an assay is considered to be a laboratory-developed test (LDT), and the laboratory carries the burden of fully validating the analytic performance of the assay. A full description of the validation process for an assay is beyond the scope of this section, but key points are briefly noted. When a laboratory conducts a validation of an assay for the detection of EGFR alterations, there are several key considerations. Samples represent-ing a cross-spectrum of EGFR alterations should be used, including those with uncommon variants. Importantly, when using an NGS-based assay, insertions and deletions of varying sizes and locations should be identified to confirm that the bioinformatics processes used can accurately detect alterations. It is important that samples representing the lowest limit of detection are also used. Given the difficulty in locating samples representing this cross-spectrum, purchased control standards may be needed. The laboratory also must consider sample types in the assay-validation process. If use of specific fixatives has the potential to alter or interfere with mutation detection, the fixatives should be individually validated. Of note, however, is that many assays have internal controls or metrics that can be used in lieu of validating every possible combination of preanalytic handling of specimens.

57REPORTING, INTERPRETATIONS, AND QUALITY ASSURANCE

Lastly, it is crucial to define the allelic (analytic) sensitivity of an assay. Regarding assays for detection of EGFR mutations, it is important to attempt to control for EGFR amplifica-tion status in validation samples because samples in which an EGFR mutation is present and amplified may misrepresent the allelic sensitivity of the assay. For NGS-based assays, allelic sensitivity may be different between single-nucleotide variants and insertions and/or deletions. A laboratory should select an assay that can account for the ability to enrich for tumor with use of techniques such as macrodissection and microdissection. Sanger sequencing, which has the least analytic sensitivity, should be used only if a laboratory has the technical capabilities to achieve sufficient tumor enrichment.

Assay Quality ManagementA comprehensive laboratory- and assay-validation program is a key element for ongoing high-quality results. Although also beyond the scope of this chapter, key points are listed for consideration. Chief among these points is the accreditation status of the laboratory. All major laboratory-accreditation organizations have requirements for maintenance of a high-quality laboratory environment and for ongoing quality management of individual assays. Other key elements include participation in proficiency testing, consistent inclusion of assay controls, and ongoing monitoring of mutation-detection rates for comparison with known populations.

StandardizationWith a wide spectrum of available assays across multiple platforms available for detection of EGFR mutations, standardization is difficult. This challenging area is noted as an ongoing issue in somatic mutation detection across a variety of tumor types, and numerous efforts are underway to establish standards for the detection of somatic mutations. However, most efforts toward generating a set of reference standards for somatic mutations are focused on a wide spectrum of alterations in tumors, focused on NGS technologies. In the context of NGS, it is crucial to use reference standards to demonstrate confident detection abilities across multiple mutation types (eg, single-nucleotide alterations, insertions, and deletions). The ability of NGS wet bench and bioinformatics (dry bench) pipelines is different between single-nucleotide alterations and insertions and/or deletions. Thus, the use of reference standards, including the availability of some commercial reference standards with known allelic fraction, is essential during validation. To the extent possible, samples containing the widest spectrum of possible EGFR alterations should be obtained for validation. Although attempts to standardize through the use of reference materials may provide some advances in the comparability of EGFR mutation detection, tumor enrichment—a cru-cial parameter in testing—is unlikely to become standardized. All human tumor samples are composed of a mixture of tumor and nontumor tissue (eg, inflammatory cells and stroma), and the extent to which the allelic sensitivity of an assay can apply to an individual sample is often dictated by the extent to which tumor enrichment is feasible. Some tumor enrichment strategies involve macrodissection, in which an area of tumor is broadly enriched based on review of a parallel H&E-stained slide. In contrast, some laboratories perform a higher degree of tumor enrichment, using microdissection, in which tumor cells are selectively isolated under a microdissecting microscope. These differences result in inherent variability


that may not be effectively mitigated through standardization of the technical components of the assays themselves. Other areas of EGFR mutation detection lend themselves to standardization as well. As noted, use of Human Genome Variation Society nomenclature is strongly recommended in testing reports, and widespread use of this approach is likely to improve the readabil-ity of results across laboratory settings. Similarly, use of recommendations, such as those in the CAP/IASLC/AMP guideline (Lindeman 2013), will improve comparability across laboratories.

Access to Testing Guidelines and AlgorithmsBy Yasushi Yatabe, Ming Sound Tsao, and Keith M. Kerr 7The testing algorithms for driver mutations or other molecular alterations that represent potential druggable targets are driven by numerous factors. These factors include the patients to be tested, the samples available, the technology in the testing laboratory, access to drugs, and, obviously, access to testing. The vast majority of patients who are candidates for testing according to current guidelines are those whose tumors are adenocarcinomas, whose tumors include an adenocar-cinoma component, or those for whom a diagnosis of adenocarcinoma cannot be reasonably excluded. (See Chapter 2 for a discussion of patient candidates for testing.) Oncogene addic-tive drivers such as EGFR mutation and ALK or ROS1 gene rearrangement are essentially found in cancers whose genesis is not related to tobacco carcinogens and in tumors that have arisen in the peripheral lung epithelium, the terminal respiratory unit (Yatabe 2005). Although all of these drivers are more common in never-smokers, they may also be found at lesser frequency in active and former smokers (Barlesi 2016, Zhang 2016). Therefore, the presence of these and other molecular drivers, such as KRAS and BRAF V600 mutations and RET rearrangements, are sought in the same patient population. These addictive oncogenic drivers are, for all practical purposes, mutually exclusive—only one of these alterations, if any, will be present in a tumor. In a white population, KRAS mutations are the most prevalent of those mentioned here, followed by EGFR mutations; RET rearrangements are among the least common (Sholl 2015, Barlesi 2016). In an East Asian population, EGFR mutations are more prevalent than KRAS mutations (Serizawa 2014, Li 2016). Sequential testing, which tests biomarkers in order of their frequency, has been promoted as a potentially more economical approach that also uses less tissue. However, this approach takes much longer to deliver comprehensive results, and rare mutations would be be unacceptable late in delivery. Most laboratories use parallel testing strategies for targeted biomarkers. For EGFR testing, this strategy would be either one or several of the stand-alone methodologies discussed in this chapter or part of a panel approach involving the use of massively parallel next-generation sequencing (NGS)


technologies. NGS allows for a greater number of biomarkers to be tested, including gene rearrangements and mutations. The tissue samples generally available for molecular testing in lung cancer are very limited, and as more biomarker testing is required, testing becomes more challenging to deliver, especially if individual stand-alone tests are being used. If a laboratory judges a sample to be inadequate for multigene NGS profiling, priority may be given to a single test for a common mutation, such as EGFR. In these circumstances, cfDNA from plasma may also provide a possible source of DNA for an EGFR mutation test and, in the future, other biomarkers. (See Chapter 3.) IHC also has a role in this complex testing scenario. Current guidelines consider testing mandatory for EGFR mutations, ALK and ROS1 rearrangements, and PD-L1 expression. IHC can be used for both ALK and ROS1 testing because the levels of these proteins are elevated when oncogenic rearrangement is present. ALK positivity by IHC is adequate test-ing to initiate treatment; however, an orthogonal molecular test is required as confirmation for ROS1 positivity by IHC (Tsao 2017, Lindeman 2017). PD-L1 testing is strictly by IHC and performed in parallel with those tests based on DNA (or RNA) extraction from the same tissue sample. Although EGFR mutation testing is based on extracted DNA, antibod-ies against the EGFR L858R mutant protein and one of the common exon 19 deletions are available for use in IHC. The limitations of IHC for EGFR testing are described in Chapter 5, and the method is not recommended in the updated joint guideline by The College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC), and the Association for Molecular Pathology (AMP) on molecular testing standards for EGFR and ALK testing (Lindeman 2017). Some laboratories use this approach for bone biopsy samples, in which decalcification may render DNA unsuitable for molecular testing. IHC based on anti-EGFR mutant protein is also sometimes used as a solution to provide more rapid results while awaiting results of NGS testing. This is a local practice decision that should be taken in the knowledge of the shortcomings of this testing approach. As previously mentioned, testing for EGFR mutations, as for ALK and ROS1 rearrange-ments, is considered mandatory because of the array of available drugs for treating patients based on these molecular markers. Another molecular driver was added to this list on June 22, 2017, when the US FDA approved dabrafenib and trametinib in combination for patients with metastatic BRAF V600E mutation-positive NSCLC. However, nearly one-half of BRAF mutations in lung cancer have mapped to sites other than V600. If drugs are not available, then the testing strategy is likely to evolve, driven by clinical requirements around a more limited access to therapy. The same may be said of access to testing itself. In some coun-tries, testing may be difficult to obtain and may be of limited spectrum. Outsourcing may be required if testing is not available locally or even within the same country. Regarding EGFR specifically, access to TKIs is generally available because of how long these agents have been available and because of their demonstrated efficacy.

Multiplex Testing for EGFR and Other Driver OncogenesAs more biomarkers are used for testing and tissue availability becomes an issue, the need for a multiplex testing approach will increase, assuming the available tissue is adequate for the methodology to be used. The average diagnostic tissue sample containing NSCLC presents

61ACCESS TO TESTING GUIDELINES AND ALGORITHMS

several challenges for testing, and there has been a concerted effort to use less-invasive procedures for tissue biopsies. Samples often include a very small tumor section, with a low number of tumor cells. In bronchial biopsy samples, the median NSCLC content ranges from 10% to 25%, depending on histologic subtype (Coghlin 2010). These samples must be processed in a way that allows for all initial and possible subsequent testing, including the initial histologic confirmation of primary lung cancer, histologic subtyping, and any required molecular testing. In addition, at each diagnostic phase, especially the early ones, it is impera-tive to take steps to preserve tissue. These steps include using block-cutting strategies to limit waste and limiting diagnostic IHC for tumor subtyping (Figure 1) (Kerr 2016, Travis 2015). Multiplex, parallel biomarker testing is the preferred approach because delivery of comprehensive results takes too long with sequential testing, especially when more biomark-ers are involved. Although EGFR mutation is the most common actionable target in NSCLC, it should not be considered in isolation (assuming therapies for other targets are accessible). It is much more practical to prepare tissue material for a single DNA extraction and to use that DNA in a single multiplex testing environment. Based on costs, reimbursement restric-tions, and drug availability, it is feasible to deliver results using several stand-alone tests

Biopsy: suspicious for lung cancer

Diagnostic stains

H&E

NSCCH&E

ALK IHC ± FISH

ROS1 IHC ± FISH

PD-L1

EGFR

12 +

H & E staining for histological diagnosis

For IHC

A. Biomarker/molecular testing sections prepared together with H&E section

H & E staining for histological diagnosis

For IHC

For molecular testing

B. Biomarker/molecular testing sections prepared after initial H&E assessment

For molecular testing

Re-sectioning

1

2

1-2

1-2

2-4

unstained sections

Figure 1. Strategies for maximizing tissue for molecular testing. As shown here, some laboratories may cut additional sec-tions in anticipation of IHC and FISH testing, but sections are cut as required in molecularly sterile conditions for molecu-lar analysis (DNA/RNA extraction). Reprinted with permission from Tsao MS, Kerr KM, Dacic S, Yatabe Y, Hirsch FR (eds). IASLC Atlas of PD-L1 Immunohistochemistry Testing in Lung Cancer. North Fort Myers, FL: Editorial Rx Press; 2017.


when material is handled carefully and the number of markers being tested is limited. The use of multiple stand-alone tests becomes cumbersome and more expensive as the number of markers required for testing increases, whereas the practicality and cost-effectiveness of an NGS approach increases along with the number of markers being tested (Konig 2015). Based on its approval of dabrafenib and trametinib in combination for treatment of patients with BRAF V600E-positive NSCLC, the US FDA also approved the Oncomine Dx Target Test (Thermo Fisher Scientific), an NGS test for multiplex detection of BRAF, ROS1, and EGFR gene mutations or alterations in NSCLC tissue. This tool is the first NGS oncology panel test approved by the FDA for multiple companion diagnostic indications. The discovery of additional targets, beyond those associated with available approved drugs, may facilitate patient access to clinical trials. In parallel to DNA-based testing, IHC-based testing is also becoming more important and should be conducted simultaneously with DNA-based testing. IHC for ALK and ROS1 alterations has been outlined previously in this chapter. In addition to IHC based on anti-mutant proteins of EGFR, antibodies also are available against BRAF protein, although use

in NSCLC is probably limited. Of course, PD-L1 IHC testing is now mandatory for NSCLC and is an integral part of this complex algorithm (Figure 2). In the future, IHC may be required to determine choice of immunotherapy, placing greater demands on the tissue resource. Multiplex IHC may well be an answer to delivering several IHC tests on a single or limited number of tissue sections. Current experience with NGS technology is mixed. Excellent results are generally

Figure 2. Once NSCLC is diagnosed and subtyped, cases are selected for biomarker testing to guide the choice of therapy according to local protocols. A parallel track of molecular testing on DNA extracted from tumor tissue and tissue-based tests using IHC and possibly FISH is now emerging as a standard approach. NGS = next-generation sequencing, NSCLC-NOS = non-small cell lung cancer not otherwise specified.

NSCLC-NOSRate <10% 25–40% of cases

morphologicallyNSCLC-NOS

Diagnose and subtype

lung cancer

Squamous, adenocarcinoma

etc

TTF1 / p40IHC

ONLY if required

Biomarker testing

dictated by histology and

protocol

Sections for Biomarker IHC & FISH

Sections for DNA

extraction

EGFR (BRAF, KRAS)

mutation(NGS panels)

ALK, ROS1PD-L1

Morphology-based tests

Molecular tests

Predominantly adenocarcinoma (see text)Selected squamous and SCLC

63ACCESS TO TESTING GUIDELINES AND ALGORITHMS

achievable for mutation testing, although individual gene failures are a reality, and it may not always be possible to complete a full mutation panel screen on all samples (Hiley 2016). NGS approaches pose more of a challenge for fusion genes, and some authorities con-sider it prudent to confirm positive results using an orthogonal methodology such as IHC, FISH, or some other approach. Nevertheless, any laboratory that adopts NGS technology for clinical multiplex testing of driver mutations must conduct analytic validation exercises as part of its quality-assurance program. The presence of the ALK protein, as determined by FISH, has been shown to be important for predicting response to ALK TKIs (Hiley 2016). Furthermore, health care regulations do not allow PD-L1 IHC testing results to be replaced by tumor mutation burden provided by NGS, therefore, integration of molecular and morphology-based testing should be indispensable (Figure 2). When stand-alone testing is used, the four-exon coverage required for comprehensive EGFR testing may not be successful, depending on sample quality and quantity and on the other gene tests being attempted. Some laboratories may prefer to use a commercial allele-specific approach for tissue samples at the poorer end of the spectrum, while favoring the more complete Sanger approach for better samples; fragment length analysis for EGFR exon 19 deletions is often very reliable in poor samples. Although KRAS is currently not a routinely actionable target outside of clinical trials, a positive result for a KRAS mutation (performed by an easy and robust stand-alone test) vastly reduces the chance of a false-negative result on EGFR testing, when the latter fails. This principle would also apply to any NGS testing approach.

Choosing a Testing Platform for Treatment DecisionsA laboratory must consider several factors when choosing a testing platform. These factors include availability of a specific sequencing platform and technical competence regarding the platform, level of funding, sample availability, desired turnaround time for results, and availability of other targeted therapies as treatment options. In clinical practice, a single-gene testing platform for EGFR mutations is considered sufficient because only EGFR TKIs have received worldwide approval for clinical use in patients with lung cancer. For single-gene testing, the most important consideration is the use of platforms with sensitivity adequate for a minimum of 5% to 10% of tumor cells in the sample, as well as the ability to detect less common sensitizing mutations on exons 18-21 of EGFR. (See Chapter 5 for a discussion of various types of tests.) However, when the sample is very small and the amount of isolated DNA is borderline adequate, the laboratory may prioritize testing for exon 19 deletions and L858R mutations because these compose 90% of all sensitizing EGFR mutations. Single-gene testing also has the advantages of shorter turnaround time and lower cost compared with NGS platforms. Notwithstanding the drawbacks of increased cost and turnaround time for results, the greatest advantage of using an NGS platform is its ability to simultaneously detect all poten-tially tractable driver mutations in one assay. This may prove to be more effective, both financially and in terms of sample utilization, because patients who have tumors that are found to be negative for EGFR mutations but who harbor other driver aberrations may be enrolled in appropriate clinical trials, which also may result in potential clinical benefit (Kim 2011, Kris 2014, Barlesi 2016, Jordan 2017).


As previously discussed, IHC using EGFR-mutant–specific antibodies has demonstrated high specificity (greater than 95%) in detecting specific types of EGFR mutations (ie, E746-A750 deletion and L858R mutation). However, the sensitivity of this platform has been shown to be moderate, at 47% to 96% (Brevet 2010, Kitamura 2010, Cooper 2013, Allo 2014, Seo 2014, Kim 2015, Ragazzi 2016). Despite the moderate sensitivity, some institutions use this platform for initial screening for EGFR mutations. However, the application of EGFR-mutant-specific IHC requires robust optimization, validation, an external quality-assurance program, and confirmation of positive results by molecular testing. Therefore, the CAP/IASLC/AMP guideline does not recommend the use of EGFR-mutation-specific IHC for routine selection of EGFR TKI therapy (Lindeman 2013). This testing should be considered only when no alternative is available. Lastly, because plasma-circulating cfDNA technology is rapidly being validated as a modestly sensitive and highly specific method for detection of EGFR mutations in patients with lung adenocarcinoma, this testing platform may soon be available in molecular diag-nostic laboratories. The update of the CAP/IASLC/AMP guideline on molecular testing in lung cancer includes a recommendation that, in some clinical settings in which tissue is limited and/or insufficient for molecular testing, physicians may use a cfDNA assay to identify EGFR mutations. (See Chapter 3 for a discussion of plasma-based testing.) In contrast, consensus is emerging that cfDNA testing will become the primary method for detecting the EGFR T790M acquired-resistance mutation in patients with lung adenocarcinoma and with disease progression or secondary clinical resistance to EGFR TKIs; testing of the tumor sample is recommended if the plasma result is negative. Furthermore, promising results have been reported on multiplex testing using cfDNA, and blood-based testing may play a significant role for molecular testing of lung cancer in the future (Thompson 2016, Paweletz 2016, Malapelle 2017b).

ConclusionAdvances in NGS techniques enable us to relatively easily obtain a molecular landscape for individual lung cancers, and therapeutic selection based on genetic alterations is clearly bene-ficial for patients. In contrast, there are some practical barriers, including sample availability for molecular testing, turnaround time according to the patients’ conditions, reimbursement for testing, and accessibility of targeted drugs. As has been noted, it is important to rank genetic alterations into several groups according to clinical benefits and drug availability. EGFR, ALK, ROS1, and BRAF V600E are currently accepted as the most actionable targets, and appropriate treatment options should be offered when a patient’s tumors is positive for one of these alterations. If the results of testing for all four genetic alterations are negative, a more comprehensive analysis, such as NGS, is warranted to inform alternative treatment choices.

Summary and Future Perspectives By Tony S. Mok, David P. Carbone, and Fred R. Hirsch 8IASLC has published an Atlas on ALK and ROS1 testing, and more recently another one on PD-L1 testing. Both are novel therapeutic targets whose assessment is essential for optimal management of advanced-stage NSCLC. These Atlases were published in a timely manner with the objective of summarizing the state-of-the-art to provide the most updated infor-mation to pathologists, laboratory scientists, and practicing physicians. Interestingly, while EGFR mutations are the first discovered and with PD-L1 are the most common molecular targets in lung cancer, we have published an Atlas on PD-L1 testing but have not addressed EGFR mutation testing until now. Compilation of this Atlas has benefited from the large amount of information developed over the past decade by a panel of experienced interna-tional experts. The chapters on clinical perspectives and EGFR testing have illustrated the importance of testing for EGFR mutations in the selection of first-line therapies. With the well-documented high response rates and prolonged high-quality progression-free survival using EGFR TKIs as first-line treatment of choice for patients with pulmonary adenocarcinoma, EGFR test-ing has become a universal standard. Upon treatment progression to first-line EGFR TKI, about 50% of patients develop EGFR exon 20 T790M mutation, thus in addition to testing before starting treatment, practicing clinicians must also test for this acquired resistance mechanism to make osimertinib, a highly effective third-generation EGFR TKI, available to their patients. Sample acquisition techniques and an expanding variety of types of mutation testing add to the complexity of this field, as we have extended our testing beyond classical tumor biopsies. Liquid biopsy using the novel source of cfDNA from plasma and urine have pro-vided new platforms for detection and monitoring of EGFR mutations. The technical details provided in this chapter are important to both pathologists and laboratory scientists per-forming these tests. The section on the comparison of different assay platforms is unique, and there is no doubt that this is the most comprehensive review on the topic. Validation, prioritization, and reporting also need to be standardized. Our expert panel taps on their vast experience and provides suggestions on validation process and quality


control. Given the expanding list of other biomarkers that need to be tested, this chapter also addresses the sequence and multiplexing of these molecular tests. The IASLC Atlas on EGFR Testing in Lung Cancer is the first and most comprehensive publication of its kind, and we have successfully addressed the essential topics related to the testing of EGFR mutations. However, many unsolved questions remain and a number of important questions need to be addressed in the future.

1. Dynamic monitoring of EGFR mutationAdvances in the detection of EGFR mutation from cfDNA in plasma or urine have opened the door for monitoring of EGFR driver and resistance mutation status during treatment. Early studies have shown that persistent EGFR mutations detectable in the blood during EGFR TKI therapy are associated with shorter progression-free survival and overall survival. However, much more research is required to determine whether dynamic moni-toring improves outcomes and should be standard practice. Monitoring is meaningful only if there are appropriate and efficacious interventions that improve overall outcomes. High sensitivity and specificity of the testing method are also essential to effective moni-toring to avoid errors in clinical therapy choices. Thus, a prospective randomized study involving the use of a high-quality testing method is warranted to address the role of dynamic monitoring.

2. Molecular prediction of SCLC transformationSCLC transformation accounts for about 10% of EGFR TKI resistance in some series. The majority of SCLC-transformed relapses are difficult to treat, and the mortality rate is high. Recent reports indicate that TP53 and RB1 loss are universal in this process of transformation and these molecular changes occur early during the course of therapy. It is crucial to better understand the molecular basis of this process, and whether it may be advantageous to identify patients at risk for this transformation for early interven-tion studies.

3. Molecular profile of osimertinib resistanceThe median progression-free survival associated with osimertinib in T790M-positive resistance is about 10 months. EGFR exon 20 C797S mutations account for about 20% of the resistance. Detection of this resistance mutation from cfDNA may be the first step to targeted therapy in this setting. Apart from this, some patients lose the T790M mutation, whereas others have disease progression in the presence of a T790M mutation. Molecular features and drivers of the tumors in these two groups of patients is expected to be different. A comprehensive approach for the serial molecular profiling of patients with osimertinib resistance will be important to improve the outcomes for these patients.

4. Understanding the basis of adaptive persistenceDramatic tumor shrinkage is often seen in tumors with the mutant EGFR driver, but there is always a larger or smaller population of cells containing the driver mutation, without evidence of a known resistance mutation that persists through the initial days of therapy. For this population, survival is long enough for acquired resistance to occur,

67SUMMARY AND FUTURE PERSPECTIVES

and patients ultimately die. Better understanding of the molecular basis of the variable depth of response in tumors with identical drivers, and how a subset of cells survives the initial days of therapy will be important to converting responses to cures.

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Testing for EGFR mutations and related developments have helped to establish a new molecular treatment paradigm, making precision medicine a reality for many thousands of patients with lung cancer. Now consensus on the establishment and optimization of EGFR testing is needed. The IASLC Atlas of EGFR Testing in Lung Cancer is a useful guidebook for this purpose.

In this text, pathologists are provided with “know-how” information on EGFR testing, and clinicians are provided with the “know-why” information about how to interpret the results. From the retrieval and handling of tumor sample to the different available assays, and from interpretation of results to reporting and quality assurance, the Atlas is a comprehensive yet user friendly compendium for the general oncology readership.

The first and most comprehensive publication of its kind, the IASLC Atlas on EGFR Testing in Lung Cancer addresses the essential topics related to the testing of EGFR mutations.

North Fort Myers, FLwww.EditorialRxPress.com

IASLC acknowledges the generous funding and support provided

by AstraZeneca for the IASLC Atlas of EGFR Testing in Lung Cancer.

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