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Updated Molecular Testing Guideline for the Selection of Lung Cancer Patients for Treatment With Targeted Tyrosine Kinase Inhibitors

Guideline From the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology
Open ArchivePublished:January 25, 2018DOI:https://doi.org/10.1016/j.jtho.2017.12.001

      Context

      In 2013, an evidence-based guideline was published by the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology to set standards for the molecular analysis of lung cancers to guide treatment decisions with targeted inhibitors. New evidence has prompted an evaluation of additional laboratory technologies, targetable genes, patient populations, and tumor types for testing.

      Objective

      To systematically review and update the 2013 guideline to affirm its validity; to assess the evidence of new genetic discoveries, technologies, and therapies; and to issue an evidence-based update.

      Design

      The College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology convened an expert panel to develop an evidence-based guideline to help define the key questions and literature search terms, review abstracts and full articles, and draft recommendations.

      Results

      Eighteen new recommendations were drafted. The panel also updated 3 recommendations from the 2013 guideline.

      Conclusions

      The 2013 guideline was largely reaffirmed with updated recommendations to allow testing of cytology samples, require improved assay sensitivity, and recommend against the use of immunohistochemistry for EGFR testing. Key new recommendations include ROS1 testing for all adenocarcinoma patients; the inclusion of additional genes (ERBB2, MET, BRAF, KRAS, and RET) for laboratories that perform next-generation sequencing panels; immunohistochemistry as an alternative to fluorescence in situ hybridization for ALK and/or ROS1 testing; use of 5% sensitivity assays for EGFR T790M mutations in patients with secondary resistance to EGFR inhibitors; and the use of cell-free DNA to “rule in” targetable mutations when tissue is limited or hard to obtain.
      Patients with advanced lung cancer have a poor prognosis, with a median survival of 1 year. However, for many patients whose tumors harbor certain specific molecular alterations (eg, activating alterations in the EGFR, ALK, and ROS1 genes), particularly in lung adenocarcinoma, targeted tyrosine kinase inhibitor (TKI) therapy provides significant improvement in survival and quality. Accordingly, patients with the types of advanced lung cancer in which these targetable molecular alterations typically occur should receive the molecular testing required to identify them, and thereby receive appropriate targeted treatments. Importantly, this testing should extend beyond those molecular alterations for which targeted therapies are approved by regulatory agencies such as the US Food and Drug Administration (FDA) to include molecular alterations for which there is compelling evidence of effective investigational targeted therapies (and, more recently, immunotherapies) from published clinical trials.
      In 2010, 3 professional societies—the College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC), and the Association for Molecular Pathology (AMP)—recruited specialists in the biology, diagnosis, and treatment of lung cancer to form a joint working group to systematically assess the evidence supporting the clinical utility of molecular analysis of lung cancer samples. In 2013, this working group published an evidence-based guideline
      • Lindeman N.I.
      • Cagle P.T.
      • Beasley M.B.
      • et al.
      Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology.
      • Lindeman N.I.
      • Cagle P.T.
      • Beasley M.B.
      • et al.
      Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology.
      • Lindeman N.I.
      • Cagle P.T.
      • Beasley M.B.
      • et al.
      Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology.
      for standard-of-care clinical practice concerning which lung cancer patients and samples should be tested, which genes should be tested, and how these tests should be designed, validated, and executed. This guideline was subsequently endorsed by the American Society of Clinical Oncology,
      • Leighl N.B.
      • Rekhtman N.
      • Biermann W.A.
      • et al.
      Molecular testing for selection of patients with lung cancer for epidermal growth factor receptor and anaplastic lymphoma kinase tyrosine kinase inhibitors: American Society of Clinical Oncology endorsement of the College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology guideline.
      and has been cited in guidelines developed by numerous professional societies around the world.

      Ettinger DS, Wood DE, Aisner DL, et al. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines): non-small cell lung cancer. Version 6.2017. National Comprehensive Cancer Network, Inc. https://www.nccn.org. Accessed June 12, 2017.

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      Molecular biomarkers for the evaluation of colorectal cancer: guideline from the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and the American Society of Clinical Oncology.
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      Guideline for the acquisition and preparation of conventional and endobronchial ultrasound-guided transbronchial needle aspiration specimens for the diagnosis and molecular testing of patients with known or suspected lung cancer.
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      Executive summary of the SEPAR recommendations for the diagnosis and treatment of non-small cell lung cancer.
      • von Laffert M.
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      • et al.
      ALK-testing in non-small cell lung cancer (NSCLC): immunohistochemistry (IHC) and/or fluorescence in-situ hybridisation (FISH)?: statement of the Germany Society for Pathology (DGP) and the Working Group Thoracic Oncology (AIO) of the German Cancer Society e.V. (Stellungnahme der Deutschen Gesellschaft fur Pathologie und der AG Thorakale Onkologie der Arbeitsgemeinschaft Onkologie/Deutsche Krebsgesellschaft e.V.).
      Scottish Intercollegiate Guidelines Network (SIGN)
      Management of lung cancer.
      However, the field has continued to advance rapidly, with the emergence of new genetic discoveries, new therapies, and new technologies, such that these same 3 organizations convened a second working group to systematically assess new evidence and to issue an evidence-based revision of the lung cancer molecular pathology practice guideline.
      The revision focuses on new recommendations in 5 specific content areas: (1) Which new genes should routinely be tested for alterations in lung cancers? (2) What methods are appropriate for lung cancer testing, with particular emphases on the use of immunohistochemistry (IHC) and next-generation sequencing (NGS)? (3) Is there a need to test patients with squamous cell, small cell, or other nonadenocarcinoma lung cancers? (4) What testing should be performed for patients with a targetable alteration who have progressed following initial response to appropriately targeted therapy? (5) What is the role of testing circulating cell-free DNA (cfDNA) in lung cancer patient management? In addition, new evidence supporting the original 2013 guideline was reviewed and used to either modify the strength of those recommendations or change them entirely. Finally, a sixth question, regarding diagnostic support for the role of immunomodulatory therapies (eg, programmed death ligand-1 or PD-L1), emerged during the revision process. Although this topic was not subject to the systematic review of evidence, the expert panel decided to issue an opinion statement addressing this question, aware that separate efforts are currently underway to develop evidence-based recommendations regarding the use of biomarkers to select patients for immunomodulatory therapies.
      One particular challenge for this evidence-based guideline revision was the rapid pace of discovery in this field. During the time between literature review and guideline drafting, major new discoveries were published and treatment advanced for BRAF-mutant lung cancers and for the use of immunotherapies. We expect that many additional advances will emerge in the fields of targeted therapy, cfDNA diagnostics, and immunotherapies in the near term. Although we make strong recommendations for the molecular biomarkers for which there was good evidence at the time we conducted our analysis, we also fully recognize the importance of emerging biomarkers to enable lung cancer patients to be eligible for clinical trials of investigational therapies. Accordingly, we have stratified the biomarkers in this guideline into 3 categories, rather than 2. The first are “must-test” biomarkers, which are standard of care for all patients with advanced lung cancer with an adenocarcinoma component who are being considered for an approved targeted therapy. Second are “should-test” biomarkers, which are used to direct patients to clinical trials and which should be included in any large sequencing panel that is performed for lung cancer patients, but which are not required for laboratories that perform only single-gene assays. All remaining candidate biomarkers are investigational and are not appropriate for clinical use at this time.

      Panel Composition

      The CAP, IASLC, and AMP convened an expert panel consisting of practicing pathologists and oncologists with expertise and experience in lung carcinoma. The CAP, IASLC, and AMP approved the appointment of the project cochairs and expert panel members. In addition, a methodologist experienced in systematic review and guideline development consulted with the panel throughout the project.

      Conflict of Interest Policy

      Prior to acceptance on the expert panel, potential members completed a joint conflict of interest disclosure process, whose policy and form require disclosure of material financial interest in, or potential for benefit of significant value from, the guideline’s development or its recommendations. The potential members completed the conflict of interest disclosure form, listing any relationship that could be interpreted as constituting an actual, potential, or apparent conflict. Potential conflicts were managed by the cochairs. All expert and advisory panel members were required to disclose conflicts prior to beginning and continuously throughout the project’s timeline. Disclosed conflicts of the expert panel members are listed in the Appendix. The CAP, IASLC, and AMP provided funding for the administration of the project; no industry funds were used in the development of the guideline. All panel members volunteered their time and were not compensated for their involvement. Please see the supplemental digital content (SDC) for full details on the conflict of interest policy.

      Objective

      The expert panel was charged with the review and update of the CAP-IASLC-AMP molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors. The panel reviewed any new studies that would change or refute the statements from the 2013 guideline. In addition, the panel also addressed additional key questions:
      • 1.
        Which new genes should be tested for lung cancer patients?
      • 2.
        What methods should be used to perform molecular testing?
      • 3.
        Is molecular testing appropriate for lung cancers that do not have an adenocarcinoma component?
      • 4.
        What testing is indicated for patients with targetable mutations who have relapsed on targeted therapy?
      • 5.
        What is the role of testing for circulating cell-free DNA for lung cancer patients?
      Key questions 1 through 3 relate to patients diagnosed with nonsquamous non–small cell lung cancer (NSCLC) of all stages. The key questions are included in full detail in the SDC.

      Methods

      A detailed account of the methods used to create this guideline can be found in the SDC, including additional scope questions.

      Systematic Literature Review and Analysis

      A systematic literature review was completed with 2 comprehensive searches. The first search was designed to assess the 2013 guideline statements and was based on the original search strategy. It included medical subject headings and keywords to address the concepts lung cancer, tumor biomarkers, and laboratory testing and was run in Ovid MEDLINE (Ovid Technologies, Inc, New York City, New York) on May 17, 2015, to locate studies published in English with publication dates from January 1, 2012 through May 17, 2015. Publication filters were applied to identify guidelines, systematic reviews, meta-analyses (MAs), and randomized clinical trials. The search was rerun on June 27, 2016, to identify relevant new literature published since May 17, 2015.
      The second search was based on new key questions that focused on additional biomarkers not included in the 2013 guideline, with specific search strategies designed for each key question. All searches were performed in Ovid MEDLINE and PubMed (US National Library of Medicine, Bethesda, Maryland) (June 28, 2015) and were limited to English-language studies. Supplemental searches were run in Scopus (Amsterdam, Netherlands) (June 25, 2015) to identify relevant publications not indexed in MEDLINE. A search for relevant clinical trials was completed using the clinicaltrials.gov Web site, and focused searches on guideline repository sites (eg, guideline.gov, g-i-n.net) and organizations’ Web sites were undertaken to identify relevant publications. Further detail about the systematic literature search, including the Ovid search strings, can be found in the SDC.

      Eligible Study Designs

      Studies were not limited to randomized controlled trials but also included other study types, including cohort designs, case series, evaluation studies, and comparative studies. Letters, commentaries, editorials, narrative reviews, case reports, studies in mouse models, in vitro studies, consensus documents, abstracts, and non-English articles were excluded a priori.

      Inclusion Criteria

      Published studies were selected for inclusion in the systematic review of evidence if they were peer-reviewed full-text articles that met the following criteria:
      • 1.
        The study population consisted of patients with nonsquamous, non–small cell lung adenocarcinoma, small cell lung carcinoma, or squamous cell lung cancer of any stage.
      • 2.
        The study evaluated, prospectively or retrospectively, sensitivity, specificity, negative predictive value, or positive predictive value of EGFR, ALK, KRAS, ROS1, RET, MET, BRAF, or ERBB2 (HER2) tests for detection of gene-specific mutation, rearrangement, translocation, amplification, overexpression, or response to a targeted gene-specific therapy.
      • 3.
        The study examined potential testing algorithms for NSCLC molecular testing.
      • 4.
        The study examined the correlation of EGFR, ALK, KRAS, ROS1, RET, MET, BRAF, or ERBB2 (HER2) status in primary or metastatic tumors from the same patients.
      • 5.
        The study included primary outcomes such as accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of tests and concordance across platforms to determine EGFR, ALK, KRAS, ROS1, RET, MET, BRAF, or ERBB2 (HER2) status or treatment response, alone or in combination.

      Quality Assessment

      An assessment of the quality of the evidence was performed for all retained studies following application of the inclusion and exclusion criteria. Using this method, studies deemed low quality would not be excluded from the systematic review, but would be retained and their methodologic strengths and weaknesses discussed where relevant. Each guideline statement includes a rating of the strength of the evidence as described in Table 1 (also in SDC Table 1). The process used to assess the quality of the evidence base is fully detailed in the SDC.
      Table 1Grades for Strength of Evidence
      Adapted from J Clin Epidemiol. 2011;64(4):401–406, Balshem H, Helfand M, Schunemann HJ, et al. GRADE guidelines: 3. Rating the quality of evidence, copyright 2011, with permission from Elsevier.262
      DesignationDescriptionQuality of Evidence
      ConvincingHigh confidence that available evidence reflects true effect. Further research is very unlikely to change the confidence in the estimate of effect.High/intermediate quality of evidence
      AdequateModerate confidence that available evidence reflects true effect. Further research is likely to have an important impact on the confidence in estimate of effect and may change the estimateIntermediate/low quality of evidence
      InadequateLittle confidence that available evidence reflects true effect. Further research is very likely to have an important impact on the confidence in the estimate of effect and is likely to change the estimate.Low/insufficient evidence and expert panel uses formal consensus process to reach recommendation
      InsufficientEvidence is insufficient to discern net effect. Any estimate of effect is very uncertain.Insufficient evidence and expert panel uses formal consensus process to reach recommendation
      a Adapted from J Clin Epidemiol. 2011;64(4):401–406, Balshem H, Helfand M, Schunemann HJ, et al. GRADE guidelines: 3. Rating the quality of evidence, copyright 2011, with permission from Elsevier.
      • Balshem H.
      • Helfand M.
      • Schunemann H.J.
      • et al.
      GRADE guidelines, 3: rating the quality of evidence.

      Assessing the Strength of Recommendations

      In order to articulate recommendation statements that were clearly written and easy to implement, the expert panel used GLIDES (Guidelines Into Decision Support) methodology and accompanying BridgeWiz software (Yale University, New Haven, Connecticut).

      Yale University School of Medicine. GuideLines Into DEcision Support (GLIDES). http://medicine.yale.edu/cmi/glides/index.aspx. Accessed June 21, 2017.

      This methodology prioritizes the use of active language; however, in some situations, the person responsible for ensuring guidance is implemented is dependent on the organization of the clinic and/or laboratory. To ensure clarity of guidance in these situations, the expert panel used passive-voice language to emphasize the recommended action. Development of recommendations required that the panel review the identified evidence and make a series of key judgments (using procedures described in the SDC). This guideline uses a 3-tier system to rate the strength of recommendations, as well as a “no recommendation” category when there is insufficient evidence to support a recommendation. Table 2 (also in SDC Table 2) summarizes the strength of evidence and net benefits and harms, as well as obligatory language that was used for each of the recommendation types.
      Table 2Strength of Recommendations
      Data derived from Andrews et al.263
      DesignationRecommendationRationale
      Strong recommendationRecommend for or against a particular molecular testing practice in lung cancer (can include must or should)Supported by convincing (high) or adequate (intermediate) quality of evidence and clear benefit that outweighs any harms.
      RecommendationRecommend for or against a particular molecular testing practice in lung cancer (can include should or may)Some limitations in quality of evidence (adequate [intermediate] or inadequate [low]), balance of benefits and harms, values, or costs, but panel concludes that there is sufficient evidence to inform a recommendation.
      Expert consensus opinionRecommend for or against a particular molecular testing practice in lung cancer (can include should or may)Serious limitations in quality of evidence (inadequate [low, very low] or insufficient), balance of benefits and harms, values, or costs, but panel consensus is that a statement is necessary.
      No recommendationNo recommendation for or against a particular molecular testing practice in lung cancerInsufficient evidence, confidence, or agreement to provide a recommendation.
      a Data derived from Andrews et al.
      • Andrews J.
      • Guyatt G.
      • Oxman A.D.
      • et al.
      GRADE guidelines, 14: going from evidence to recommendations: the significance and presentation of recommendations.

      Guideline Revision

      This guideline will be reviewed every 4 years or earlier in the event of publication of substantive and high-quality evidence that could potentially alter the original guideline statements. If necessary, the entire panel will reconvene to discuss potential changes and, if indicated, recommend revision of the guideline to CAP, IASLC, and AMP.

      Disclaimer

      Practice guidelines and consensus statements reflect the best available evidence and expert consensus supported in practice. They are intended to assist physicians and patients in clinical decision making and to identify questions and settings for further research. With the rapid flow of scientific information, new evidence may emerge between the time a practice guideline or consensus statement is developed and when it is published or read. Guidelines and statements are not continually updated and may not reflect the most recent evidence. Guidelines and statements address only the topics specifically identified therein and are not applicable to other interventions, diseases, or stages of diseases. Furthermore, guidelines and consensus statements cannot account for individual variation among patients and cannot be considered inclusive of all proper methods of care or exclusive of other treatments. It is the responsibility of the treating physician or other health care provider, relying on independent experience and knowledge, to determine the best course of treatment for the patient. Accordingly, adherence to any practice guideline or consensus statement is voluntary, with the ultimate determination regarding its application to be made by the physician in light of each patient’s individual circumstances and preferences. The CAP, IASLC, and AMP make no warranty, express or implied, regarding guidelines and statements and specifically exclude any warranties of merchantability and fitness for a particular use or purpose. The CAP, IASLC, and AMP assume no responsibility for any injury or damage to persons or property arising out of or related to any use of this statement or for any errors or omissions.

      Results

      For the reaffirmation of the 2013 guideline recommendations, a total of 610 studies met the search term requirements. Following a review of the 610 abstracts, the full texts of 77 studies that met the inclusion criteria and were likely to refute or change the 2013 recommendations were reviewed. A total of 21 articles were included for data extraction. Excluded articles were available as discussion or background references.
      For the new key questions, 1654 articles met the search term requirements. Based on review of these abstracts, 488 articles met the inclusion criteria and continued to full-text review. Articles that addressed any of the new key questions were moved to a second-level full-text–review phase. A total of 118 articles were included for data extraction. Excluded articles were available as discussion or background references.
      The panel convened 5 times (3 times by teleconference and 2 face-to-face meetings) to develop the scope, draft recommendations, review and respond to solicited feedback, and assess the quality of evidence that supports the final recommendations. A nominal group technique was used by the panel for consensus decision making to encourage unique input with balanced participation among group members. An open comment period was held from June 28 to August 2, 2016, during which the 2013 guideline statements and new draft recommendations and statements were posted for public comment. The public comment period was posted on the AMP Web site at www.amp.org. All 2013 recommendations received strong agreement (95%–99%) from the open comment period participants. There were 20 new draft statements with strong agreement, ranging from 86% to 97%, from the open comment period participants (refer to Outcomes in the SDC for full details). The expert panel members were assigned to review the public comments in small groups. The panel modified the draft statements and recommended the deletion of 1 expert consensus opinion and a no recommendation statement based on the feedback during the considered judgment process. The final recommendations were approved by the expert panel with a vote. The panel considered benefits and harms, required resources, feasibility, and acceptability throughout the entire process, although neither cost nor cost-effectiveness analyses were performed. A description of the benefits and harms of implementing the guideline statements is included in the SDC (SDC Table 3).
      Table 3Summary of the Updated Statements With Strength of Recommendations
      Supplemental Table 4b includes a list of the 2013 reaffirmed statements.
      2013 Statement2018 Statement
      Expert consensus opinion: Cytologic samples are also suitable for EGFR and ALK testing, with cell blocks being preferred over smear preparations.Recommendation: Pathologists may use either cell blocks or other cytologic preparations as suitable specimens for lung cancer biomarker molecular testing.
      Expert consensus opinion: Laboratories should use EGFR test methods that are able to detect mutations in specimens with at least 50% cancer cell content, although laboratories are strongly encouraged to use (or have available at an external reference laboratory) more sensitive tests that are able to detect mutations in specimens with as little as 10% cancer cells.Expert consensus opinion: Laboratories should use, or have available at an external reference laboratory, clinical lung cancer biomarker molecular testing assays that are able to detect molecular alterations in specimens with as little as 20% cancer cells.
      Recommendation: Immunohistochemistry for total EGFR is not recommended for selection of EGFR TKI therapy.Strong recommendation: Laboratories should not use total EGFR expression by IHC testing to select patients for EGFR-targeted TKI therapy.
      Abbreviations: IHC, immunohistochemistry; TKI, tyrosine kinase inhibitor.
      a Supplemental Table 4b includes a list of the 2013 reaffirmed statements.
      Each organization instituted a review process to approve the guideline. For the CAP, an independent review panel representing the Council on Scientific Affairs was assembled to review and approve the guideline. The independent review panel was masked to the expert panel and vetted through the conflict of interest process. The IASLC approval process required review and approval by the IASLC Board of Directors. The AMP approval process required content review by an independent subject matter expert panel, led by the Publications & Communications chair, with representation from the Clinical Practice Committee and Solid Tumors Subdivision leadership, and organizational approval by the AMP Executive Committee.

      Guideline Statements

      Reaffirmation of 2013 Recommendations

      The 2013 guideline recommended universal testing of lung cancer patients with advanced-stage cancers with an adenocarcinoma component, using molecular diagnosis for activating “hot-spot” mutations in EGFR exons 18 to 21 with at least 1% prevalence (ie, codons 709 and 719, exon 19 deletion 768, and exon 20 insertions 790, 858, and 861), and using fluorescence in situ hybridization (FISH) for rearrangements involving ALK. Any methodology or testing algorithm with suitable analytic sensitivity (ability to detect mutations in formalin-fixed samples with 50% or more malignant cells) and turnaround time (10 days between sample receipt and reporting of all results), with appropriate validation and deployment under the Clinical Laboratory Improvement Act of 1988, was acceptable.
      The 2013 guideline recommended against applying clinical parameters (eg, tobacco exposure, age, gender, ethnicity) to select patients for testing, testing pure squamous carcinomas, using KRAS negativity as a determinant of anti-EGFR therapy, using IHC for EGFR or ALK testing, and using FISH for EGFR testing.
      The 2013 guideline left several decisions open to each institution to set policy, such as whether or not to test early-stage patients, whether or not to use clinical predictors to select patients with minimally sampled squamous carcinoma biopsies such that a mixed adenosquamous carcinoma could not be excluded, and whether or not to use a simultaneous or sequential testing approach. Of these, the question concerning testing early-stage disease remains open, and awaits data from more clinical trials before an evidence-based recommendation can be made. Although the American Society of Clinical Oncology Clinical Practice Guidelines Committee highlighted consideration of molecular testing for early-stage lung cancer patients,
      • Leighl N.B.
      • Rekhtman N.
      • Biermann W.A.
      • et al.
      Molecular testing for selection of patients with lung cancer for epidermal growth factor receptor and anaplastic lymphoma kinase tyrosine kinase inhibitors: American Society of Clinical Oncology endorsement of the College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology guideline.
      our opinion remains that each institution should set its own policy regarding testing patients with early-stage disease, balancing the benefit of having results on record from testing a high-quality resection sample for alterations that are likely to become necessary at a time of future progression when a high-quality sample could be hard to obtain against the cost of testing patients for whom a subset will be surgically cured and never need the test result. Accordingly, the testing recommended below applies to patients with advanced-stage (stages IIIB and IV) lung cancer.
      Following review of literature published since 2013, the original recommendations are largely reaffirmed. Several statements have gained strength with the publication of additional supporting evidence (SDC Tables 4a, 4b, and 5). Some warranted a complete reevaluation in this revision, and will appear subsequently (Table 3); these include the use of IHC for ALK, the use of multigene NGS panels, and the question of testing nonadenocarcinoma samples.
      Of the remaining 2013 recommendations, the following changes are made:
      1. Any Cytology Sample With Adequate Cellularity and Preservation May Be Tested
      The original recommendation preferred cell blocks over smears. A recent systematic review
      • Ellison G.
      • Zhu G.
      • Moulis A.
      • Dearden S.
      • Speake G.
      • McCormack R.
      EGFR mutation testing in lung cancer: a review of available methods and their use for analysis of tumour tissue and cytology samples.
      identified by the literature search has indicated that numerous studies have been published showing excellent performance of smear preparations, such that this preference is no longer appropriate. It is incumbent upon any laboratory that tests cytopathology specimens to perform appropriate validation studies of these as separate sample types, distinct from tissue and blood samples.
      2. Analytic Methods Must Be Able to Detect Mutation in a Sample With 20% or More Malignant Cell Content
      Although the original studies demonstrating response of EGFR-mutated lung cancers to treatment with EGFR inhibitors used unmodified Sanger sequencing with a sensitivity limit of 50% tumor cellularity, this is insufficient in practice because many lung cancer samples are small and comprise a majority of benign stromal cells, and most of the larger phase III clinical trials that confirmed the clinical utility of EGFR mutation testing used polymerase chain reaction (PCR)–based methods that were more sensitive than unmodified Sanger sequencing. Given the widespread availability of technology capable of reliably detecting lower-frequency mutational events in small samples, it is no longer appropriate to offer a low-sensitivity test that cannot test tumors with 20% to 50% tumor content and requires patients to undergo more procedures, and potentially more invasive procedures, solely to procure a tissue sample with high tumor content.
      3. It Is Not Appropriate to Use IHC for EGFR Mutation Testing
      There is no role whatsoever for IHC against total EGFR protein as a determinant of treatment with an EGFR kinase inhibitor. The targetable mutations lead to activation of the cytoplasmic kinase of this transmembrane protein, but that has no bearing on the extent of expression at the cell surface, which is what is detected by the total EGFR immunostain. Although EGFR expression by IHC was performed for some of the very early studies of EGFR kinase inhibitors in the start of this century, clinical responses were seen in patients with mutations but absent/weak IHC expression, and poor responses were seen in patients with strong IHC expression but no mutations.
      Following the discovery of EGFR mutations, antibodies were developed for IHC directed at the most common mutated forms of the protein, most notably the L858R substitution and the 746 to 750 ELREA deletion. The original guideline allowed for the use of the mutant-specific EGFR antibodies by IHC in a setting with extremely limited material. Although published evidence for these antibodies shows good accuracy for the L858R activating mutation and for some of the exon 19 deletions, these antibodies have poor sensitivity for other exon 19 deletions, insensitivity to less common mutations (eg, codon 719 mutations), and false-positive results with exon 20 insertions.
      • Kitamura A.
      • Hosoda W.
      • Sasaki E.
      • Mitsudomi T.
      • Yatabe Y.
      Immunohistochemical detection of EGFR mutation using mutation-specific antibodies in lung cancer.
      Overall, the performance is suboptimal for reliable detection of EGFR mutations. Given that advances in molecular diagnostic technology now enable analysis of very limited samples as well as circulating tumor DNA (see below), at this time there is no role for routine use of mutant-specific IHC in selecting anti-EGFR treatment for lung cancer patients.

      New Recommendations

      Question 1: Which New Genes Should Be Tested for Lung Cancer Patients?

      In the 2013 guideline, genes fell into 1 of 2 categories: testing is necessary (EGFR, ALK), or testing is investigational. One gene, KRAS, was considered conditionally necessary in the context of sequential testing algorithms because of its ease of analysis and mutual exclusivity with EGFR and ALK. By 2018, however, we believe that there are now 3 categories into which genes should be placed. One set of genes must be offered by all laboratories that test lung cancers, as an absolute minimum: EGFR, ALK, and ROS1. A second group of genes should be included in any expanded panel that is offered for lung cancer patients: BRAF, MET, RET, ERBB2 (HER2), and KRAS, if adequate material is available. KRAS testing may also be offered as a single-gene test to exclude patients from expanded panel testing. All other genes are considered investigational at the time of publication.
      In this context, institutions providing care for lung cancer patients have a choice: (1) offer a comprehensive cancer panel that includes all of the genes in the first 2 categories (EGFR, ALK, ROS1, BRAF, MET, ERBB2 [HER2], KRAS, RET) for all appropriate patients, or (2) offer targeted testing for the genes in the must-test category (EGFR, ALK, ROS1) for all appropriate patients and offer as a second test an expanded panel containing the second-category genes (BRAF, MET, ERBB2 [HER2], and RET) for patients who are suitable candidates for clinical trials, possibly after performing a single-gene KRAS test to exclude patients with KRAS-mutant cancers from expanded panel testing. Table 4 includes a list of the recommendation statements with the strength of recommendations.
      Table 4Summary of 2018 Guideline Statements
      Guideline StatementsStrength of Recommendation
      Key Question 1: Which new genes should be tested for lung cancer patients?
      1. ROS1 testing must be performed on all lung adenocarcinoma patients, irrespective of clinical characteristics.Strong recommendation
      2. ROS1 IHC may be used as a screening test in lung adenocarcinoma patients; however, positive ROS1 IHC results should be confirmed by a molecular or cytogenetic method.Expert consensus opinion
      3. BRAF molecular testing is currently not indicated as a routine stand-alone assay outside the context of a clinical trial. It is appropriate to include BRAF as part of larger testing panels performed either initially or when routine EGFR, ALK, and ROS1 testing are negative.Expert consensus opinion
      4. RET molecular testing is not recommended as a routine stand-alone assay outside the context of a clinical trial. It is appropriate to include RET as part of larger testing panels performed either initially or when routine EGFR, ALK, and ROS1 testing are negative.Expert consensus opinion
      5. ERBB2 (HER2) molecular testing is not indicated as a routine stand-alone assay outside the context of a clinical trial. It is appropriate to include ERBB2 (HER2) mutation analysis as part of a larger testing panel performed either initially or when routine EGFR, ALK, and ROS1 testing are negative.Expert consensus opinion
      6. KRAS molecular testing is not indicated as a routine stand-alone assay as a sole determinant of targeted therapy. It is appropriate to include KRAS as part of larger testing panels performed either initially or when routine EGFR, ALK, and ROS1 testing are negative.Expert consensus opinion
      7. MET molecular testing is not indicated as a routine stand-alone assay outside the context of a clinical trial. It is appropriate to include MET as part of larger testing panels performed either initially or when routine EGFR, ALK, and ROS1 testing are negative.Expert consensus opinion
      Key Question 2: What methods should be used to perform molecular testing?
      8. IHC is an equivalent alternative to FISH for ALK testing.Recommendation
      9. Multiplexed genetic sequencing panels are preferred over multiple single-gene tests to identify other treatment options beyond EGFR, ALK, and ROS1.Expert consensus opinion
      10. Laboratories should ensure test results that are unexpected, discordant, equivocal, or otherwise of low confidence are confirmed or resolved using an alternative method or sample.Expert consensus opinion
      Key Question 3: Is molecular testing appropriate for lung cancers that do not have an adenocarcinoma component?
      11. Physicians may use molecular biomarker testing in tumors with histologies other than adenocarcinoma when clinical features indicate a higher probability of an oncogenic driver.Expert consensus opinion
      Key Question 4: What testing is indicated for patients with targetable mutations who have relapsed on targeted therapy?
      12. In lung adenocarcinoma patients who harbor sensitizing EGFR mutations and have progressed after treatment with an EGFR-targeted tyrosine kinase inhibitor, physicians must use EGFR T790M mutational testing when selecting patients for third-generation EGFR-targeted therapy.Strong recommendation
      13. Laboratories testing for EGFR T790M mutation in patients with secondary clinical resistance to EGFR-targeted kinase inhibitors should deploy assays capable of detecting EGFR T790M mutations in as little as 5% of viable cells.Recommendation
      14. There is currently insufficient evidence to support a recommendation for or against routine testing for ALK mutational status for lung adenocarcinoma patients with sensitizing ALK mutations who have progressed after treatment with an ALK-targeted tyrosine kinase inhibitor.No recommendation
      Key Question 5: What is the role of testing for circulating cell-free DNA for lung cancer patients?
      15. There is currently insufficient evidence to support the use of circulating cell-free plasma DNA molecular methods for the diagnosis of primary lung adenocarcinoma.No recommendation
      16. In some clinical settings in which tissue is limited and/or insufficient for molecular testing, physicians may use a cell-free plasma DNA assay to identify EGFR mutations.Recommendation
      17. Physicians may use cell-free plasma DNA methods to identify EGFR T790M mutations in lung adenocarcinoma patients with progression or secondary clinical resistance to EGFR-targeted tyrosine kinase inhibitors; testing of the tumor sample is recommended if the plasma result is negative.Expert consensus opinion
      18. There is currently insufficient evidence to support the use of circulating tumor cell 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.No recommendation
      Abbreviations: FISH, fluorescence in situ hybridization; IHC, immunohistochemistry; TKI, tyrosine kinase inhibitor.
      1. Strong Recommendation
      ROS1 testing must be performed on all lung advanced-stage adenocarcinoma patients, irrespective of clinical characteristics.
      The strength of evidence was convincing to support the use of ROS1 molecular (ie, reverse transcription PCR [RT-PCR] or sequencing) or cytogenetic (ie, FISH or other in situ hybridization) testing to identify ROS1 rearrangements. The strength of evidence supporting the use of any clinical characteristic to identify patients who should receive ROS1 testing was adequate. This recommendation is evidence based and supported by 9 studies,
      • Bergethon K.
      • Shaw A.T.
      • Ou S.H.
      • et al.
      ROS1 rearrangements define a unique molecular class of lung cancers.
      • Go H.
      • Kim D.W.
      • Kim D.
      • et al.
      Clinicopathologic analysis of ROS1-rearranged non-small-cell lung cancer and proposal of a diagnostic algorithm.
      • Mazieres J.
      • Zalcman G.
      • Crino L.
      • et al.
      Crizotinib therapy for advanced lung adenocarcinoma and a ROS1 rearrangement: results from the EUROS1 cohort.
      • Shaw A.T.
      • Ou S.H.
      • Bang Y.J.
      • et al.
      Crizotinib in ROS1-rearranged non-small-cell lung cancer.
      • Cai W.
      • Li X.
      • Su C.
      • et al.
      ROS1 fusions in Chinese patients with non-small-cell lung cancer.
      • Chen Y.F.
      • Hsieh M.S.
      • Wu S.G.
      • et al.
      Clinical and the prognostic characteristics of lung adenocarcinoma patients with ROS1 fusion in comparison with other driver mutations in East Asian populations.
      • Warth A.
      • Muley T.
      • Dienemann H.
      • et al.
      ROS1 expression and translocations in non-small-cell lung cancer: clinicopathological analysis of 1478 cases.
      • Lee S.E.
      • Lee B.
      • Hong M.
      • et al.
      Comprehensive analysis of RET and ROS1 rearrangement in lung adenocarcinoma.
      • Scheffler M.
      • Schultheis A.
      • Teixido C.
      • et al.
      ROS1 rearrangements in lung adenocarcinoma: prognostic impact, therapeutic options and genetic variability.
      6 of which informed on the association between ROS1 rearrangement and patient or tumor characteristics
      • Bergethon K.
      • Shaw A.T.
      • Ou S.H.
      • et al.
      ROS1 rearrangements define a unique molecular class of lung cancers.
      • Go H.
      • Kim D.W.
      • Kim D.
      • et al.
      Clinicopathologic analysis of ROS1-rearranged non-small-cell lung cancer and proposal of a diagnostic algorithm.
      • Cai W.
      • Li X.
      • Su C.
      • et al.
      ROS1 fusions in Chinese patients with non-small-cell lung cancer.
      • Chen Y.F.
      • Hsieh M.S.
      • Wu S.G.
      • et al.
      Clinical and the prognostic characteristics of lung adenocarcinoma patients with ROS1 fusion in comparison with other driver mutations in East Asian populations.
      • Warth A.
      • Muley T.
      • Dienemann H.
      • et al.
      ROS1 expression and translocations in non-small-cell lung cancer: clinicopathological analysis of 1478 cases.
      • Lee S.E.
      • Lee B.
      • Hong M.
      • et al.
      Comprehensive analysis of RET and ROS1 rearrangement in lung adenocarcinoma.
      and consisted of 1 prospective cohort study (PCS),
      • Chen Y.F.
      • Hsieh M.S.
      • Wu S.G.
      • et al.
      Clinical and the prognostic characteristics of lung adenocarcinoma patients with ROS1 fusion in comparison with other driver mutations in East Asian populations.
      1 prospective-retrospective cohort study (PRCS),
      • Go H.
      • Kim D.W.
      • Kim D.
      • et al.
      Clinicopathologic analysis of ROS1-rearranged non-small-cell lung cancer and proposal of a diagnostic algorithm.
      and 4 retrospective cohort studies (RCSs).
      • Bergethon K.
      • Shaw A.T.
      • Ou S.H.
      • et al.
      ROS1 rearrangements define a unique molecular class of lung cancers.
      • Cai W.
      • Li X.
      • Su C.
      • et al.
      ROS1 fusions in Chinese patients with non-small-cell lung cancer.
      • Warth A.
      • Muley T.
      • Dienemann H.
      • et al.
      ROS1 expression and translocations in non-small-cell lung cancer: clinicopathological analysis of 1478 cases.
      • Lee S.E.
      • Lee B.
      • Hong M.
      • et al.
      Comprehensive analysis of RET and ROS1 rearrangement in lung adenocarcinoma.
      The 3 remaining studies assessed clinical outcomes of patients treated with the ROS1-targeted therapy crizotinib
      • Mazieres J.
      • Zalcman G.
      • Crino L.
      • et al.
      Crizotinib therapy for advanced lung adenocarcinoma and a ROS1 rearrangement: results from the EUROS1 cohort.
      • Shaw A.T.
      • Ou S.H.
      • Bang Y.J.
      • et al.
      Crizotinib in ROS1-rearranged non-small-cell lung cancer.
      • Scheffler M.
      • Schultheis A.
      • Teixido C.
      • et al.
      ROS1 rearrangements in lung adenocarcinoma: prognostic impact, therapeutic options and genetic variability.
      and included 1 nonrandomized clinical trial
      • Shaw A.T.
      • Ou S.H.
      • Bang Y.J.
      • et al.
      Crizotinib in ROS1-rearranged non-small-cell lung cancer.
      and 3 RCSs.
      • Mazieres J.
      • Zalcman G.
      • Crino L.
      • et al.
      Crizotinib therapy for advanced lung adenocarcinoma and a ROS1 rearrangement: results from the EUROS1 cohort.
      • Scheffler M.
      • Schultheis A.
      • Teixido C.
      • et al.
      ROS1 rearrangements in lung adenocarcinoma: prognostic impact, therapeutic options and genetic variability.
      All included studies were assessed for quality and none were found to have methodologic flaws that would raise concerns about the studies’ findings (SDC Table 6). Refer to SDC Table 7 for a summary of findings from studies supporting the use of ROS1 molecular or cytogenetic testing to enable selection of patients for ROS1-targeted therapy.
      Although relatively rare, accounting for less than 2% of non–small cell lung carcinomas
      • Bergethon K.
      • Shaw A.T.
      • Ou S.H.
      • et al.
      ROS1 rearrangements define a unique molecular class of lung cancers.
      • Go H.
      • Kim D.W.
      • Kim D.
      • et al.
      Clinicopathologic analysis of ROS1-rearranged non-small-cell lung cancer and proposal of a diagnostic algorithm.
      • Cai W.
      • Li X.
      • Su C.
      • et al.
      ROS1 fusions in Chinese patients with non-small-cell lung cancer.
      and 2% to 3% of lung adenocarcinomas,
      • Bergethon K.
      • Shaw A.T.
      • Ou S.H.
      • et al.
      ROS1 rearrangements define a unique molecular class of lung cancers.
      • Cai W.
      • Li X.
      • Su C.
      • et al.
      ROS1 fusions in Chinese patients with non-small-cell lung cancer.
      • Chen Y.F.
      • Hsieh M.S.
      • Wu S.G.
      • et al.
      Clinical and the prognostic characteristics of lung adenocarcinoma patients with ROS1 fusion in comparison with other driver mutations in East Asian populations.
      structural rearrangements involving the ROS1 gene generate an oncogenic fusion that can be treated successfully with targeted inhibitors. A single phase I clinical trial of 50 NSCLC patients demonstrated that the presence of a ROS1 rearrangement by FISH or RT-PCR predicts response to targeted inhibition using crizotinib, with a response rate of 72% and median progression-free survival of 19.2 months.
      • Shaw A.T.
      • Ou S.H.
      • Bang Y.J.
      • et al.
      Crizotinib in ROS1-rearranged non-small-cell lung cancer.
      Based on this trial, the FDA approved the expanded use of crizotinib in patients with ROS1-rearranged NSCLC in 2016. A European multi-institutional retrospective study of 32 patients with ROS1-rearranged NSCLC treated with crizotinib demonstrated an 80% response rate and 9.1-month progression-free survival.
      • Mazieres J.
      • Zalcman G.
      • Crino L.
      • et al.
      Crizotinib therapy for advanced lung adenocarcinoma and a ROS1 rearrangement: results from the EUROS1 cohort.
      Overall survival for patients with ROS1-rearranged tumors irrespective of use of targeted therapy appears longer than that for patients with other molecular alterations undergoing targeted therapy.
      • Scheffler M.
      • Schultheis A.
      • Teixido C.
      • et al.
      ROS1 rearrangements in lung adenocarcinoma: prognostic impact, therapeutic options and genetic variability.
      • Kim H.R.
      • Lim S.M.
      • Kim H.J.
      • et al.
      The frequency and impact of ROS1 rearrangement on clinical outcomes in never smokers with lung adenocarcinoma.
      As with ALK, ROS1 activation is driven by structural variants, with multiple different partners fusing to the C-terminal portion of ROS1 containing the cytoplasmic tyrosine kinase and driving downstream signaling through MAPK, JAK/STAT, and PI3K pathways. Common fusion partners include SLC34A2, CD74, and TPM3, among others. The role of wild-type ROS1 is still being elucidated, but it shares similar structure with ALK, albeit with significant differences, notably absence of a dimerization domain, an extracellular domain with some resemblance to cell adhesion molecules, and no clear ligand.
      As with EGFR mutations and ALK rearrangements, light to never smoking history has been associated with an increased incidence of ROS1 rearrangements in patients with lung adenocarcinoma.
      • Bergethon K.
      • Shaw A.T.
      • Ou S.H.
      • et al.
      ROS1 rearrangements define a unique molecular class of lung cancers.
      • Lee S.E.
      • Lee B.
      • Hong M.
      • et al.
      Comprehensive analysis of RET and ROS1 rearrangement in lung adenocarcinoma.
      However, this association has not been consistently observed across studies.
      • Cai W.
      • Li X.
      • Su C.
      • et al.
      ROS1 fusions in Chinese patients with non-small-cell lung cancer.
      Other clinical characteristics, such as younger age, female sex, and non-Asian ethnicity, have been associated with ROS1 rearrangement in isolated studies only.
      • Bergethon K.
      • Shaw A.T.
      • Ou S.H.
      • et al.
      ROS1 rearrangements define a unique molecular class of lung cancers.
      • Go H.
      • Kim D.W.
      • Kim D.
      • et al.
      Clinicopathologic analysis of ROS1-rearranged non-small-cell lung cancer and proposal of a diagnostic algorithm.
      • Chen Y.F.
      • Hsieh M.S.
      • Wu S.G.
      • et al.
      Clinical and the prognostic characteristics of lung adenocarcinoma patients with ROS1 fusion in comparison with other driver mutations in East Asian populations.
      Therefore, clinical characteristics should not be used to either select or exclude patients from testing for ROS1 rearrangements. ROS1 rearrangements occur in a mutually exclusive fashion with other oncogenic driver alterations (such as EGFR and KRAS mutation and ALK rearrangement). In recognition of the rarity of ROS1 rearrangement, it may be reasonable to perform sequential testing of EGFR and ALK followed by ROS1 testing. Indeed, the frequency of ROS1 rearrangements is enriched to 5% to 10% in otherwise driver (ie, EGFR, ALK, KRAS, BRAF)-negative lung adenocarcinomas.
      • Go H.
      • Kim D.W.
      • Kim D.
      • et al.
      Clinicopathologic analysis of ROS1-rearranged non-small-cell lung cancer and proposal of a diagnostic algorithm.
      • Lee S.E.
      • Lee B.
      • Hong M.
      • et al.
      Comprehensive analysis of RET and ROS1 rearrangement in lung adenocarcinoma.
      Notably, in the United States in 2016, crizotinib therapy in ROS1-rearranged tumors does not require the use of an FDA-approved companion diagnostic. Published methods that have established clinical utility of testing ROS1 in order to choose ROS1-targeted therapy have relied primarily upon FISH and RT-PCR. Outside the United States, a diagnostic test using RT-PCR was used for an international phase II clinical trial,

      Kemner A. Phase II safety and efficacy study of crizotinib in East Asian patients with ROS1 positive, ALK negative advanced NSCLC (NCT01945021). https://clinicaltrials.gov/ct2/show/results/NCT01945021?term=NCT01945021&rank=1. Accessed June 12, 2017.

      involving mainly East Asian countries, for selection of tumors with ROS1 rearrangement. This assay has been approved as an in vitro diagnostic in Europe and China, and may be recognized as a companion diagnostic test in some countries. Although targeted RT-PCR assays may be challenging because of variation in ROS1 break points (typically introns 31–35) and partner genes, capture-based sequencing strategies for RNA or DNA may be used, provided they are properly validated on known positive samples. Within the United States, FISH methods have been published more frequently. Fluorescence in situ hybridization testing should be performed with a break-apart probe design given the multiple fusion partners, and should show rearrangement, defined as signals split by at least 1 probe diameter, in 15% or more of tumor cells.

      US Food and Drug Administration. XALKORI prescribing information. http://www.accessdata.fda.gov/drugsatfda_docs/label/2016/202570s016lbl.pdf. Accessed June 12, 2017.

      2. Expert Consensus Opinion
      ROS1 IHC may be used as a screening test in advanced-stage lung adenocarcinoma patients; however, positive ROS1 IHC results should be confirmed by a molecular or cytogenetic method.
      The strength of evidence is inadequate supporting the use of IHC as a screening assay for ROS1 molecular testing. This statement is evidence based and supported by 6 studies,
      • Cha Y.J.
      • Lee J.S.
      • Kim H.R.
      • et al.
      Screening of ROS1 rearrangements in lung adenocarcinoma by immunohistochemistry and comparison with ALK rearrangements.
      • Mescam-Mancini L.
      • Lantuejoul S.
      • Moro-Sibilot D.
      • et al.
      On the relevance of a testing algorithm for the detection of ROS1-rearranged lung adenocarcinomas.
      • Sholl L.M.
      • Sun H.
      • Butaney M.
      • et al.
      ROS1 immunohistochemistry for detection of ROS1-rearranged lung adenocarcinomas.
      • Yoshida A.
      • Tsuta K.
      • Wakai S.
      • et al.
      Immunohistochemical detection of ROS1 is useful for identifying ROS1 rearrangements in lung cancers.
      • Boyle T.A.
      • Masago K.
      • Ellison K.E.
      • Yatabe Y.
      • Hirsch F.R.
      ROS1 immunohistochemistry among major genotypes of non-small-cell lung cancer.
      • Shan L.
      • Lian F.
      • Guo L.
      • et al.
      Detection of ROS1 gene rearrangement in lung adenocarcinoma: comparison of IHC, FISH and real-time RT-PCR.
      consisting of 2 PCSs,
      • Mescam-Mancini L.
      • Lantuejoul S.
      • Moro-Sibilot D.
      • et al.
      On the relevance of a testing algorithm for the detection of ROS1-rearranged lung adenocarcinomas.
      • Sholl L.M.
      • Sun H.
      • Butaney M.
      • et al.
      ROS1 immunohistochemistry for detection of ROS1-rearranged lung adenocarcinomas.
      1 PRCS,
      • Cha Y.J.
      • Lee J.S.
      • Kim H.R.
      • et al.
      Screening of ROS1 rearrangements in lung adenocarcinoma by immunohistochemistry and comparison with ALK rearrangements.
      and 3 RCSs.
      • Yoshida A.
      • Tsuta K.
      • Wakai S.
      • et al.
      Immunohistochemical detection of ROS1 is useful for identifying ROS1 rearrangements in lung cancers.
      • Boyle T.A.
      • Masago K.
      • Ellison K.E.
      • Yatabe Y.
      • Hirsch F.R.
      ROS1 immunohistochemistry among major genotypes of non-small-cell lung cancer.
      • Shan L.
      • Lian F.
      • Guo L.
      • et al.
      Detection of ROS1 gene rearrangement in lung adenocarcinoma: comparison of IHC, FISH and real-time RT-PCR.
      Five studies compared ROS1 IHC with a FISH reference test
      • Cha Y.J.
      • Lee J.S.
      • Kim H.R.
      • et al.
      Screening of ROS1 rearrangements in lung adenocarcinoma by immunohistochemistry and comparison with ALK rearrangements.
      • Mescam-Mancini L.
      • Lantuejoul S.
      • Moro-Sibilot D.
      • et al.
      On the relevance of a testing algorithm for the detection of ROS1-rearranged lung adenocarcinomas.
      • Sholl L.M.
      • Sun H.
      • Butaney M.
      • et al.
      ROS1 immunohistochemistry for detection of ROS1-rearranged lung adenocarcinomas.
      • Yoshida A.
      • Tsuta K.
      • Wakai S.
      • et al.
      Immunohistochemical detection of ROS1 is useful for identifying ROS1 rearrangements in lung cancers.
      • Shan L.
      • Lian F.
      • Guo L.
      • et al.
      Detection of ROS1 gene rearrangement in lung adenocarcinoma: comparison of IHC, FISH and real-time RT-PCR.
      and 1 study compared ROS1 IHC with an RT-PCR reference test.
      • Boyle T.A.
      • Masago K.
      • Ellison K.E.
      • Yatabe Y.
      • Hirsch F.R.
      ROS1 immunohistochemistry among major genotypes of non-small-cell lung cancer.
      Using reported true-positive, false-positive, true-negative, and false-negative data from studies comparing IHC with FISH, an MA was conducted to determine a pooled estimate of sensitivity and specificity for ROS1 IHC (Figure 1). All included studies were assessed for quality and none were found to have methodologic flaws that would raise concerns about the studies’ findings (SDC Table 8). Refer to SDC Table 9 for a summary of findings from studies supporting the use of IHC as a screening assay for ROS1 molecular testing.
      Figure thumbnail gr1
      Figure 1Forest plot of sensitivity and specificity for immunohistochemistry (IHC)-based determination of ROS1 rearrangement positivity compared with fluorescence in situ hybridization. Pooled estimate of sensitivity and specificity based on bivariate analysis of included studies. All included studies used an IHC staining intensity of at least 2+ with a D4D6 antibody to define ROS1 rearrangement positivity. Abbreviations: FN, false-negative; FP, false-positive; TN, true-negative; TP, true-positive.
      In light of the relative rarity of ROS1 rearrangement events in NSCLC, screening by IHC may be preferable to FISH or molecular techniques in some settings. Interpretation of ROS1 IHC is challenging, however, as expression can be seen in a patchy pattern, typically at weak intensity, in up to a third of tumors that do not have an underlying rearrangement.
      • Sholl L.M.
      • Sun H.
      • Butaney M.
      • et al.
      ROS1 immunohistochemistry for detection of ROS1-rearranged lung adenocarcinomas.
      • Yoshida A.
      • Tsuta K.
      • Wakai S.
      • et al.
      Immunohistochemical detection of ROS1 is useful for identifying ROS1 rearrangements in lung cancers.
      • Wiesweg M.
      • Eberhardt W.E.
      • Reis H.
      • et al.
      High prevalence of concomitant oncogene mutations in prospectively identified patients with ROS1-positive metastatic lung cancer.
      Although some studies suggest that ROS1 expression in the absence of a rearrangement may have prognostic significance,
      • Wiesweg M.
      • Eberhardt W.E.
      • Reis H.
      • et al.
      High prevalence of concomitant oncogene mutations in prospectively identified patients with ROS1-positive metastatic lung cancer.
      focal or patchy expression in tumor cells is rarely associated with a ROS1 rearrangement and therefore is unlikely to predict response to ROS1-targeted therapy. Moreover, the pattern of staining can vary among fusion types, including granular to globular staining in CD74-ROS1 fusions, weak membranous staining in EZR-ROS1 fusions, and vesicular localization staining in GOPC-ROS1 fusions.
      • Yoshida A.
      • Tsuta K.
      • Wakai S.
      • et al.
      Immunohistochemical detection of ROS1 is useful for identifying ROS1 rearrangements in lung cancers.
      A single commercially available antibody clone (D4D6, Cell Signaling Technology, Danvers, Massachusetts) has been used in studies published to date. Most retrospective studies of ROS1 IHC using the D4D6 antibody demonstrate a sensitivity of 100% relative to FISH or RT-PCR.
      • Chen Y.F.
      • Hsieh M.S.
      • Wu S.G.
      • et al.
      Clinical and the prognostic characteristics of lung adenocarcinoma patients with ROS1 fusion in comparison with other driver mutations in East Asian populations.
      • Cha Y.J.
      • Lee J.S.
      • Kim H.R.
      • et al.
      Screening of ROS1 rearrangements in lung adenocarcinoma by immunohistochemistry and comparison with ALK rearrangements.
      • Mescam-Mancini L.
      • Lantuejoul S.
      • Moro-Sibilot D.
      • et al.
      On the relevance of a testing algorithm for the detection of ROS1-rearranged lung adenocarcinomas.
      • Sholl L.M.
      • Sun H.
      • Butaney M.
      • et al.
      ROS1 immunohistochemistry for detection of ROS1-rearranged lung adenocarcinomas.
      • Yoshida A.
      • Tsuta K.
      • Wakai S.
      • et al.
      Immunohistochemical detection of ROS1 is useful for identifying ROS1 rearrangements in lung cancers.
      • Boyle T.A.
      • Masago K.
      • Ellison K.E.
      • Yatabe Y.
      • Hirsch F.R.
      ROS1 immunohistochemistry among major genotypes of non-small-cell lung cancer.
      • Shan L.
      • Lian F.
      • Guo L.
      • et al.
      Detection of ROS1 gene rearrangement in lung adenocarcinoma: comparison of IHC, FISH and real-time RT-PCR.
      Tumors lacking ROS1 expression can be safely interpreted as lacking a ROS1 fusion. However, the specificity of ROS1 IHC is more variable, ranging from 92% to 100% using different methods and interpretive cutoffs.
      • Chen Y.F.
      • Hsieh M.S.
      • Wu S.G.
      • et al.
      Clinical and the prognostic characteristics of lung adenocarcinoma patients with ROS1 fusion in comparison with other driver mutations in East Asian populations.
      • Cha Y.J.
      • Lee J.S.
      • Kim H.R.
      • et al.
      Screening of ROS1 rearrangements in lung adenocarcinoma by immunohistochemistry and comparison with ALK rearrangements.
      • Mescam-Mancini L.
      • Lantuejoul S.
      • Moro-Sibilot D.
      • et al.
      On the relevance of a testing algorithm for the detection of ROS1-rearranged lung adenocarcinomas.
      • Sholl L.M.
      • Sun H.
      • Butaney M.
      • et al.
      ROS1 immunohistochemistry for detection of ROS1-rearranged lung adenocarcinomas.
      • Yoshida A.
      • Tsuta K.
      • Wakai S.
      • et al.
      Immunohistochemical detection of ROS1 is useful for identifying ROS1 rearrangements in lung cancers.
      • Boyle T.A.
      • Masago K.
      • Ellison K.E.
      • Yatabe Y.
      • Hirsch F.R.
      ROS1 immunohistochemistry among major genotypes of non-small-cell lung cancer.
      • Shan L.
      • Lian F.
      • Guo L.
      • et al.
      Detection of ROS1 gene rearrangement in lung adenocarcinoma: comparison of IHC, FISH and real-time RT-PCR.
      Meta-analysis of 5 studies identified by the literature search determined a pooled sensitivity of 96% (95% CI, 71%–99%) and specificity of 94% (95% CI, 89%–96%) for IHC compared with FISH when the D4D6 antibody with a staining intensity of at least 2+ (as defined within the study) was used (Figure 1). Several cutoffs have been proposed using intensity alone or H score (intensity × percentage of tumor cells staining). In most studies, FISH- or molecularly confirmed ROS1-rearranged tumors have at least moderate-intensity ROS1 protein expression, but published evidence is insufficient to recommend one specific cutoff or scoring system,
      • Cha Y.J.
      • Lee J.S.
      • Kim H.R.
      • et al.
      Screening of ROS1 rearrangements in lung adenocarcinoma by immunohistochemistry and comparison with ALK rearrangements.
      • Yoshida A.
      • Tsuta K.
      • Wakai S.
      • et al.
      Immunohistochemical detection of ROS1 is useful for identifying ROS1 rearrangements in lung cancers.
      and each laboratory must validate its own interpretive cutoff from known positive and negative samples.
      Because of imperfect specificity and challenges related to interpretation of nonspecific expression, we recommend that all ROS1 IHC positive results undergo confirmation by FISH or a molecular method (ie, RT-PCR, NGS) prior to considering a patient for ROS1-targeted therapy. Given the high sensitivity of IHC, however, tumors that clearly lack ROS1 staining can be interpreted as negative for ROS1 fusion.

      Additional Genes

      Of the genes newly included in this guideline, only ROS1 testing must be offered to all appropriate lung cancer patients. Testing for the following genes should be included with any expanded multigene panel testing performed for lung cancer patients, whether or not the panel is offered for all lung cancer patients, or if the panel is reserved as a second-line test for EGFR/ALK/ROS1 wild-type patients seeking clinical trials.
      3. Expert Consensus Opinion
      BRAF molecular testing is currently not indicated as a routine stand-alone assay outside the context of a clinical trial. It is appropriate to include BRAF as part of larger testing panels performed either initially or when routine EGFR, ALK, and ROS1 testing is negative.
      The strength of evidence was inadequate to support the use of BRAF molecular testing. This statement was evidence based and supported by 9 studies: 4 PCSs
      • Hsu K.H.
      • Ho C.C.
      • Hsia T.C.
      • et al.
      Identification of five driver gene mutations in patients with treatment-naive lung adenocarcinoma in Taiwan.
      • Kinno T.
      • Tsuta K.
      • Shiraishi K.
      • et al.
      Clinicopathological features of nonsmall cell lung carcinomas with BRAF mutations.
      • Li H.
      • Pan Y.
      • Li Y.
      • et al.
      Frequency of well-identified oncogenic driver mutations in lung adenocarcinoma of smokers varies with histological subtypes and graduated smoking dose.
      • Li S.
      • Li L.
      • Zhu Y.
      • et al.
      Coexistence of EGFR with KRAS, or BRAF, or PIK3CA somatic mutations in lung cancer: a comprehensive mutation profiling from 5125 Chinese cohorts.
      and 3 RCSs,
      • Cardarella S.
      • Ogino A.
      • Nishino M.
      • et al.
      Clinical, pathologic, and biologic features associated with BRAF mutations in non-small cell lung cancer.
      • Marchetti A.
      • Felicioni L.
      • Malatesta S.
      • et al.
      Clinical features and outcome of patients with non-small-cell lung cancer harboring BRAF mutations.
      • Brustugun O.T.
      • Khattak A.M.
      • Tromborg A.K.
      • et al.
      BRAF-mutations in non-small cell lung cancer.
      all of which informed on the association between BRAF mutation and patient or tumor characteristics,
      • Hsu K.H.
      • Ho C.C.
      • Hsia T.C.
      • et al.
      Identification of five driver gene mutations in patients with treatment-naive lung adenocarcinoma in Taiwan.
      • Kinno T.
      • Tsuta K.
      • Shiraishi K.
      • et al.
      Clinicopathological features of nonsmall cell lung carcinomas with BRAF mutations.
      • Li H.
      • Pan Y.
      • Li Y.
      • et al.
      Frequency of well-identified oncogenic driver mutations in lung adenocarcinoma of smokers varies with histological subtypes and graduated smoking dose.
      • Li S.
      • Li L.
      • Zhu Y.
      • et al.
      Coexistence of EGFR with KRAS, or BRAF, or PIK3CA somatic mutations in lung cancer: a comprehensive mutation profiling from 5125 Chinese cohorts.
      • Cardarella S.
      • Ogino A.
      • Nishino M.
      • et al.
      Clinical, pathologic, and biologic features associated with BRAF mutations in non-small cell lung cancer.
      • Marchetti A.
      • Felicioni L.
      • Malatesta S.
      • et al.
      Clinical features and outcome of patients with non-small-cell lung cancer harboring BRAF mutations.
      • Brustugun O.T.
      • Khattak A.M.
      • Tromborg A.K.
      • et al.
      BRAF-mutations in non-small cell lung cancer.
      and 2 additional nonrandomized clinical trials that assessed the activity of a BRAF inhibitor in p.V600E mutant patients.
      • Planchard D.
      • Kim T.M.
      • Mazieres J.
      • et al.
      Dabrafenib in patients with BRAF(V600E)-positive advanced non-small-cell lung cancer: a single-arm, multicentre, open-label, phase 2 trial.
      • Planchard D.
      • Besse B.
      • Groen H.J.
      • et al.
      Dabrafenib plus trametinib in patients with previously treated BRAF(V600E)-mutant metastatic non-small cell lung cancer: an open-label, multicentre phase 2 trial.
      All included studies were assessed for quality and none were found to have methodologic flaws that would raise concerns about the studies’ findings (SDC Table 10). Refer to SDC Table 11 for a summary of findings from studies supporting the use of BRAF molecular testing.
      Activating mutations in BRAF, especially p.V600E, lead to oncogenic signaling through MAPK, and are rare recurrent alterations in lung adenocarcinoma, seen in 0.5% to 4.9% of tumors.
      • Hsu K.H.
      • Ho C.C.
      • Hsia T.C.
      • et al.
      Identification of five driver gene mutations in patients with treatment-naive lung adenocarcinoma in Taiwan.
      • Kinno T.
      • Tsuta K.
      • Shiraishi K.
      • et al.
      Clinicopathological features of nonsmall cell lung carcinomas with BRAF mutations.
      • Li H.
      • Pan Y.
      • Li Y.
      • et al.
      Frequency of well-identified oncogenic driver mutations in lung adenocarcinoma of smokers varies with histological subtypes and graduated smoking dose.
      • Li S.
      • Li L.
      • Zhu Y.
      • et al.
      Coexistence of EGFR with KRAS, or BRAF, or PIK3CA somatic mutations in lung cancer: a comprehensive mutation profiling from 5125 Chinese cohorts.
      • Marchetti A.
      • Felicioni L.
      • Malatesta S.
      • et al.
      Clinical features and outcome of patients with non-small-cell lung cancer harboring BRAF mutations.
      In lung cancer, data from a 2016 phase II single-arm clinical trial
      • Planchard D.
      • Kim T.M.
      • Mazieres J.
      • et al.
      Dabrafenib in patients with BRAF(V600E)-positive advanced non-small-cell lung cancer: a single-arm, multicentre, open-label, phase 2 trial.
      showed that (1) single-agent dabrafenib given in second line to stage IV BRAF p.V600E mutant NSCLC had a partial response rate of 33% and disease control rate of 58% and (2) combination dabrafenib-trametinib therapy given in second line to stage IV BRAF p.V600E mutant lung adenocarcinoma had a partial response rate of 63% and disease control rate of 75%.
      Based on these data, the FDA conferred a breakthrough therapy designation for the combination treatment in BRAF p.V600E mutation–positive NSCLC, and FDA approval was granted in 2017. Hence, this was the most controversial of all recommendations among the working panel. Although there was a strong opinion in the working group that BRAF mutation analysis should be performed at the time of initial molecular testing in lung adenocarcinoma, the published evidence available at the time of publication lacked controlled prospective trials, and therefore lacked the strength to warrant an international recommendation for single-gene testing for BRAF for all lung adenocarcinoma patients. We anticipate the publication of stronger evidence supporting the utility of BRAF inhibition in BRAF-mutant lung cancer, and our opinion is that BRAF testing will be proven necessary. We expect that the next revision of this guideline will include a recommendation to include single-gene testing for BRAF alongside EGFR, ALK, and ROS1, but we are unable to make that recommendation in the spring of 2017. Although stand-alone single-gene testing for BRAF is not currently recommended, if a panel strategy is used, either initially or for patients who are known wild type for EGFR, ALK, and ROS1, then BRAF should be included.
      As with EGFR and KRAS mutations, selected hot-spot mutations in BRAF exert an oncogenic effect. The V-raf murine sarcoma homolog b (BRAF) gene encodes for a nonreceptor serine-threonine kinase in the MAPK kinase signaling pathway, between RAS and MEK. The most common BRAF mutation in NSCLC is the c.1799T>A (p.V600E) point mutation that is the predominant mutation in many other cancers, including melanoma, papillary thyroid cancer, colorectal cancer, hairy cell leukemia, and ganglioglioma. However, in contrast to other cancers with BRAF mutations, lung cancers frequently have non-p.V600E BRAF mutations, including other mutations at codon 600 (eg, p.V600K) and nearby codons in exon 15, and substitutions at codons 466 and 469 in exon 11.
      Like many other targetable oncogenes in lung cancer, BRAF mutations are more frequent in adenocarcinomas than in squamous cell carcinomas. BRAF p.V600E mutation is more frequent in females
      • Li S.
      • Li L.
      • Zhu Y.
      • et al.
      Coexistence of EGFR with KRAS, or BRAF, or PIK3CA somatic mutations in lung cancer: a comprehensive mutation profiling from 5125 Chinese cohorts.
      • Marchetti A.
      • Felicioni L.
      • Malatesta S.
      • et al.
      Clinical features and outcome of patients with non-small-cell lung cancer harboring BRAF mutations.
      and never smokers
      • Marchetti A.
      • Felicioni L.
      • Malatesta S.
      • et al.
      Clinical features and outcome of patients with non-small-cell lung cancer harboring BRAF mutations.
      in some studies, but several studies failed to show these associations.
      • Hsu K.H.
      • Ho C.C.
      • Hsia T.C.
      • et al.
      Identification of five driver gene mutations in patients with treatment-naive lung adenocarcinoma in Taiwan.
      • Kinno T.
      • Tsuta K.
      • Shiraishi K.
      • et al.
      Clinicopathological features of nonsmall cell lung carcinomas with BRAF mutations.
      • Cardarella S.
      • Ogino A.
      • Nishino M.
      • et al.
      Clinical, pathologic, and biologic features associated with BRAF mutations in non-small cell lung cancer.
      • Ilie M.
      • Long E.
      • Hofman V.
      • et al.
      Diagnostic value of immunohistochemistry for the detection of the BRAFV600E mutation in primary lung adenocarcinoma Caucasian patients.
      One distinction between BRAF mutations and other targetable oncogenes is that non-p.V600E BRAF mutations (particularly the exon 11 mutations) may coexist with mutations in KRAS,
      • Hsu K.H.
      • Ho C.C.
      • Hsia T.C.
      • et al.
      Identification of five driver gene mutations in patients with treatment-naive lung adenocarcinoma in Taiwan.
      • Li S.
      • Li L.
      • Zhu Y.
      • et al.
      Coexistence of EGFR with KRAS, or BRAF, or PIK3CA somatic mutations in lung cancer: a comprehensive mutation profiling from 5125 Chinese cohorts.
      • Cardarella S.
      • Ogino A.
      • Nishino M.
      • et al.
      Clinical, pathologic, and biologic features associated with BRAF mutations in non-small cell lung cancer.
      • Smit E.
      BRAF mutations in non-small-cell lung cancer.
      whereas the p.V600E mutations are mutually exclusive of KRAS, EGFR, or ALK alterations.
      Single-gene assays for BRAF are in wide use for other cancer types, particularly for melanoma patients being considered for targeted therapy, but most of these methods cannot detect the exon 11 mutations that are seen in lung cancer. Although the evidence supporting utility of BRAF testing was restricted to the p.V600E mutations, our opinion is that testing for BRAF, done as part of a large panel or for clinical trial enrollment, should use a method that evaluates at a minimum exons 11 and 15.
      A similar challenge arises concerning the use of mutation-specific IHC using antibodies against the p.V600E mutant protein (VE1), which have been widely used in melanoma diagnosis. Reported data on small numbers of lung cancer cases
      • Ilie M.
      • Long E.
      • Hofman V.
      • et al.
      Diagnostic value of immunohistochemistry for the detection of the BRAFV600E mutation in primary lung adenocarcinoma Caucasian patients.
      • Sasaki H.
      • Shimizu S.
      • Tani Y.
      • et al.
      Usefulness of immunohistochemistry for the detection of the BRAF V600E mutation in Japanese lung adenocarcinoma.
      demonstrate the VE1 clone can stain between 90% and 100% of p.V600E-mutant adenocarcinomas. In 1 of these studies, all non-p.V600E cases were negative on IHC testing,
      • Ilie M.I.
      • Bence C.
      • Hofman V.
      • et al.
      Discrepancies between FISH and immunohistochemistry for assessment of the ALK status are associated with ALK “borderline”-positive rearrangements or a high copy number: a potential major issue for anti-ALK therapeutic strategies.
      whereas in another, a single non-p.V600–mutated case out of 21, with a unique 599 insertion T mutation, showed positive staining. There is currently insufficient evidence to support a recommendation either for or against BRAF p.V600E IHC (VE1) testing in NSCLC.
      4. Expert Consensus Opinion
      RET molecular testing is not recommended as a routine stand-alone assay outside the context of a clinical trial. It is appropriate to include RET as part of larger testing panels performed either initially or when routine EGFR, ALK, and ROS1 testing is negative.
      The strength of evidence to support the use of RET molecular testing was inadequate. This statement is evidence based and supported by 3 studies,
      • Lee S.E.
      • Lee B.
      • Hong M.
      • et al.
      Comprehensive analysis of RET and ROS1 rearrangement in lung adenocarcinoma.
      • Wang R.
      • Hu H.
      • Pan Y.
      • et al.
      RET fusions define a unique molecular and clinicopathologic subtype of non-small-cell lung cancer.
      • Tsai T.H.
      • Wu S.G.
      • Hsieh M.S.
      • Yu C.J.
      • Yang J.C.
      • Shih J.Y.
      Clinical and prognostic implications of RET rearrangements in metastatic lung adenocarcinoma patients with malignant pleural effusion.
      consisting of 1 PCS
      • Wang R.
      • Hu H.
      • Pan Y.
      • et al.
      RET fusions define a unique molecular and clinicopathologic subtype of non-small-cell lung cancer.
      and 2 RCSs.
      • Lee S.E.
      • Lee B.
      • Hong M.
      • et al.
      Comprehensive analysis of RET and ROS1 rearrangement in lung adenocarcinoma.
      • Tsai T.H.
      • Wu S.G.
      • Hsieh M.S.
      • Yu C.J.
      • Yang J.C.
      • Shih J.Y.
      Clinical and prognostic implications of RET rearrangements in metastatic lung adenocarcinoma patients with malignant pleural effusion.
      All included studies were assessed for quality and none were found to have methodologic flaws that would raise concerns about the studies’ findings (SDC Table 12). Refer to SDC Table 13 for a summary of findings from studies supporting the use of RET molecular testing.
      Structural variants causing RET fusions are rare, being found in 0.6% to 0.9% of NSCLCs and in 1.2% to 2% of adenocarcinomas.
      • Wang R.
      • Hu H.
      • Pan Y.
      • et al.
      RET fusions define a unique molecular and clinicopathologic subtype of non-small-cell lung cancer.
      • Kohno T.
      • Ichikawa H.
      • Totoki Y.
      • et al.
      KIF5B-RET fusions in lung adenocarcinoma.
      • Takeuchi K.
      • Soda M.
      • Togashi Y.
      • et al.
      RET, ROS1 and ALK fusions in lung cancer.
      • Suehara Y.
      • Arcila M.
      • Wang L.
      • et al.
      Identification of KIF5B-RET and GOPC-ROS1 fusions in lung adenocarcinomas through a comprehensive mRNA-based screen for tyrosine kinase fusions.
      • Lipson D.
      • Capelletti M.
      • Yelensky R.
      • et al.
      Identification of new ALK and RET gene fusions from colorectal and lung cancer biopsies.
      The potential to treat RET-positive lung cancers with inhibitors of the RET kinase is being explored in phase II clinical trials,
      • Yoh K.
      • Seto T.
      • Satouchi M.
      • et al.
      Vandetanib in patients with previously treated RET-rearranged advanced non-small-cell lung cancer (LURET): an open-label, multicentre phase 2 trial.
      • Drilon A.
      • Rekhtman N.
      • Arcila M.
      • et al.
      Cabozantinib in patients with advanced RET-rearranged non-small-cell lung cancer: an open-label, single-centre, phase 2, single-arm trial.
      although small series and case reports have shown promise.
      • Gautschi O.
      • Milia J.
      • Filleron T.
      • et al.
      Targeting RET in patients with RET-rearranged lung cancers: results from the global, multicenter RET registry.
      • Gautschi O.
      • Zander T.
      • Keller F.A.
      • et al.
      A patient with lung adenocarcinoma and RET fusion treated with vandetanib.
      Given the rarity of RET rearrangements and limited evidence of therapeutic benefit, testing for RET alterations is not recommended as a stand-alone test for all lung adenocarcinoma patients. However, any large multigene panel test developed for lung cancer patients, either for initial workup or for patients who are wild type for EGFR, ALK, and ROS1, should include RET.
      As with ALK and ROS1 rearrangements, RET is activated by rearrangements that fuse the tyrosine kinase domain of RET with coiled-coil dimerization domains of one of a variety of recurring partner genes, including KIF5B (the most common, at 90%),
      • Kohno T.
      • Ichikawa H.
      • Totoki Y.
      • et al.
      KIF5B-RET fusions in lung adenocarcinoma.
      • Drilon A.
      • Wang L.
      • Hasanovic A.
      • et al.
      Response to cabozantinib in patients with RET fusion-positive lung adenocarcinomas.
      • Thunnissen E.
      • van der Oord K.
      • den Bakker M.
      Prognostic and predictive biomarkers in lung cancer: a review.
      CCDC6,
      • Takeuchi K.
      • Soda M.
      • Togashi Y.
      • et al.
      RET, ROS1 and ALK fusions in lung cancer.
      • Li F.
      • Feng Y.
      • Fang R.
      • et al.
      Identification of RET gene fusion by exon array analyses in “pan-negative” lung cancer from never smokers.
      NCOA4,
      • Wang R.
      • Hu H.
      • Pan Y.
      • et al.
      RET fusions define a unique molecular and clinicopathologic subtype of non-small-cell lung cancer.
      and TRIM33.
      • Drilon A.
      • Wang L.
      • Hasanovic A.
      • et al.
      Response to cabozantinib in patients with RET fusion-positive lung adenocarcinomas.
      RET rearrangement is mutually exclusive with aberrations in EGFR, KRAS, ALK, HER2, and BRAF in lung cancer.
      • Wang R.
      • Hu H.
      • Pan Y.
      • et al.
      RET fusions define a unique molecular and clinicopathologic subtype of non-small-cell lung cancer.
      • Kohno T.
      • Ichikawa H.
      • Totoki Y.
      • et al.
      KIF5B-RET fusions in lung adenocarcinoma.
      • Takeuchi K.
      • Soda M.
      • Togashi Y.
      • et al.
      RET, ROS1 and ALK fusions in lung cancer.
      RET fusion occurs more frequently in never smokers than ever smokers.
      • Lee S.E.
      • Lee B.
      • Hong M.
      • et al.
      Comprehensive analysis of RET and ROS1 rearrangement in lung adenocarcinoma.
      • Wang R.
      • Hu H.
      • Pan Y.
      • et al.
      RET fusions define a unique molecular and clinicopathologic subtype of non-small-cell lung cancer.
      • Kohno T.
      • Ichikawa H.
      • Totoki Y.
      • et al.
      KIF5B-RET fusions in lung adenocarcinoma.
      • Suehara Y.
      • Arcila M.
      • Wang L.
      • et al.
      Identification of KIF5B-RET and GOPC-ROS1 fusions in lung adenocarcinomas through a comprehensive mRNA-based screen for tyrosine kinase fusions.
      • Drilon A.
      • Wang L.
      • Hasanovic A.
      • et al.
      Response to cabozantinib in patients with RET fusion-positive lung adenocarcinomas.
      Patients with RET fusion harboring tumors are usually younger than patients with an EGFR mutation and have an equal gender distribution.
      • Lipson D.
      • Capelletti M.
      • Yelensky R.
      • et al.
      Identification of new ALK and RET gene fusions from colorectal and lung cancer biopsies.
      RET fusion proteins have been detected in adenocarcinoma
      • Lee S.E.
      • Lee B.
      • Hong M.
      • et al.
      Comprehensive analysis of RET and ROS1 rearrangement in lung adenocarcinoma.
      • Wang R.
      • Hu H.
      • Pan Y.
      • et al.
      RET fusions define a unique molecular and clinicopathologic subtype of non-small-cell lung cancer.
      • Kohno T.
      • Ichikawa H.
      • Totoki Y.
      • et al.
      KIF5B-RET fusions in lung adenocarcinoma.
      and in adenosquamous carcinoma.
      • Wang R.
      • Hu H.
      • Pan Y.
      • et al.
      RET fusions define a unique molecular and clinicopathologic subtype of non-small-cell lung cancer.
      Histologic subtypes in adenocarcinomas include those with mucinous/signet ring cells and those with a cribriform
      • Lee S.E.
      • Lee B.
      • Hong M.
      • et al.
      Comprehensive analysis of RET and ROS1 rearrangement in lung adenocarcinoma.
      • Wang R.
      • Hu H.
      • Pan Y.
      • et al.
      RET fusions define a unique molecular and clinicopathologic subtype of non-small-cell lung cancer.
      • Takeuchi K.
      • Soda M.
      • Togashi Y.
      • et al.
      RET, ROS1 and ALK fusions in lung cancer.
      or solid growth pattern.
      • Lee S.E.
      • Lee B.
      • Hong M.
      • et al.
      Comprehensive analysis of RET and ROS1 rearrangement in lung adenocarcinoma.
      • Wang R.
      • Hu H.
      • Pan Y.
      • et al.
      RET fusions define a unique molecular and clinicopathologic subtype of non-small-cell lung cancer.
      However, no clinical or histologic features (other than excluding from testing pure squamous histology cases) should be used to select a patient for RET testing.
      Multiple methods have been applied for RET analysis, including break-apart FISH analysis,
      • Sasaki H.
      • Shimizu S.
      • Tani Y.
      • et al.
      RET expression and detection of KIF5B/RET gene rearrangements in Japanese lung cancer.
      IHC,
      • Lee S.E.
      • Lee B.
      • Hong M.
      • et al.
      Comprehensive analysis of RET and ROS1 rearrangement in lung adenocarcinoma.
      RT-PCR,
      • Sasaki H.
      • Shimizu S.
      • Tani Y.
      • et al.
      RET expression and detection of KIF5B/RET gene rearrangements in Japanese lung cancer.
      and NGS.
      • Lee S.E.
      • Lee B.
      • Hong M.
      • et al.
      Comprehensive analysis of RET and ROS1 rearrangement in lung adenocarcinoma.
      RET FISH is particularly challenging, however, because of the narrow spacing between the split probe signals seen in the common fusion types, and a pattern of split RET signals separated by as little as 1 signal diameter distance is interpreted as positive.
      • Lee S.E.
      • Lee B.
      • Hong M.
      • et al.
      Comprehensive analysis of RET and ROS1 rearrangement in lung adenocarcinoma.
      Similar to ALK rearrangement testing by FISH, the threshold for RET FISH positivity for rearrangement is 15% of cells with split signals or single 3′ probe signals. In another study, a 4-colored RET FISH assay was used
      • Wang R.
      • Hu H.
      • Pan Y.
      • et al.
      RET fusions define a unique molecular and clinicopathologic subtype of non-small-cell lung cancer.
      ; samples were positive for RET rearrangement or KIF5B-RET fusion if more than 20% of tumor cells exhibited split red-green signals or touching golden-green signals, respectively.
      One recent retrospective study used RET IHC (anti-RET antibody ab134100, Abcam, Cambridge, United Kingdom) showing diffusely granular cytoplasm staining and occasionally membranous or perinuclear staining, with moderate to strong intensity. A sensitivity of 100% and specificity of 88% were reported,
      • Lee S.E.
      • Lee B.
      • Hong M.
      • et al.
      Comprehensive analysis of RET and ROS1 rearrangement in lung adenocarcinoma.
      although corroborating evidence is not strong enough to warrant a recommendation.
      Although multiplex RT-PCR may be successful for common fusions involving KIF5B-RET and CCDC6-RET,
      • Sasaki H.
      • Shimizu S.
      • Tani Y.
      • et al.
      RET expression and detection of KIF5B/RET gene rearrangements in Japanese lung cancer.
      as with ALK and ROS1, targeted RT-PCR alone is usually insufficient to detect new partners or isoforms. However, although the diversity of treatable rearrangements in ALK and ROS1 has matured sufficiently through years of testing and clinical trials, such that targeted RT-PCR assays for these genes can be designed with adequate clinical sensitivity, the diversity of treatable RET rearrangements is earlier in evolution. A capture-based sequencing approach, involving DNA or RNA, may be more sensitive and more readily integrated into a large multigene panel.
      • Zheng Z.
      • Liebers M.
      • Zhelyazkova B.
      • et al.
      Anchored multiplex PCR for targeted next-generation sequencing.
      5. Expert Consensus Opinion
      ERBB2 (HER2) molecular testing is not indicated as a routine stand-alone assay outside the context of a clinical trial. It is appropriate to include ERBB2 (HER2) mutation analysis as part of a larger testing panel performed either initially or when routine EGFR, ALK, and ROS1 testing is negative.
      The strength of evidence was inadequate to support the use of ERBB2 (HER2) molecular testing. This recommendation was evidence based and supported by 10 studies, 9 that reported on the association between ERBB2 (HER2) and patient or tumor characteristics
      • Hsu K.H.
      • Ho C.C.
      • Hsia T.C.
      • et al.
      Identification of five driver gene mutations in patients with treatment-naive lung adenocarcinoma in Taiwan.
      • Aleric I.
      • Razumovic J.J.
      • Koprivica B.
      HER-2/neu oncogene and estrogen receptor expression in non small cell lung cancer patients.
      • Calikusu Z.
      • Yildirim Y.
      • Akcali Z.
      • Sakalli H.
      • Bal N.
      • Ozyilkan O.
      Prognostic significance of the C-erbB-2 expression in Turkish non-small cell lung cancer patients.
      • Liu L.
      • Shao X.
      • Gao W.
      • et al.
      The role of human epidermal growth factor receptor 2 as a prognostic factor in lung cancer: a meta-analysis of published data.
      • Tomizawa K.
      • Suda K.
      • Onozato R.
      • et al.
      Prognostic and predictive implications of HER2/ERBB2/neu gene mutations in lung cancers.
      • Arcila M.E.
      • Chaft J.E.
      • Nafa K.
      • et al.
      Prevalence, clinicopathologic associations, and molecular spectrum of ERBB2 (HER2) tyrosine kinase mutations in lung adenocarcinomas.
      • Yoshizawa A.
      • Sumiyoshi S.
      • Sonobe M.
      • et al.
      HER2 status in lung adenocarcinoma: a comparison of immunohistochemistry, fluorescence in situ hybridization (FISH), dual-ISH, and gene mutations.
      • Shan L.
      • Qiu T.
      • Ling Y.
      • et al.
      Prevalence and clinicopathological characteristics of HER2 and BRAF mutation in Chinese patients with lung adenocarcinoma.
      • Suzuki M.
      • Shiraishi K.
      • Yoshida A.
      • et al.
      HER2 gene mutations in non-small cell lung carcinomas: concurrence with Her2 gene amplification and Her2 protein expression and phosphorylation.
      and 1 that assessed the use of ERBB2-targeted therapy (dacomitinib)
      • Kris M.G.
      • Camidge D.R.
      • Giaccone G.
      • et al.
      Targeting HER2 aberrations as actionable drivers in lung cancers: phase II trial of the pan-HER tyrosine kinase inhibitor dacomitinib in patients with HER2-mutant or amplified tumors.
      in patients with ERBB2 (HER2) mutations and amplifications. All included studies were assessed for quality and none were found to have methodologic flaws that would raise concerns about the studies’ findings (SDC Table 14). Refer to SDC Table 15 for a summary of findings from studies supporting the use of ERBB2 (HER2) molecular testing.
      Alterations in the human epidermal growth factor receptor 2 gene (HER2, ERBB2) have emerged as oncogenic drivers and as potential therapeutic targets in lung cancer.
      • Arcila M.E.
      • Chaft J.E.
      • Nafa K.
      • et al.
      Prevalence, clinicopathologic associations, and molecular spectrum of ERBB2 (HER2) tyrosine kinase mutations in lung adenocarcinomas.
      • Shan L.
      • Qiu T.
      • Ling Y.
      • et al.
      Prevalence and clinicopathological characteristics of HER2 and BRAF mutation in Chinese patients with lung adenocarcinoma.
      • Suzuki M.
      • Shiraishi K.
      • Yoshida A.
      • et al.
      HER2 gene mutations in non-small cell lung carcinomas: concurrence with Her2 gene amplification and Her2 protein expression and phosphorylation.
      • Li C.
      • Sun Y.
      • Fang R.
      • et al.
      Lung adenocarcinomas with HER2-activating mutations are associated with distinct clinical features and HER2/EGFR copy number gains.
      Sequence alterations and gene amplification occur in this setting and constitute approximately 2% to 3% and 2% to 5% of reported recurrent alterations, respectively. Therapeutic targeting of HER2 (the protein product of the ERBB2 gene) remains an area of active investigation at this time. Earlier clinical trials selecting patients based on protein expression by IHC or ERBB2 amplification by FISH did not demonstrate a clear benefit.
      • Gatzemeier U.
      • Groth G.
      • Butts C.
      • et al.
      Randomized phase II trial of gemcitabine-cisplatin with or without trastuzumab in HER2-positive non-small-cell lung cancer.
      • Krug L.M.
      • Miller V.A.
      • Patel J.
      • et al.
      Randomized phase II study of weekly docetaxel plus trastuzumab versus weekly paclitaxel plus trastuzumab in patients with previously untreated advanced nonsmall cell lung carcinoma.
      An additional phase II trial using ERBB2 mutation and ERBB2 amplification for patient selection demonstrated durable responses to dacomitinib, but only in patients with specific HER2 mutations.
      • Kris M.G.
      • Camidge D.R.
      • Giaccone G.
      • et al.
      Targeting HER2 aberrations as actionable drivers in lung cancers: phase II trial of the pan-HER tyrosine kinase inhibitor dacomitinib in patients with HER2-mutant or amplified tumors.
      In-frame insertions in exon 20 and substitutions at S310 are the most common mutations seen, and are typically mutually exclusive with other recurrent alterations, including mutations in EGFR, KRAS, and BRAF, as well as rearrangements involving ALK and ROS1. Insertions in exon 20 are variable, with most being a 12–base pair duplication of codons 775–778 encoding amino acids YVMA,
      • Arcila M.E.
      • Chaft J.E.
      • Nafa K.
      • et al.
      Prevalence, clinicopathologic associations, and molecular spectrum of ERBB2 (HER2) tyrosine kinase mutations in lung adenocarcinomas.
      and are more commonly observed in younger patients and patients with no smoking history. De novo ERBB2 amplification may occur with or without ERBB2 mutation,
      • Yoshizawa A.
      • Sumiyoshi S.
      • Sonobe M.
      • et al.
      HER2 status in lung adenocarcinoma: a comparison of immunohistochemistry, fluorescence in situ hybridization (FISH), dual-ISH, and gene mutations.
      • Suzuki M.
      • Shiraishi K.
      • Yoshida A.
      • et al.
      HER2 gene mutations in non-small cell lung carcinomas: concurrence with Her2 gene amplification and Her2 protein expression and phosphorylation.
      • Li C.
      • Sun Y.
      • Fang R.
      • et al.
      Lung adenocarcinomas with HER2-activating mutations are associated with distinct clinical features and HER2/EGFR copy number gains.
      with highly variable reported rates of co-occurrence from 0% to 87%.
      • Arcila M.E.
      • Chaft J.E.
      • Nafa K.
      • et al.
      Prevalence, clinicopathologic associations, and molecular spectrum of ERBB2 (HER2) tyrosine kinase mutations in lung adenocarcinomas.
      • Suzuki M.
      • Shiraishi K.
      • Yoshida A.
      • et al.
      HER2 gene mutations in non-small cell lung carcinomas: concurrence with Her2 gene amplification and Her2 protein expression and phosphorylation.
      • Li C.
      • Sun Y.
      • Fang R.
      • et al.
      Lung adenocarcinomas with HER2-activating mutations are associated with distinct clinical features and HER2/EGFR copy number gains.
      Although differences in methods and criteria defining amplification levels may be responsible for these observed discrepancies and require standardization, the higher prevalence of ERBB2 amplification independent of ERBB2 mutation suggests that mutation and amplification could represent distinct markers and therapeutic targets in lung cancer.
      • Li B.T.
      • Ross D.S.
      • Aisner D.L.
      • et al.
      HER2 amplification and HER2 mutation are distinct molecular targets in lung cancers.
      ERBB2 amplification has also been reported rarely as a secondary event in patients with sensitizing EGFR mutations and as a potential mechanism of resistance following treatment with EGFR inhibitors.
      • Takezawa K.
      • Pirazzoli V.
      • Arcila M.E.
      • et al.
      HER2 amplification: a potential mechanism of acquired resistance to EGFR inhibition in EGFR-mutant lung cancers that lack the second-site EGFRT790M mutation.
      In this context and with current evidence, routine stand-alone testing for ERBB2 mutations is not indicated outside a clinical trial. Nevertheless, when broader testing is performed through a multiplex assay or NGS, it is appropriate to include ERBB2 as part of the testing, as it may identify patients to be directed to clinical trials—in this context, testing for sequence alterations in ERBB2, particularly insertions/duplications in exon 20, which have been associated with response to treatment with targeted inhibitors of ERBB2 in case reports and small series.
      • Kris M.G.
      • Camidge D.R.
      • Giaccone G.
      • et al.
      Targeting HER2 aberrations as actionable drivers in lung cancers: phase II trial of the pan-HER tyrosine kinase inhibitor dacomitinib in patients with HER2-mutant or amplified tumors.
      • Chuang J.C.
      • Stehr H.
      • Liang Y.
      • et al.
      ERBB2-mutated metastatic non-small cell lung cancer: response and resistance to targeted therapies.
      6. Expert Consensus Opinion
      KRAS molecular testing is not indicated as a routine stand-alone assay as a sole determinant of targeted therapy. It is appropriate to include KRAS molecular testing as part of larger testing panels performed either initially or when routine EGFR, ALK, and ROS1 testing is negative.
      The strength of evidence was adequate to support the use of KRAS molecular testing when selecting patients for targeted therapy. The strength of evidence supporting the use of any clinical characteristic to identify patients who should receive KRAS testing was inadequate. This statement is evidence based and supported by 7 studies,
      • Hsu K.H.
      • Ho C.C.
      • Hsia T.C.
      • et al.
      Identification of five driver gene mutations in patients with treatment-naive lung adenocarcinoma in Taiwan.
      • Li H.
      • Pan Y.
      • Li Y.
      • et al.
      Frequency of well-identified oncogenic driver mutations in lung adenocarcinoma of smokers varies with histological subtypes and graduated smoking dose.
      • Li S.
      • Li L.
      • Zhu Y.
      • et al.
      Coexistence of EGFR with KRAS, or BRAF, or PIK3CA somatic mutations in lung cancer: a comprehensive mutation profiling from 5125 Chinese cohorts.
      • Fiala O.
      • Pesek M.
      • Finek J.
      • Benesova L.
      • Belsanova B.
      • Minarik M.
      The dominant role of G12C over other KRAS mutation types in the negative prediction of efficacy of epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer.
      • Mao C.
      • Qiu L.X.
      • Liao R.Y.
      • et al.
      KRAS mutations and resistance to EGFR-TKIs treatment in patients with non-small cell lung cancer: a meta-analysis of 22 studies.
      • Meng D.
      • Yuan M.
      • Li X.
      • et al.
      Prognostic value of K-RAS mutations in patients with non-small cell lung cancer: a systematic review with meta-analysis.
      • Yeung S.F.
      • Tong J.H.
      • Law P.P.
      • et al.
      Profiling of oncogenic driver events in lung adenocarcinoma revealed MET mutation as independent prognostic factor.
      comprising 2 MAs,
      • Mao C.
      • Qiu L.X.
      • Liao R.Y.
      • et al.
      KRAS mutations and resistance to EGFR-TKIs treatment in patients with non-small cell lung cancer: a meta-analysis of 22 studies.
      • Meng D.
      • Yuan M.
      • Li X.
      • et al.
      Prognostic value of K-RAS mutations in patients with non-small cell lung cancer: a systematic review with meta-analysis.
      4 PCSs,
      • Hsu K.H.
      • Ho C.C.
      • Hsia T.C.
      • et al.
      Identification of five driver gene mutations in patients with treatment-naive lung adenocarcinoma in Taiwan.
      • Li H.
      • Pan Y.
      • Li Y.
      • et al.
      Frequency of well-identified oncogenic driver mutations in lung adenocarcinoma of smokers varies with histological subtypes and graduated smoking dose.
      • Li S.
      • Li L.
      • Zhu Y.
      • et al.
      Coexistence of EGFR with KRAS, or BRAF, or PIK3CA somatic mutations in lung cancer: a comprehensive mutation profiling from 5125 Chinese cohorts.
      • Fiala O.
      • Pesek M.
      • Finek J.
      • Benesova L.
      • Belsanova B.
      • Minarik M.
      The dominant role of G12C over other KRAS mutation types in the negative prediction of efficacy of epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer.
      and 1 RCS.
      • Yeung S.F.
      • Tong J.H.
      • Law P.P.
      • et al.
      Profiling of oncogenic driver events in lung adenocarcinoma revealed MET mutation as independent prognostic factor.
      Five studies attempted to identify associations between patient or tumor characteristics and KRAS mutational status.
      • Hsu K.H.
      • Ho C.C.
      • Hsia T.C.
      • et al.
      Identification of five driver gene mutations in patients with treatment-naive lung adenocarcinoma in Taiwan.
      • Li S.
      • Li L.
      • Zhu Y.
      • et al.
      Coexistence of EGFR with KRAS, or BRAF, or PIK3CA somatic mutations in lung cancer: a comprehensive mutation profiling from 5125 Chinese cohorts.
      • Fiala O.
      • Pesek M.
      • Finek J.
      • Benesova L.
      • Belsanova B.
      • Minarik M.
      The dominant role of G12C over other KRAS mutation types in the negative prediction of efficacy of epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer.
      • Mao C.
      • Qiu L.X.
      • Liao R.Y.
      • et al.
      KRAS mutations and resistance to EGFR-TKIs treatment in patients with non-small cell lung cancer: a meta-analysis of 22 studies.
      • Yeung S.F.
      • Tong J.H.
      • Law P.P.
      • et al.
      Profiling of oncogenic driver events in lung adenocarcinoma revealed MET mutation as independent prognostic factor.
      Two MAs
      • Mao C.
      • Qiu L.X.
      • Liao R.Y.
      • et al.
      KRAS mutations and resistance to EGFR-TKIs treatment in patients with non-small cell lung cancer: a meta-analysis of 22 studies.
      • Meng D.
      • Yuan M.
      • Li X.
      • et al.
      Prognostic value of K-RAS mutations in patients with non-small cell lung cancer: a systematic review with meta-analysis.
      reported on overall survival and EGFR-TKI response rates when KRAS mutation–positive patients were treated with standard care. All included studies were assessed for quality and none were found to have methodologic flaws that would raise concerns about the studies’ findings (SDC Table 16). Refer to SDC Table 17 for a summary of findings from studies supporting the use of KRAS molecular testing.
      KRAS mutations are reported in 20% to 30% of lung adenocarcinomas. KRAS mutations are encountered more frequently in people with tobacco exposure, but have been reported in approximately 5% of lung cancer patients who have never used tobacco. Most studies indicate an increased incidence in males and those of white or African ancestry, in comparison with females and those of Asian ancestry. KRAS mutations occur most frequently in codon 12 and 13, much less commonly in codon 61, and rarely in codon 146, and can readily be detected by quick targeted hot-spot assays (ie, real-time PCR, droplet digital PCR, or pyrosequencing) interrogating these codons, as well as incorporated into larger panel tests. They are typically mutually exclusive with other driver mutations such as EGFR mutations and ALK rearrangements.
      • Hsu K.H.
      • Ho C.C.
      • Hsia T.C.
      • et al.
      Identification of five driver gene mutations in patients with treatment-naive lung adenocarcinoma in Taiwan.
      • Li H.
      • Pan Y.
      • Li Y.
      • et al.
      Frequency of well-identified oncogenic driver mutations in lung adenocarcinoma of smokers varies with histological subtypes and graduated smoking dose.
      • Fiala O.
      • Pesek M.
      • Finek J.
      • Benesova L.
      • Belsanova B.
      • Minarik M.
      The dominant role of G12C over other KRAS mutation types in the negative prediction of efficacy of epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer.
      • Mao C.
      • Qiu L.X.
      • Liao R.Y.
      • et al.
      KRAS mutations and resistance to EGFR-TKIs treatment in patients with non-small cell lung cancer: a meta-analysis of 22 studies.
      • Yeung S.F.
      • Tong J.H.
      • Law P.P.
      • et al.
      Profiling of oncogenic driver events in lung adenocarcinoma revealed MET mutation as independent prognostic factor.
      • Leidner R.S.
      • Fu P.
      • Clifford B.
      • et al.
      Genetic abnormalities of the EGFR pathway in African American patients with non-small-cell lung cancer.
      • Reinersman J.M.
      • Johnson M.L.
      • Riely G.J.
      • et al.
      Frequency of EGFR and KRAS mutations in lung adenocarcinomas in African Americans.
      • Shigematsu H.
      • Lin L.
      • Takahashi T.
      • et al.
      Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers.
      • Riely G.J.
      • Kris M.G.
      • Rosenbaum D.
      • et al.
      Frequency and distinctive spectrum of KRAS mutations in never smokers with lung adenocarcinoma.
      • Thu K.L.
      • Vucic E.A.
      • Chari R.
      • et al.
      Lung adenocarcinoma of never smokers and smokers harbor differential regions of genetic alteration and exhibit different levels of genomic instability.
      • Broet P.
      • Dalmasso C.
      • Tan E.H.
      • et al.
      Genomic profiles specific to patient ethnicity in lung adenocarcinoma.
      Therapies directed against mutated KRAS have not been proven clinically effective. For example, although promising results (37% objective response rate) were obtained in a phase II study of selumetinib, an inhibitor of MEK1 (downstream of KRAS), plus docetaxel
      • Janne P.A.
      • Shaw A.T.
      • Pereira J.R.
      • et al.
      Selumetinib plus docetaxel for KRAS-mutant advanced non-small-cell lung cancer: a randomised, multicentre, placebo-controlled, phase 2 study.
      in KRAS-mutant advanced lung cancer, this combination failed to demonstrate an outcome benefit in the Selumetinib Evaluation as Combination Therapy-1 (SELECT-1) phase III trial,
      • Janne P.A.
      • van den Heuvel M.M.
      • Barlesi F.
      • et al.
      Selumetinib plus docetaxel compared with docetaxel alone and progression-free survival in patients with KRAS-mutant advanced non-small cell lung cancer: the SELECT-1 randomized clinical trial.
      and a phase II study of selumetinib + erlotinib in KRAS-mutant lung cancers failed to show response to selumetinib independent of erlotinib.
      • Carter C.A.
      • Rajan A.
      • Keen C.
      • et al.
      Selumetinib with and without erlotinib in KRAS mutant and KRAS wild-type advanced nonsmall-cell lung cancer.
      Hence, intense research investigation into therapeutic strategies against this common mutation continues, and it is appropriate to include KRAS in a larger testing panel used for directing patients to investigational therapies.
      Another application of KRAS mutation testing is in a sequential testing algorithm, with a positive result greatly diminishing the likelihood of another, targetable oncogenic alteration. If the KRAS test is performed prior to EGFR, ALK, or ROS1 testing, however, the laboratory must ensure that sufficient tumor is available for EGFR, ALK, and ROS1 testing within the recommended time frame, particularly in the event of a negative KRAS result. Similarly, the presence of a KRAS mutation renders unlikely the other oncogenes recommended for larger panels, such as RET, ERBB2 (HER2), and BRAF. In this context, a rapid, targeted assay for KRAS may have value in helping to determine whether or not an EGFR/ALK/ROS1 wild-type patient would benefit from expanded panel testing, in that panel testing would be less likely to benefit KRAS-mutant cancer patients. This model may, however, change as technology evolves, as newer ultrasensitive methods have shown co-occurrence of driver oncogenes, including KRAS, in subpopulations within tumors that previously had not been detected by less sensitive methods.
      • Guibert N.
      • Barlesi F.
      • Descourt R.
      • et al.
      Characteristics and outcomes of patients with lung cancer harboring multiple molecular alterations: results from the IFCT study Biomarkers France.
      • Scheffler M.
      • Ihle M.A.
      • Hein R.
      • et al.
      Genetic heterogeneity of KRAS-mutated NSCLC: co-occurrence of potentially targetable aberrations and evolutionary background.
      7. Expert Consensus Opinion
      MET molecular testing is not indicated as a routine stand-alone assay outside the context of a clinical trial. It is appropriate to include MET as part of larger testing panels performed either initially or when routine EGFR, ALK, and ROS1 testing is negative.
      The strength of evidence is inadequate supporting the use of MET molecular testing. This statement was evidence based and supported by 7 studies,
      • Guo B.
      • Cen H.
      • Tan X.
      • Liu W.
      • Ke Q.
      Prognostic value of MET gene copy number and protein expression in patients with surgically resected non-small cell lung cancer: a meta-analysis of published literatures.
      • Jin Y.
      • Sun P.L.
      • Kim H.
      • et al.
      MET gene copy number gain is an independent poor prognostic marker in Korean stage I lung adenocarcinomas.
      • Spigel D.R.
      • Ervin T.J.
      • Ramlau R.A.
      • et al.
      Randomized phase II trial of Onartuzumab in combination with erlotinib in patients with advanced non-small-cell lung cancer.
      • Kowalczuk O.
      • Kozlowski M.
      • Niklinska W.
      • Kisluk J.
      • Niklinska B.J.
      • Niklinski J.
      Increased MET gene copy number but not mRNA level predicts postoperative recurrence in patients with non-small cell lung cancer.
      • Jurmeister P.
      • Lenze D.
      • Berg E.
      • et al.
      Parallel screening for ALK, MET and ROS1 alterations in non-small cell lung cancer with implications for daily routine testing.
      • Noro R.
      • Seike M.
      • Zou F.
      • et al.
      MET FISH-positive status predicts short progression-free survival and overall survival after gefitinib treatment in lung adenocarcinoma with EGFR mutation.
      • Weingertner N.
      • Meyer N.
      • Voegeli A.C.
      • et al.
      Correlation between MET protein expression and MET gene copy number in a Caucasian cohort of non-small cell lung cancers according to the new IASLC/ATS/ERS classification.
      comprising 1 MA,
      • Guo B.
      • Cen H.
      • Tan X.
      • Liu W.
      • Ke Q.
      Prognostic value of MET gene copy number and protein expression in patients with surgically resected non-small cell lung cancer: a meta-analysis of published literatures.
      1 phase II randomized controlled trial,
      • Spigel D.R.
      • Ervin T.J.
      • Ramlau R.A.
      • et al.
      Randomized phase II trial of Onartuzumab in combination with erlotinib in patients with advanced non-small-cell lung cancer.
      1 PCS,
      • Kowalczuk O.
      • Kozlowski M.
      • Niklinska W.
      • Kisluk J.
      • Niklinska B.J.
      • Niklinski J.
      Increased MET gene copy number but not mRNA level predicts postoperative recurrence in patients with non-small cell lung cancer.
      and 4 RCSs.
      • Jin Y.
      • Sun P.L.
      • Kim H.
      • et al.
      MET gene copy number gain is an independent poor prognostic marker in Korean stage I lung adenocarcinomas.
      • Jurmeister P.
      • Lenze D.
      • Berg E.
      • et al.
      Parallel screening for ALK, MET and ROS1 alterations in non-small cell lung cancer with implications for daily routine testing.
      • Noro R.
      • Seike M.
      • Zou F.
      • et al.
      MET FISH-positive status predicts short progression-free survival and overall survival after gefitinib treatment in lung adenocarcinoma with EGFR mutation.
      • Weingertner N.
      • Meyer N.
      • Voegeli A.C.
      • et al.
      Correlation between MET protein expression and MET gene copy number in a Caucasian cohort of non-small cell lung cancers according to the new IASLC/ATS/ERS classification.
      All included studies were assessed for quality and none were found to have methodologic flaws that would raise concerns about the studies’ finding (SDC Table 18). Refer to SDC Table 19 for a summary of findings from studies supporting the use of MET molecular testing.
      Initially reported as a mechanism of secondary resistance to EGFR TKI therapy in EGFR-mutant lung cancer,
      • Engelman J.A.
      • Zejnullahu K.
      • Mitsudomi T.
      • et al.
      MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling.
      • Piotrowska Z.
      • Sequist L.V.
      Epidermal growth factor receptor-mutant lung cancer: new drugs, new resistance mechanisms, and future treatment options.
      both the understanding of the mechanism of activation of MET and the utility of MET testing in lung cancer have gone through several phases. MET copy gain was initially recognized in association with secondary resistance to EGFR inhibitors,
      • Engelman J.A.
      • Zejnullahu K.
      • Mitsudomi T.
      • et al.
      MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling.
      prompting the development of targeted therapies that showed disappointing results.
      • Spigel D.R.
      • Edelman M.J.
      • O’Byrne K.
      • et al.
      Results from the phase III randomized trial of onartuzumab plus erlotinib versus erlotinib in previously treated stage IIIB or IV non-small-cell lung cancer: METLung.
      More recently, interest in targeting MET has been rekindled by the discovery of activating mutations that may respond to targeted inhibition.
      The MET gene encodes for the receptor for hepatocyte growth factor (HGFR), and its activation has pleotropic functions in promoting cell survival, proliferation, motility, invasion, and epithelial-mesenchymal transition.
      • Trusolino L.
      • Bertotti A.
      • Comoglio P.M.
      MET signalling: principles and functions in development, organ regeneration and cancer.
      • Gherardi E.
      • Birchmeier W.
      • Birchmeier C.
      • Vande Woude G.
      Targeting MET in cancer: rationale and progress.
      • Sierra J.R.
      • Tsao M.S.
      c-MET as a potential therapeutic target and biomarker in cancer.
      • Feng Y.
      • Thiagarajan P.S.
      • Ma P.C.
      MET signaling: novel targeted inhibition and its clinical development in lung cancer.
      HGFR can become activated and drive oncogenesis through several different mechanisms, including (1) amplification resulting in high expression of the receptor,
      • Lutterbach B.
      • Zeng Q.
      • Davis L.J.
      • et al.
      Lung cancer cell lines harboring MET gene amplification are dependent on Met for growth and survival.