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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

      Objective

      To establish evidence-based recommendations for the molecular analysis of lung cancers that are that are required to guide EGFR- and ALK-directed therapies, addressing which patients and samples should be tested, and when and how testing should be performed.

      Participants

      Three cochairs without conflicts of interest were selected, one from each of the 3 sponsoring professional societies: College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Writing and advisory panels were constituted from additional experts from these societies.

      Evidence

      Three unbiased literature searches of electronic databases were performed to capture articles published published from January 2004 through February 2012, yielding 1533 articles whose abstracts were screened to identify 521 pertinent articles that were then reviewed in detail for their relevance to the recommendations. Evidence was formally graded for each recommendation.

      Consensus Process

      Initial recommendations were formulated by the cochairs and panel members at a public meeting. Each guideline section was assigned to at least 2 panelists. Drafts were circulated to the writing panel (version 1), advisory panel (version 2), and the public (version 3) before submission (version 4).

      Conclusions

      The 37 guideline items address 14 subjects, including 15 recommendations (evidence grade A/B). The major recommendations are to use testing for EGFR mutations and ALK fusions to guide patient selection for therapy with an epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) inhibitor, respectively, in all patients with advanced-stage adenocarcinoma, regardless of sex, race, smoking history, or other clinical risk factors, and to prioritize EGFR and ALK testing over other molecular predictive tests. As scientific discoveries and clinical practice outpace the completion of randomized clinical trials, evidence-based guidelines developed by expert practitioners are vital for communicating emerging clinical standards. Already, new treatments targeting genetic alterations in other, less common driver oncogenes are being evaluated in lung cancer, and testing for these may be addressed in future versions of these guidelines.

      BACKGROUND—EGFR MUTATIONSAND ALK FUSIONS

      Lung cancer is the leading cause of cancer-related mortality, accounting for approximately 1.4 million deaths per year worldwide and approximately 160 000 deaths per year in the United States, which is approximately 25% to 30% of all US cancer deaths and more than the next 3 cancers (colon, prostate, breast) combined.
      • World Health Organization
      Fortunately, the past decade has seen major advances in our understanding of the pathogenesis and management of lung cancers, adenocarcinoma in particular. Specifically, the discovery of the biologic and therapeutic importance of acquired genetic alterations in 2 genes that encode pharmacologically targetable tyrosine kinases involved in growth factor receptor signaling, epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK), has changed the way these cancers are diagnosed and treated.
      As gefitinib and erlotinib, small-molecule competitive inhibitors of the EGFR tyrosine kinase, were being evaluated in clinical trials of advanced-stage lung cancer in the early part of the last decade, unusual prolonged responses to these medications were recognized in a subset of patients.
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      Bronchioloalveolar pathologic subtype and smoking history predict sensitivity to gefitinib in advanced non-small-cell lung cancer.
      This unusual clinical behavior, not seen previously with standard chemotherapy, led to investigations that identified a correlation between activating somatic mutation in the EGFR gene and clinical response to gefitinib and erlotinib. This initial exciting observation has led to sustained and continuing laboratory and clinical investigations into the mechanism and clinical consequences of EGFR mutations in lung cancer. In unselected advanced non–small cell lung cancer (NSCLC) patients, gefitinib and erlotinib produce response rates of 8% to 9%, with a median time to progression of 2.2 months to 3.0 months.
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      Biological and clinical features in predicting efficacy of epidermal growth factor receptor tyrosine kinase inhibitors: a systematic review and meta-analysis.
      In contrast, advanced NSCLC patients selected on the basis of activating EGFR mutations in their tumors show response rates (RRs) of 68%, with a mean progression-free survival (PFS) and time to progression of 12 months (Table 1).
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      Erlotinib in previously treated non-small-cell lung cancer.
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      Gefitinib in advanced non-small cell lung cancer: does it deserve a second chance?.
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      Gefitinib plus best supportive care in previously treated patients with refractory advanced non-small-cell lung cancer: results from a randomised, placebo-controlled, multicentre study (Iressa Survival Evaluation in Lung Cancer).
      TABLE 1Different Outcomes in All Stages of Non–Small Cell Lung Cancer Patients With and Without EGFR Mutations, Treated With Tyrosine Kinase Inhibitor
      Mean ± SDPercentage
      OutcomeEGFR Mutation PositiveEGFR Mutation NegativeEGFR Mutation PositiveEGFR Mutation Negativen (N)WMD (95% CI)RR (95% CI)P Value
      Response rate, %
      References 7, 17, 28, 48, 53, 56, 58, 110, 129, 133, 170, 191–198, 202, 211, 250–278.
      681151 (3644)5.16 (4.41–6.04)<.001
      Disease control rate, %
      References 7, 17, 53, 56, 115, 129, 133, 191, 193–195, 202, 250–254, 258, 259, 261, 266, 267, 273–275, 277–279.
      864228 (2204)1.99 (1.73–2.29)<.001
      Time to progression/progression- free survival, moc12.0 ± 7.863.4 ± 2.5927 (2347)8.66 (6.31–11.00)<.001
      Median survival time, mod23.3 ± 18.412.1 ± 13.927 (2489)10.66 (8.36–12.96)<.001
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      Mutation in the tyrosine kinase domain of epidermal growth factor receptor is a predictive and prognostic factor for gefitinib treatment in patients with non-small cell lung cancer.
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      Epidermal growth factor receptor gene mutations and increased copy numbers predict gefitinib sensitivity in patients with recurrent non-small-cell lung cancer.
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      First-SIGNAL: first-line single-agent iressa versus gemcitabine and cisplatin trial in never-smokers with adenocarcinoma of the lung.
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      Prediction of epidermal growth factor receptor mutations in the plasma/pleural effusion to efficacy of gefitinib treatment in advanced non-small cell lung cancer.
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      Predictors of the response to gefitinib in refractory non-small cell lung cancer.
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      Randomized phase II study of gefitinib versus erlotinib in patients with advanced non-small cell lung cancer who failed previous chemotherapy.
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      Novel heteroduplex method using small cytology specimens with a remarkably high success rate for analysing EGFR gene mutations with a significant correlation to gefitinib efficacy in non-small-cell lung cancer.
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      Brain metastases from lung cancer responding to erlotinib: the importance of EGFR mutation.
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      EGFR mutations and HER2/3 protein expression and clinical outcome in Chinese advanced non-small cell lung cancer patients treated with gefitinib.
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      Specific EGFR mutations predict treatment outcome of stage IIIB/IV patients with chemotherapy-naive non-small-cell lung cancer receiving first-line gefitinib monotherapy.
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      Gefitinib therapy in patients with advanced non-small cell lung cancer with or without testing for epidermal growth factor receptor (EGFR) mutations.
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      Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK.
      • Douillard JY
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      Molecular predictors of outcome with gefitinib and docetaxel in previously treated non-small-cell lung cancer: data from the randomized phase III INTEREST trial.
      • Han SW
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      Predictive and prognostic impact of epidermal growth factor receptor mutation in non-small-cell lung cancer patients treated with gefitinib.
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      Mutation in the tyrosine kinase domain of epidermal growth factor receptor is a predictive and prognostic factor for gefitinib treatment in patients with non-small cell lung cancer.
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      Epidermal growth factor receptor gene mutations and increased copy numbers predict gefitinib sensitivity in patients with recurrent non-small-cell lung cancer.
      • Miller VA
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      Molecular characteristics of bronchioloalveolar carcinoma and adenocarcinoma, bronchioloalveolar carcinoma subtype, predict response to erlotinib.
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      First-line gefitinib for patients with advanced non-small-cell lung cancer harboring epidermal growth factor receptor mutations without indication for chemotherapy.
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      Activating mutations within the EGFR kinase domain: a molecular predictor of disease-free survival in resected pulmonary adenocarcinoma.
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      KRAS mutation is an important predictor of resistance to therapy with epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer.
      • Hirsch FR
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      • et al.
      Combination of EGFR gene copy number and protein expression predicts outcome for advanced non-small-cell lung cancer patients treated with gefitinib.
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      Predictors of gefitinib outcomes in advanced non-small cell lung cancer (NSCLC): study of a comprehensive panel of molecular markers.
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      Epidermal growth factor receptor activating mutations in Spanish gefitinib-treated non-small-cell lung cancer patients.
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      First-SIGNAL: first-line single-agent iressa versus gemcitabine and cisplatin trial in never-smokers with adenocarcinoma of the lung.
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      Complex mutation patterns of epidermal growth factor receptor gene associated with variable responses to gefitinib treatment in patients with non-small cell lung cancer.
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      Prediction of epidermal growth factor receptor mutations in the plasma/pleural effusion to efficacy of gefitinib treatment in advanced non-small cell lung cancer.
      • Kim KS
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      • Kim YC
      • et al.
      Predictors of the response to gefitinib in refractory non-small cell lung cancer.
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      Novel heteroduplex method using small cytology specimens with a remarkably high success rate for analysing EGFR gene mutations with a significant correlation to gefitinib efficacy in non-small-cell lung cancer.
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      Brain metastases from lung cancer responding to erlotinib: the importance of EGFR mutation.
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      Predictive factors associated with prolonged survival in patients with advanced non-small-cell lung cancer (NSCLC) treated with gefitinib.
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      Response to treatment and survival of patients with non-small cell lung cancer undergoing somatic EGFR mutation testing.
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      Gefitinib for non-small-cell lung cancer patients with epidermal growth factor receptor gene mutations screened by peptide nucleic acid-locked nucleic acid PCR clamp.
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      • Han Y
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      • et al.
      EGFR mutations and HER2/3 protein expression and clinical outcome in Chinese advanced non-small cell lung cancer patients treated with gefitinib.
      • Yang CH
      • Yu CJ
      • Shih JY
      • et al.
      Specific EGFR mutations predict treatment outcome of stage IIIB/IV patients with chemotherapy-naive non-small-cell lung cancer receiving first-line gefitinib monotherapy.
      • Wu J-Y
      • Shih J-Y
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      • Yang C-H
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      • Yang P-C
      Gefitinib therapy in patients with advanced non-small cell lung cancer with or without testing for epidermal growth factor receptor (EGFR) mutations.
      .
      Abbreviations: CI, confidence interval; n, number of studies; N, number of patients; RR, relative risk; SD, standard deviation; WMD, weighted mean difference.
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      Mutation in the tyrosine kinase domain of epidermal growth factor receptor is a predictive and prognostic factor for gefitinib treatment in patients with non-small cell lung cancer.
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      KRAS mutation is an important predictor of resistance to therapy with epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer.
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      Combination of EGFR gene copy number and protein expression predicts outcome for advanced non-small-cell lung cancer patients treated with gefitinib.
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      Epidermal growth factor receptor mutations and gene amplification in non-small-cell lung cancer: molecular analysis of the IDEAL/INTACT gefitinib trials.
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      • et al.
      Are there any ethnic differences in molecular predictors of erlotinib efficacy in advanced non-small cell lung cancer?.
      • Cappuzzo F
      • Ligorio C
      • Janne PA
      • et al.
      Prospective study of gefitinib in epidermal growth factor receptor fluorescence in situ hybridization-positive/phospho-Akt-positive or never smoker patients with advanced non-small-cell lung cancer: the ONCOBELL trial.
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      Erlotinib in first-line therapy for non-small cell lung cancer: a prospective phase II study.
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      • Agulnik JS
      • Ang C
      • et al.
      Epidermal growth factor receptor mutations detected by denaturing high-performance liquid chromatography in nonsmall cell lung cancer: impact on response to therapy with epidermal growth factor receptor-tyrosine kinase inhibitors.
      • Cortes-Funes H
      • Gomez C
      • Rosell R
      • et al.
      Epidermal growth factor receptor activating mutations in Spanish gefitinib-treated non-small-cell lung cancer patients.
      • Han J-Y
      • Park K
      • Kim S-W
      • et al.
      First-SIGNAL: first-line single-agent iressa versus gemcitabine and cisplatin trial in never-smokers with adenocarcinoma of the lung.
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      Clinical significance of epidermal growth factor receptor gene mutations on treatment outcome after first-line cytotoxic chemotherapy in Japanese patients with non-small cell lung cancer.
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      • Chang WC
      • et al.
      Complex mutation patterns of epidermal growth factor receptor gene associated with variable responses to gefitinib treatment in patients with non-small cell lung cancer.
      • Jackman DM
      • Yeap BY
      • Lindeman NI
      • et al.
      Phase II clinical trial of chemotherapy-naive patients > or = 70 years of age treated with erlotinib for advanced non-small-cell lung cancer.
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      Prediction of epidermal growth factor receptor mutations in the plasma/pleural effusion to efficacy of gefitinib treatment in advanced non-small cell lung cancer.
      • Kim KS
      • Jeong JY
      • Kim YC
      • et al.
      Predictors of the response to gefitinib in refractory non-small cell lung cancer.
      • Kim ST
      • Uhm JE
      • Lee J
      • et al.
      Randomized phase II study of gefitinib versus erlotinib in patients with advanced non-small cell lung cancer who failed previous chemotherapy.
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      Mutations of epidermal growth factor receptor of non-small cell lung cancer were associated with sensitivity to gefitinib in recurrence after surgery.
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      Phase II study of erlotinib for chemotherapy-naive patients with advanced or metastatic non-small cell lung cancer who are ineligible for platinum doublets.
      • Oshita F
      • Matsukuma S
      • Yoshihara M
      • et al.
      Novel heteroduplex method using small cytology specimens with a remarkably high success rate for analysing EGFR gene mutations with a significant correlation to gefitinib efficacy in non-small-cell lung cancer.
      • Porta R
      • Sanchez-Torres JM
      • Paz-Ares L
      • et al.
      Brain metastases from lung cancer responding to erlotinib: the importance of EGFR mutation.
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      Molecular characteristics predict clinical outcomes: prospective trial correlating response to the EGFR tyrosine kinase inhibitor gefitinib with the presence of sensitizing mutations in the tyrosine binding domain of the EGFR gene.
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      Epidermal growth factor receptor gene amplification and gefitinib sensitivity in patients with recurrent lung cancer.
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      • Negoro S
      • Funada Y
      • et al.
      Predictive factors associated with prolonged survival in patients with advanced non-small-cell lung cancer (NSCLC) treated with gefitinib.
      • Sequist LV
      • Joshi VA
      • Janne PA
      • et al.
      Response to treatment and survival of patients with non-small cell lung cancer undergoing somatic EGFR mutation testing.
      • Sutani A
      • Nagai Y
      • Udagawa K
      • et al.
      Gefitinib for non-small-cell lung cancer patients with epidermal growth factor receptor gene mutations screened by peptide nucleic acid-locked nucleic acid PCR clamp.
      • Taron M
      • Ichinose Y
      • Rosell R
      • et al.
      Activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor are associated with improved survival in gefitinib-treated chemorefractory lung adenocarcinomas.
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      • Sugio K
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      Epidermal growth factor receptor mutations are associated with gefitinib sensitivity in non-small cell lung cancer in Japanese.
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      • Wu YL
      • Zhang GC
      • Zhou Q
      • Xu CR
      • Guo AL
      EGFR/KRAS mutations and gefitinib therapy in Chinese NSCLC patients.
      • Xu JM
      • Han Y
      • Duan HQ
      • et al.
      EGFR mutations and HER2/3 protein expression and clinical outcome in Chinese advanced non-small cell lung cancer patients treated with gefitinib.
      • Yang CH
      • Yu CJ
      • Shih JY
      • et al.
      Specific EGFR mutations predict treatment outcome of stage IIIB/IV patients with chemotherapy-naive non-small-cell lung cancer receiving first-line gefitinib monotherapy.
      • Wu J-Y
      • Shih J-Y
      • Chen K-Y
      • Yang C-H
      • Yu C-J
      • Yang P-C
      Gefitinib therapy in patients with advanced non-small cell lung cancer with or without testing for epidermal growth factor receptor (EGFR) mutations.
      .
      b References
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      • et al.
      Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma.
      • Shaw AT
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      • Mino-Kenudson M
      • et al.
      Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK.
      • Han SW
      • Kim TY
      • Hwang PG
      • et al.
      Predictive and prognostic impact of epidermal growth factor receptor mutation in non-small-cell lung cancer patients treated with gefitinib.
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      • Li LH
      • et al.
      Mutation in the tyrosine kinase domain of epidermal growth factor receptor is a predictive and prognostic factor for gefitinib treatment in patients with non-small cell lung cancer.
      • Janne PA
      • Borras AM
      • Kuang Y
      • et al.
      A rapid and sensitive enzymatic method for epidermal growth factor receptor mutation screening.
      • Takano T
      • Ohe Y
      • Sakamoto H
      • et al.
      Epidermal growth factor receptor gene mutations and increased copy numbers predict gefitinib sensitivity in patients with recurrent non-small-cell lung cancer.
      • Kawada I
      • Soejima K
      • Watanabe H
      • et al.
      An alternative method for screening EGFR mutation using RFLP in non-small cell lung cancer patients.
      • Massarelli E
      • Varella-Garcia M
      • Tang X
      • et al.
      KRAS mutation is an important predictor of resistance to therapy with epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer.
      • Hirsch FR
      • Varella-Garcia M
      • Cappuzzo F
      • et al.
      Combination of EGFR gene copy number and protein expression predicts outcome for advanced non-small-cell lung cancer patients treated with gefitinib.
      • Endoh H
      • Yatabe Y
      • Kosaka T
      • Kuwano H
      • Mitsudomi T
      PTEN and PIK3CA expression is associated with prolonged survival after gefitinib treatment in EGFR-mutated lung cancer patients.
      • Hirsch FR
      • Kabbinavar F
      • Eisen T
      • et al.
      A randomized, phase II, biomarker-selected study comparing erlotinib to erlotinib intercalated with chemotherapy in first-line therapy for advanced non-small-cell lung cancer.
      • Endo K
      • Sasaki H
      • Yano M
      • et al.
      Evaluation of the epidermal growth factor receptor gene mutation and copy number in non-small cell lung cancer with gefitinib therapy.
      • Ahn MJ
      • Park BB
      • Ahn JS
      • et al.
      Are there any ethnic differences in molecular predictors of erlotinib efficacy in advanced non-small cell lung cancer?.
      • Cappuzzo F
      • Ligorio C
      • Janne PA
      • et al.
      Prospective study of gefitinib in epidermal growth factor receptor fluorescence in situ hybridization-positive/phospho-Akt-positive or never smoker patients with advanced non-small-cell lung cancer: the ONCOBELL trial.
      • Choi DR
      • Lee DH
      • Choi C-M
      • Kim S-W
      • Suh C
      • Lee J-S
      Erlotinib in first-line therapy for non-small cell lung cancer: a prospective phase II study.
      • Cohen V
      • Agulnik JS
      • Ang C
      • et al.
      Epidermal growth factor receptor mutations detected by denaturing high-performance liquid chromatography in nonsmall cell lung cancer: impact on response to therapy with epidermal growth factor receptor-tyrosine kinase inhibitors.
      • Cortes-Funes H
      • Gomez C
      • Rosell R
      • et al.
      Epidermal growth factor receptor activating mutations in Spanish gefitinib-treated non-small-cell lung cancer patients.
      • Hsieh MH
      • Fang YF
      • Chang WC
      • et al.
      Complex mutation patterns of epidermal growth factor receptor gene associated with variable responses to gefitinib treatment in patients with non-small cell lung cancer.
      • Jackman DM
      • Yeap BY
      • Lindeman NI
      • et al.
      Phase II clinical trial of chemotherapy-naive patients > or = 70 years of age treated with erlotinib for advanced non-small-cell lung cancer.
      • Jian G
      • Songwen Z
      • Ling Z
      • et al.
      Prediction of epidermal growth factor receptor mutations in the plasma/pleural effusion to efficacy of gefitinib treatment in advanced non-small cell lung cancer.
      • Oshita F
      • Matsukuma S
      • Yoshihara M
      • et al.
      Novel heteroduplex method using small cytology specimens with a remarkably high success rate for analysing EGFR gene mutations with a significant correlation to gefitinib efficacy in non-small-cell lung cancer.
      • Porta R
      • Sanchez-Torres JM
      • Paz-Ares L
      • et al.
      Brain metastases from lung cancer responding to erlotinib: the importance of EGFR mutation.
      • Taron M
      • Ichinose Y
      • Rosell R
      • et al.
      Activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor are associated with improved survival in gefitinib-treated chemorefractory lung adenocarcinomas.
      • Uramoto H
      • Sugio K
      • Oyama T
      • et al.
      Epidermal growth factor receptor mutations are associated with gefitinib sensitivity in non-small cell lung cancer in Japanese.
      • Wang Z
      • Wu YL
      • Zhang GC
      • Zhou Q
      • Xu CR
      • Guo AL
      EGFR/KRAS mutations and gefitinib therapy in Chinese NSCLC patients.
      • Yang CH
      • Yu CJ
      • Shih JY
      • et al.
      Specific EGFR mutations predict treatment outcome of stage IIIB/IV patients with chemotherapy-naive non-small-cell lung cancer receiving first-line gefitinib monotherapy.
      • Wu J-Y
      • Shih J-Y
      • Chen K-Y
      • Yang C-H
      • Yu C-J
      • Yang P-C
      Gefitinib therapy in patients with advanced non-small cell lung cancer with or without testing for epidermal growth factor receptor (EGFR) mutations.
      • Argiris A
      • Hensing T
      • Yeldandi A
      • et al.
      Combined analysis of molecular and clinical predictors of gefitinib activity in advanced non-small cell lung cancer: epidermal growth factor receptor mutations do not tell the whole story.
      .
      In 2009, the first randomized clinical trial (the Iressa Pan-Asia Study [IPASS]) showed that, for advanced NSCLC patients with an activating EGFR mutation, initial treatment with an EGFR tyrosine kinase inhibitor (TKI) was superior to standard platinum-based chemotherapy.
      • Mok TS
      • Wu YL
      • Thongprasert S
      • et al.
      Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma.
      In this study, which enrolled East Asian patients with stage IIIB/IV lung adenocarcinoma who never smoked tobacco (or only smoked lightly), the patients whose tumors contained an activating EGFR mutation and who received gefitinib had a significantly longer PFS than those receiving chemotherapy (hazard ratio [HR] for progression or death, 0.48; P <.001).
      • Mok TS
      • Wu YL
      • Thongprasert S
      • et al.
      Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma.
      Subsequently, 5 additional randomized controlled trials confirmed this association between activating EGFR mutations and objective response to gefitinib and/or erlotinib therapy (Table 2). However, in spite of these impressive differences in PFS, no study has shown an advantage in overall survival for EGFR-mutation–bearing patients treated initially with an EGFR TKI in comparison to chemotherapy. This is likely to be at least partly due to the crossover design of these studies, in that a large fraction of the patients with EGFR-mutated tumors treated initially with chemotherapy crossed over to the EGFR TKI treatment arm, confounding the interpretation of overall survival data.
      TABLE 2Randomized Clinical Trial Data on Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor (TKI) Therapy Versus Chemotherapy as First-Line Therapy for Patients With EGFR-Mutated Lung Cancers
      StudyNo. of Patients With EGFR-Mutated Lung CancersResponse Rate (EGFR TKI Versus Chemotherapy), %Progression-Free Survival (EGFR TKI Versus Chemotherapy), mo
      EURTAC
      • Rosell R
      • Carcereny E
      • Gervais R
      Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial.
      173 (86 erlotinib and 87 chemo)58 versus 159.7 versus 5.2 (HR 0.37)
      OPTIMAL
      • Zhou C
      • Wu Y-L
      • Chen G
      • et al.
      Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomised, phase 3 study.
      154 (82 erlotinib and 72 chemo)83 versus 3613.1 versus 4.6 (HR 0.16)
      NEJ 002
      • Maemondo M
      • Inoue A
      • Kobayashi K
      • et al.
      Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR.
      228 (114 gefitinib and 114 chemo)74 versus 3110.8 versus 5.4 (HR 0.30)
      WJTOG3405
      • Mitsudomi T
      • Morita S
      • Yatabe Y
      • et al.
      Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial.
      117 (58 gefitinib and 59 chemo)62 versus 329.2 versus 6.3 (HR 0.49)
      IPASS
      • Mok TS
      • Wu YL
      • Thongprasert S
      • et al.
      Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma.
      • Fukuoka M
      • Wu YL
      • Thongprasert S
      • et al.
      Biomarker analyses and final overall survival results from a phase III, randomized, open-label, first-line study of gefitinib versus carboplatin/paclitaxel in clinically selected patients with advanced non-small-cell lung cancer in Asia (IPASS).
      261 (132 gefitinib and 129 chemo)71 versus 479.5 versus 6.3 (HR 0.48)
      LUX LUNG3
      • Yang JC-H
      • Schuler MH
      • Yamamoto N
      • et al.
      LUX-Lung 3: a randomized, open-label, phase III study of afatinib versus pemetrexed and cisplatin as first-line treatment for patients with advanced adenocarcinoma of the lung harboring EGFR-activating mutations.
      345 (230 afatinib and 115 chemo)56 versus 2311.1 versus 6.9 (HR 0.58)
      Abbreviations: Chemo, chemotherapy; HR, hazard ratio.
      Three years after the initial discoveries of EGFR mutations in lung cancer, in 2007, Soda and coworkers
      • Soda M
      • Choi YL
      • Enomoto M
      • et al.
      Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer.
      reported that an inversion on chromosome arm 2p resulted in the creation of an EML4-ALK fusion gene in lung cancer. The fusion gene was identified in 5 of 75 (7%) NSCLC patients examined. Subsequent studies have indicated that the prevalence of this gene fusion event is about 2% to 7% of all NSCLCs seen in the United States, with enrichment in adenocarcinomas in never smokers or light smokers.
      • Boland JM
      • Erdogan S
      • Vasmatzis G
      • et al.
      Anaplastic lymphoma kinase immunoreactivity correlates with ALK gene rearrangement and transcriptional up-regulation in non-small cell lung carcinomas.
      • Inamura K
      • Takeuchi K
      • Togashi Y
      • et al.
      EML4-ALK fusion is linked to histological characteristics in a subset of lung cancers.
      • Inamura K
      • Takeuchi K
      • Togashi Y
      • et al.
      EML4-ALK lung cancers are characterized by rare other mutations, a TTF-1 cell lineage, an acinar histology, and young onset.
      • Koivunen JP
      • Mermel C
      • Zejnullahu K
      • et al.
      EML4-ALK fusion gene and efficacy of an ALK kinase inhibitor in lung cancer.
      • Kwak EL
      • Bang YJ
      • Camidge DR
      • et al.
      Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer.
      • Paik JH
      • Choe G
      • Kim H
      • et al.
      Screening of anaplastic lymphoma kinase rearrangement by immunohistochemistry in non-small cell lung cancer: correlation with fluorescence in situ hybridization.
      • Rodig SJ
      • Mino-Kenudson M
      • Dacic S
      • et al.
      Unique clinicopathologic features characterize ALK-rearranged lung adenocarcinoma in the western population.
      • Salido M
      • Pijuan L
      • Martinez-Aviles L
      • et al.
      Increased ALK gene copy number and amplification are frequent in non-small cell lung cancer.
      • Shaw AT
      • Yeap BY
      • Mino-Kenudson M
      • et al.
      Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK.
      • Shaw AT
      • Yeap BY
      • Solomon BJ
      • et al.
      Effect of crizotinib on overall survival in patients with advanced non-small-cell lung cancer harbouring ALK gene rearrangement: a retrospective analysis.
      • Shinmura K
      • Kageyama S
      • Tao H
      • et al.
      EML4-ALK fusion transcripts, but no NPM-, TPM3-, CLTC-, ATIC-, or TFG-ALK fusion transcripts, in non-small cell lung carcinomas.
      • Kim H
      • Yoo S-B
      • Choe J-Y
      • et al.
      Detection of ALK gene rearrangement in non-small cell lung cancer: a comparison of fluorescence in situ hybridization and chromogenic in situ hybridization with correlation of ALK protein expression.
      Testing for this ALK gene fusion has been facilitated by the commercial availability of a dual-probe “break-apart” fluorescence in situ hybridization (FISH) assay for ALK rearrangements that was already in clinical use to detect ALK fusions in lymphomas and certain sarcomas.
      • Rodig SJ
      • Mino-Kenudson M
      • Dacic S
      • et al.
      Unique clinicopathologic features characterize ALK-rearranged lung adenocarcinoma in the western population.
      A recent report of a large clinical series indicated that ALK rearrangements were seen in about 5% of 1500 NSCLC patients screened.
      • Kwak EL
      • Bang YJ
      • Camidge DR
      • et al.
      Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer.
      Moreover, ALK rearrangement-positive patients treated with a novel ALK inhibitor, crizotinib, showed an overall response rate of 57%, with 72% having a PFS of 6 months or greater.
      • Kwak EL
      • Bang YJ
      • Camidge DR
      • et al.
      Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer.
      The US Food and Drug Administration (FDA) has approved crizotinib for advanced-stage, ALK-positive lung cancer as is also recommended by recent guidelines from professional organizations, including the American Society of Clinical Oncology (ASCO), European Society for Medical Oncology, and National Comprehensive Cancer Network (NCCN).
      Given the considerable published data on EGFR-mutated lung cancer and the rapid pace of work on ALK, representatives of 3 professional organizations with interest in the diagnosis and management of lung cancer—the College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC), and the Association for Molecular Pathology (AMP)—convened to systematically review the published data and develop evidence-based recommendations for the molecular testing of lung cancers for these 2 critical predictive biomarkers in a clinical practice guideline (CPG).
      CPGs are systematically developed statements intended to assist practitioners and patients in making decisions about appropriate health care options for specific clinical circumstances. Attributes of good CPGs include validity, reliability, reproducibility, clinical applicability, clinical flexibility, clarity, multidisciplinary process, review of evidence, and documentation. Specifically, utilization of CPG recommendations may provide improvements in outcomes and in medical practice; minimize inappropriate practice variation; provide decision support tools for practitioners, a reference for medical education, criteria for self-evaluation, and indicators and criteria for external quality review; and assist with reimbursement and coverage decisions. Finally, the process of CPG development can also identify areas where further research is needed.

      Clinical Practice Guideline Questions

      This CPG addresses 5 principal and 14 corollary questions regarding molecular testing in NSCLC:
      • I.
        When should molecular testing for NSCLC be performed?
        • 1.
          Which patients should be tested for EGFR mutations and ALK rearrangements?
        • 2.
          When should a patient specimen be tested for EGFR mutations or ALK rearrangements?
        • 3.
          How rapidly should test results be available?
      • II.
        How should EGFR testing be performed?
        • 4.
          How should specimens be processed for EGFR mutation testing?
        • 5.
          What are the specimen requirements for EGFR testing?
        • 6.
          How should EGFR testing be performed?
        • 7.
          What is the role of KRAS analysis in selecting patients for targeted therapy with EGFR TKIs?
        • 8.
          What additional testing considerations are important in the setting of secondary or acquired EGFR TKI resistance?
      • III.
        How should ALK testing be performed?
        • 9.
          What methods should be used for ALK testing?
      • IV.
        Should other genes be routinely tested in lung adenocarcinoma?
        • 10.
          Are other molecular markers suitable for testing in lung cancer?
      • V.
        How should molecular testing of lung adenocarcinomas be implemented and operationalized?
        • 11.
          Must all adenocarcinomas be tested for both EGFR and ALK?
        • 12.
          How should EGFR and ALK results be reported?
        • 13.
          How should EGFR and ALK testing be validated?
        • 14.
          How should quality assurance be maintained?

      Disclaimer

      Clinical 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 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, 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. 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. 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.
      In formulating recommendations for molecular testing in lung cancer, CAP, IASLC, and AMP considered these tenets of guideline development, emphasizing review of data from appropriately conducted and analyzed clinical trials. Practice guidelines are not intended to supplant physician judgment with respect to particular patients or special clinical situations. The literature and expert review process was directed toward evaluating and selecting the best science for the best possible patient care; a cost analysis was not performed for this guideline.

      METHODS

      A detailed account of the methods used to create this guideline can be found in the supplemental digital content at http://links.lww.com/JTO/A430.

      Panel Composition

      The CAP Pathology and Laboratory Quality Center, and representatives from the IASLC and AMP, jointly convened an expert author panel and scientific advisory panel consisting of experts in clinical pathology and oncology, and research and development relevant to molecular testing in NSCLC. A conference with the expert author and advisory panels was held in December 2010, at which representatives from industry, public health policy and regulatory affairs, patient advocacy, and commercial drug and/or diagnostic device manufacturers were invited to participate. Representatives from the FDA, the National Cancer Institute, ASCO, and the NCCN attended the conference. The opinions of panel members associated with official government agencies represent their individual views and not necessarily those of the agency with which they are affiliated. All members of the expert (author) panel were required to disclose financial and personal conflicts of interest (see below).

      Systematic Literature Review and Analysis

      The literature search strategy involved searching the following electronic databases from January 2004 through February 2012: Ovid MEDLINE, Ovid MEDLINE In-Process & Other Non-indexed Citations, and the Wiley Cochrane Library. The following keywords and MeSH terms were used in the search: lung neoplasms; lung cancer; carcinoma, non-small-cell lung; EGFR; Epidermal growth factor receptor; ALK; KRAS; BRAF; mutation; amplification; gene copy number; rearrangement; fusion; translocation; inversion; immunohistochemistry; IHC; and FISH. All searches were limited to the English language.

      Eligible Study Designs

      Systematic reviews with or without meta-analyses, randomized controlled trials, cohort studies, case-control studies, case series, and method comparisons were eligible for this study. Also included were testing guidelines and proficiency testing strategies of various US and international organizations.

      Inclusion Criteria

      Articles were eligible for inclusion if they met the following criteria:
      • 1.
        The study compared, prospectively or retrospectively, the sensitivity, specificity, negative predictive value, or positive predictive value of EGFR or ALK tests for detection of an EGFR mutation, ALK rearrangement, or response to a targeted EGFR or ALK TKI; the study described technical comparisons across various assay platforms; the study examined potential testing algorithms for NSCLC molecular testing; or the study examined the correlation of EGFR or ALK status in primary versus metastatic tumors from the same patients.
      • 2.
        The study population consisted of patients with a diagnosis of NSCLC.
      • 3.
        The primary outcomes included the sensitivity, specificity, positive predictive value, and negative predictive value of tests to determine EGFR or ALK status or treatment response, alone and in combination; concordance across platforms; and accuracy in determining EGFR or ALK status and benefit from anti-EGFR or ALK TKI therapy.

      Exclusion Criteria

      Letters, commentaries, editorials, reviews, and case reports were excluded.

      Tests Examined

      Additional test methods considered included EGFR copy number by FISH or bright-field chromogenic in situ hybridization, immunohistochemistry for expression of ALK (kinase domain or carboxy-terminal) or mutated EGFR protein, and reverse transcription–polymerase chain reaction (RT-PCR) detection of EML4-ALK fusion transcript. Alterations in other genes, including KRAS, BRAF, and MET, were also considered.

      Outcomes of Interest

      The primary outcomes of interest were the correlations between EGFR mutation or ALK rearrangement and benefit from EGFR or ALK TKI therapies, respectively. Other outcomes of interest included accuracy in determining EGFR or ALK status, concordance across technical platforms, sensitivity, and specificity of different tests. After careful consideration of each of these, the expert panel and advisory panel agreed that the primary recommendations of this guideline should focus on EGFR mutation assays and ALK FISH assays.
      The panel reviewed the results of randomized controlled trials in lung cancer, evaluating therapies targeting EGFR or ALK, such as gefitinib, erlotinib, and crizotinib. The panel also reviewed unblinded trials comparing various testing methods, describing test characteristics, and defining strategies for quality assurance of testing in the literature.

      Environmental Scan

      At the December 2010 conference, individuals representing regulatory agencies (FDA) also provided information about the regulatory framework. Individuals involved with quality assurance in the United States (CAP), the Netherlands, and Canada (Province of Ontario) also provided information about programs to measure and improve EGFR and ALK testing. This information was used to help the panel specify testing requirements and exclusions, and the necessary quality assurance monitoring that will make the testing less variable and more accurate.

      Quality Assessment and Grading of the Included Evidence

      Grading of recommendations was based on overall ratings of individual components of the evidence, such as strength of evidence, its consistency, clinical impact, generalizability, and applicability to the international health care system.
      • Kim H
      • Yoo S-B
      • Choe J-Y
      • et al.
      Detection of ALK gene rearrangement in non-small cell lung cancer: a comparison of fluorescence in situ hybridization and chromogenic in situ hybridization with correlation of ALK protein expression.
      • Hillier S
      • Grimmer-Somers K
      • Merlin T
      • et al.
      FORM: an Australian method for formulating and grading recommendations in evidence-based clinical guidelines.
      • Merlin T
      • Weston A
      • Tooher R
      Extending an evidence hierarchy to include topics other than treatment: revising the Australian ‘levels of evidence’.
      For strength of the evidence, we considered the level of evidence based on its hierarchy, number of studies and number of patients, magnitude of effect from the weighted mean difference or risk ratio, statistical precision measured as a point estimate or confidence interval, and methodologic quality of included studies.
      • Merlin T
      • Weston A
      • Tooher R
      Extending an evidence hierarchy to include topics other than treatment: revising the Australian ‘levels of evidence’.
      The quality of systematic reviews, randomized control trials, and cohort studies was assessed by using the AMSTAR (Assessment of Multiple Systematic Reviews) instrument and SIGN (Scottish Intercollegiate Guidelines Network) 50 checklists, respectively.
      • Shea BJ
      • Grimshaw JM
      • Wells GA
      • et al.
      Development of AMSTAR: a measurement tool to assess the methodological quality of systematic reviews.
      ,
      • Scottish Intercollegiate Guidelines Network
      The overall grade of the recommendation was obtained by rating all components of the evidence. The overall grade indicates the strength of the body of evidence to assist the users of clinical practice guidelines in making appropriate and informed clinical judgments.
      • Shea BJ
      • Grimshaw JM
      • Wells GA
      • et al.
      Development of AMSTAR: a measurement tool to assess the methodological quality of systematic reviews.
      Grade A or B evidence supports “recommendations,” which are generally based on a body of evidence that can be trusted to guide clinical practice in all or in most situations. Grade C evidence is insufficient for a “recommendation” and provides support for “suggestions,” for which care should be taken in application. Grade D evidence is weak and does not provide support for “recommendations” or “suggestions.” Expert consensus opinion was used where grade C or above evidence was lacking.

      Revision Dates

      This guideline will be reviewed regularly, as mandated by publication of substantive and high-quality medical evidence that could potentially alter the original guideline recommendations. If necessary, the entire panel will reconvene to discuss potential changes. When appropriate, panel members will recommend revision of the guideline to their respective organizations for review and approval.

      Conflict of Interest Policy

      Before acceptance on the expert panel, potential member authors from all guideline partnering organizations completed the CAP conflict of interest process, whose policy and form requires disclosure of material financial interest in, or potential for benefit of significant value from, the guideline's development or its recommendations beginning 12 months prior and ending when the guideline was submitted for publication (see “Appendix”). The potential members completed the conflict of interest disclosure form conservatively, listing any relationship that could be interpreted as constituting an actual, potential, or apparent conflict. Regarding members declaring potentially perceived or real conflict, guideline cochairs agreed that these individuals would best serve as advisory panel members for the guideline, but not authors on the expert panel. CAP, IASLC, and AMP provided funding for this project; no industry funds were used in the development of the guideline.
      APPENDIXDisclosed Interests And Activities
      NameInterest/Activity TypeEntity
      Mary Beth Beasley, MDBoard or Advisory BoardPulmonary Pathology Society Board of Trustees
      Lecture fees paid by entityInternational Association for the Study of Lung Cancer
      Expert witnessWillcox and Savage, Asbestos Litigation
      McGuire Woods, Cooch and Taylor, Asbestos Litigation
      Cetrulo and Capone, Asbestos Litigation
      Rassmussen, Dickey, Willis and Moore, Asbestos Litigation
      Informa Health Care
      RoyaltiesLippincott, Williams & Wilkins
      Leadership in associationsPulmonary Pathology Society, Secretary
      College of American Pathologists, Chair, Surgical Pathology Resource Committee
      Philip T. Cagle, MDLecture fees paid by entityEmory University
      New York University
      Oklahoma State Association of Pathologists
      University of Michigan
      Massachusetts General Hospital
      RoyaltiesSpringer Publishing
      Lippincott, Williams & Wilkins
      Leadership in associationsArchives of Pathology & Laboratory Medicine, Editor-in-Chief
      Dr. Cagle recused himself from the 3 journals' peer review process for this guideline, a process completed subsequent to the approval of the guideline by the 3 societies.
      Texas Society of Pathologists, Chair of Education Council
      Pulmonary Pathology Society, Past President
      Pulmonary Pathology Society, Chair of Nominating Committee
      GrantsPennsylvania Breast Cancer Coalition
      Department of Defense
      Dhananjay A. Chitale, MDSusan G. Komen for the Cure
      National Institutes of Health
      ConsultancyNovartis
      Lecture fees paid by entityMillenium
      David J. Kwiatkowski, MD, PhDConsultancyArqule/Daiichi Sankyo (April 2010)
      NanoString (September 2012)
      Marc Ladanyi, MDLecture fees paid by entityGenzyme (March 2010)
      Infinity (July 2010)
      Sequenom (November 2009)
      Remedica Medical Education (June 2012)
      Family and business partnersWife: Continuing medical education activities for Abbott
      Institutional financial interestMemorial Sloan-Kettering Cancer Center licensed patent for EGFR T790M testing to MolecularMD. M.L. is not a patent holder.
      Leadership in associationsAssociation for Molecular Pathology, Strategic Opportunities Committee
      United States and Canadian Academy of Pathology, Translational Genomics in Anatomic Pathology Task Force
      American Association for Cancer Research, 2012 Annual Meeting Program Committee
      Neal I. Lindeman, MDInstitutional financial interestPartners Health Care has a patent on EGFR Mutation Testing. N.I.L. is not a patent holder.
      Leadership in associationsAcademy of Clinical Laboratory Physicians and Scientists, Paul E. Strandjord Young Investigator Award Coordinator
      Association for Molecular Pathology, Solid Tumor Representative, Clinical Practices Committee
      Association for Molecular Pathology, Nominating Committee, Solid Tumors Subdivision Representative
      AACC, Nominating Committee, Molecular Division
      AACC, Short Course/Interactive Workshops Director for AACC Annual Meeting Organizing Committee
      College of American Pathologists, Molecular Oncology Resource Committee
      Juan-Sebastian Saldivar, MDAdditional employmentSequenom Center for Molecular Medicine
      Existence Genetics
      Institutional financial interestCity of Hope functions as a reference laboratory, providing somatic testing for NSCLC
      Jeremy A. Squire, PhDConsultancyCymoGen Dx, LLC
      Board or Advisory BoardApplied Spectral Imaging
      Patents received or pendingDevelopment of PTEN FISH Probe through Queens' University Commercialization Office (Parteq)
      RoyaltiesParteq-Queen's Commercialization Office
      Leadership in associationsNCIC (National Cancer Institute of Canada) Clinical Trials Group, Director of Laboratory Translational Research
      Erik Thunnissen, MD, PhDBoard or Advisory BoardMerck
      Eli Lilly
      Pfizer
      Lecture fees paid by entityMerck
      Pfizer
      Eli Lilly
      Leadership in associationsEuropean Society of Pathology lung external quality assessment
      Paul A. Bunn Jr, MD, SteeringConsultancyEli Lilly
      CommitteeOSI/Genentech/Roche
      Astra Zeneca
      Boehringer Ingelheim
      Pfizer
      Amgen
      GlaxoSmithKline
      Bristol-Myers-Squibb
      Sanofi-Aventis
      Board or Advisory BoardOSI/Genentech/Roche
      Astra Zeneca
      Boehringer Ingelheim
      Amgen
      Bristol-Myers-Squibb
      Sanofi-Aventis
      Eli Lilly
      Patents received or pendingPatent relating to use of EGFR FISH results as predictive biomarkers for EGFR therapy
      EmploymentUniversity of Colorado Denver
      International Association for the Study of Lung Cancer
      Leadership in associationsInternational Association for the Study of Lung Cancer, Executive Director
      Jan A. Nowak, MD, SteeringLecture fees paid by entityWilliam Beaumont Symposium
      CommitteeEmploymentNorthShore University HealthSystem
      Leadership in associationsAMP, Past President 2010
      AMP, Professional Relations Committee Member
      AMP, Economic Affairs Committee Member
      Abbreviations: AACC, American Association for Clinical Chemistry; AMP, Association for Molecular Pathology; EGFR, epidermal growth factor receptor; FISH, fluorescence in situ hybridization; NSCLC, non–small cell lung cancer; PTEN, phosphate and tensin homolog.
      a Dr. Cagle recused himself from the 3 journals' peer review process for this guideline, a process completed subsequent to the approval of the guideline by the 3 societies.

      OUTCOMES

      CAP/IASLC/AMP Expert Panel LiteratureReview and Analysis

      The expert author panel cochairs (N.I.L, P.T.C., M.L.) reviewed 1533 potentially relevant abstracts identified in the original literature searches to select studies pertinent to the guideline: 2 cochairs independently reviewed each abstract, and disagreements were resolved by the third cochair. Full-text articles (521) were then reviewed for all selected abstracts by 2 members of the expert author panel; discrepancies were resolved by a cochair. Evidence tables were developed from selected studies that met the criteria for inclusion. A third literature review was performed by the authors of each section of the guideline, to verify that the highest levels of evidence supported each of their recommendations and, if not, to reevaluate the recommendation and modify or defend it.

      Consensus Development Based on Evidence

      The entire panel met in December 2010 (Chicago, Illinois); additional work on the guideline was completed through electronic mail and monthly teleconferences of the cochairs and/or expert panel. The purposes of the panel meeting were to refine the questions addressed by the guideline, solicit input and testimony from the nonwriting advisory panel, and make writing assignments for the respective sections. All members of the expert panel participated in the preparation of the draft guideline, which was then disseminated for review by the entire panel. Feedback from external reviewers was also solicited. The content of the guideline and the manuscript were reviewed by an independent review panel and approved by the CAP Transformation Program Office Steering Committee, by the IASLC Board of Directors, and by the AMP Clinical Practice Committee and Executive Council. The recommendations are summarized in Table 3.
      TABLE 3Summary of Guideline Recommendations
      Section I: When Should Molecular Testing of Lung Cancers Be Performed?
       Question 1: Which Patients Should Be Tested for EGFR Mutations and ALK Rearrangements?
        1.1a: Recommendation: EGFR molecular testing should be used to select patients for EGFR- targeted TKI therapy, and patients with lung adenocarcinoma should not be excluded from testing on the basis of clinical characteristics.
        1.1b: Recommendation: ALK molecular testing should be used to select patients for ALK-targeted TKI therapy, and patients with lung adenocarcinoma should not be excluded from testing on the basis of clinical characteristics.
        1.2: Recommendation: In the setting of lung cancer resection specimens, EGFR and ALK testing is recommended for adenocarcinomas and mixed lung cancers with an adenocarcinoma component, regardless of histologic grade. In the setting of fully excised lung cancer specimens, EGFR and ALK testing is not recommended in lung cancers that lack any adenocarcinoma component, such as pure squamous cell carcinomas, pure small cell carcinomas, or large cell carcinomas lacking any immunohistochemistry (IHC) evidence of adenocarcinoma differentiation.
        1.3: Recommendation: In the setting of more limited lung cancer specimens (biopsies, cytology) where an adenocarcinoma component cannot be completely excluded, EGFR and ALK testing may be performed in cases showing squamous or small cell histology but clinical criteria (eg, young age, lack of smoking history) may be useful in selecting a subset of these samples for testing.
        1.4: Recommendation: To determine EGFR and ALK status for initial treatment selection, primary tumors or metastatic lesions are equally suiTABLE for testing.
        1.5: Expert consensus opinion: For patients with multiple, apparently separate, primary lung adenocarcinomas, each tumor may be tested but testing of multiple different areas within a single tumor is not necessary.
       Question 2: When Should a Patient Specimen Be Tested for EGFR Mutation or ALK Rearrangement?
        2.1a: Recommendation: EGFR mutation testing should be ordered at the time of diagnosis for patients presenting with advanced-stage disease (stage IV according to the 7th edition TNM staging system) who are suiTABLE for therapy or at time of recurrence or progression in patients who originally presented with lower-stage disease but were not previously tested.
        2.1b: Suggestion: ALK rearrangement testing should be ordered at the time of diagnosis for patients presenting with advanced-stage disease (stage IV according to the 7th edition TNM staging system) who are suiTABLE for therapy or at time of recurrence or progression in patients who originally presented with lower-stage disease but were not previously tested.
        2.2a: Expert consensus opinion: EGFR testing of tumors at diagnosis from patients presenting with stage I, II, or III disease is encouraged but the decision to do so should be made locally by each laboratory, in collaboration with its oncology team.
        2.2b: Expert consensus opinion: ALK testing of tumors at diagnosis from patients presenting with stage I, II, or III disease is encouraged, but the decision to do so should be made locally by each laboratory, in collaboration with its oncology team.
        2.3: Recommendation: Tissue should be prioritized for EGFR and ALK testing.
       Question 3: How Rapidly Should Test Results Be Available?
       3.1: Expert consensus opinion: EGFR and ALK results should be available within 2 weeks (10 working days) of receiving the specimen in the testing laboratory.
       3.2: Expert consensus opinion: Laboratories with average turnaround times beyond 2 weeks need to make available a more rapid test—either in-house or through a reference laboratory—in instances of clinical urgency.
       3.3: Expert consensus opinion: Laboratory departments should establish processes to ensure that specimens that have a final histopathologic diagnosis are sent to outside molecular pathology laboratories within 3 working days of receiving requests and to intramural molecular pathology laboratories within 24 hours.
      Section II: How Should EGFR Testing Be Performed?
       Question 4: How Should Specimens Be Processed for EGFR Mutation Testing?
        4.1: Expert consensus opinion: Pathologists should use formalin-fixed, paraffin-embedded (FFPE) specimens or fresh, frozen, or alcohol-fixed specimens for PCR-based EGFR mutation tests. Other tissue treatments (eg, acidic or heavy metal fixatives, or decalcifying solutions) should be avoided in specimens destined for EGFR testing.
        4.2: Expert consensus opinion: Cytologic samples are also suiTABLE for EGFR and ALK testing, with cell blocks being preferred over smear preparations.
       Question 5: What Are the Specimen Requirements for EGFR Testing?
        5.1: Expert consensus opinion: Pathologists should determine the adequacy of specimens for EGFR testing by assessing cancer cell content and DNA quantity and quality.
        5.2: Expert consensus opinion: Each laboratory should establish the minimum proportion and number of cancer cells needed for mutation detection during validation.
        5.3: Expert consensus opinion: A pathologist should assess the tumor content of each specimen and either perform, or guide a trained technologist to perform, microdissection for tumor cell enrichment as needed.
       Question 6: How Should EGFR Testing Be Performed?
        6.1: Recommendation: Laboratories may use any validated EGFR testing method with sufficient performance characteristics.
        6.2: Expert consensus opinion: Laboratories should use EGFR test methods that are able to detect mutations in specimens with at least 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.
        6.3: Expert consensus opinion: Clinical EGFR mutation testing should be able to detect all individual mutations that have been reported with a frequency of at least 1% of EGFR-mutated lung adenocarcinomas.
        6.4: Recommendation: Immunohistochemistry for total EGFR is not recommended for selection of EGFR TKI therapy.
        6.5: Recommendation: EGFR copy number analysis (ie, FISH or CISH) is not recommended for selection of EGFR TKI therapy.
       Question 7: What Is the Role of KRAS Analysis in Selecting Patients for Targeted Therapy With EGFR TKIs?
        7.1: Recommendation: KRAS mutation testing is not recommended as a sole determinant of EGFR TKI therapy.
       Question 8: What Additional Testing Considerations Are Important in the Setting of Secondary or Acquired EGFR TKI Resistance?
        8.1: Recommendation: If a laboratory performs testing on specimens from patients with acquired resistance to EGFR kinase inhibitors, such tests should be able to detect the secondary EGFR T790M mutation in as few as 5% of cells.
      Section III: How Should ALK Testing Be Performed?
       Question 9: What Methods Should be Used for ALK testing?
        9.1: Recommendation: Laboratories should use an ALK FISH assay using dual-labeled break-apart probes for selecting patients for ALK TKI therapy; ALK immunohistochemistry, if carefully validated, may be considered as a screening methodology to select specimens for ALK FISH testing.
        9.2: Recommendation: RT-PCR is not recommended as an alternative to FISH for selecting patients for ALK inhibitor therapy.
        9.3: Expert consensus opinion: A pathologist should be involved in the selection of sections for ALK FISH testing, by assessing tumor architecture, cytology, and specimen quality.
        9.4: Expert consensus opinion: A pathologist should participate in the interpretation of ALK FISH slides, either by performing the analysis directly or by reviewing the interpretations of cytogeneticists or technologists with specialized training in solid tumor FISH analysis.
        9.5: Expert consensus opinion: Testing for secondary mutations in ALK associated with acquired resistance to ALK inhibitors is not currently required for clinical management.
      Section IV: Should Other Genes Be Routinely Tested in Lung Adenocarcinoma?
       Question 10: Are Other Molecular Markers SuiTABLE for Testing in Lung Cancer?
        10.1a: Recommendation: Testing for EGFR should be prioritized over other molecular markers in lung adenocarcinoma.
       10.1b: Suggestion: After EGFR testing, testing for ALK should be prioritized over other proposed molecular markers in lung adenocarcinoma, for which published evidence is insufficient to support testing guideline development at the present time.
       Section V: How Should Molecular Testing of Lung Adenocarcinomas Be Implemented and Operationalized?
       Question 11: Must All Adenocarcinomas Be Tested for Both EGFR and ALK?
        11.1: Expert consensus opinion: Laboratories may implement testing algorithms to enhance the efficiency of molecular testing of lung adenocarcinomas, provided the overall turnaround time requirements are met.
       Question 12: How Should EGFR and ALK Results Be Reported?
        12.1: Expert consensus opinion: EGFR mutation testing reports and ALK FISH reports should include a results and interpretation section readily understandable by oncologists and by nonspecialist pathologists.
       Question 13: How Should EGFR and ALK Testing Be Validated?
        13.1: Expert consensus opinion: EGFR and ALK testing validation should follow the same guidelines as for other molecular diagnostics and FISH tests.
       Question 14: How Should Quality Assurance Be Maintained?
        14.1: Expert consensus opinion: Laboratories should follow similar quality control and quality assurance policies and procedures for EGFR and ALK testing in lung cancers as for other clinical laboratory assays. In particular, laboratories performing EGFR and ALK testing for TKI therapy should enroll in proficiency testing, if available.
      Abbreviations: CISH, chromogenic in situ hybridization; EGFR, epidermal growth factor receptor; FISH, fluorescence in situ hybridization; PCR, polymerase chain reaction; RT-PCR, reverse transcription–polymerase chain reaction; TKI, tyrosine kinase inhibitor; TNM, tumor, node, metastasis.

      SECTION I: WHEN SHOULD MOLECULAR TESTING FOR NSCLC BE PERFORMED?

      Question 1: Which Patients Should Be Tested for EGFR Mutations and ALK Rearrangements?

      • 1.1a:
        Recommendation.EGFR molecular testing should be used to select patients for EGFR-targeted TKI therapy, and patients with lung adenocarcinoma should not be excluded from testing on the basis of clinical characteristics.
      • 1.1b:
        Recommendation.ALK molecular testing should be used to select patients for ALK-targeted TKI therapy, and patients with lung adenocarcinoma should not be excluded from testing on the basis of clinical characteristics.
      Evidence Grade: EGFR: A; ALK: B.—Clinical characteristics (eg, age, sex, ethnicity, smoking history) are not sufficiently sensitive or specific to be used to select or exclude patients for treatment or testing. Ethnicity, smoking history, and sex have all been associated with the presence of EGFR mutations in NSCLC. Multiple studies have established that EGFR mutations are more common in women than men, in patients who have never smoked tobacco than in patients who have smoked tobacco, and in East Asians than in other ethnic groups.
      • Mok TS
      • Wu YL
      • Thongprasert S
      • et al.
      Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma.
      ,
      • Shigematsu H
      • Lin L
      • Takahashi T
      • et al.
      Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers.
      • Mitsudomi T
      • Morita S
      • Yatabe Y
      • et al.
      Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial.
      • Rosell R
      • Moran T
      • Queralt C
      • et al.
      Screening for epidermal growth factor receptor mutations in lung cancer.
      • Douillard JY
      • Shepherd FA
      • Hirsh V
      • et al.
      Molecular predictors of outcome with gefitinib and docetaxel in previously treated non-small-cell lung cancer: data from the randomized phase III INTEREST trial.
      • Morinaga R
      • Okamoto I
      • Fujita Y
      • et al.
      Association of epidermal growth factor receptor (EGFR) gene mutations with EGFR amplification in advanced non-small cell lung cancer.
      In contrast to EGFR-mutated lung cancer, ALK gene fusions do not show sharp differences in prevalence according to sex and ethnic origin, but do show a similar strong association with patients who have never smoked tobacco and younger age.
      • Inamura K
      • Takeuchi K
      • Togashi Y
      • et al.
      EML4-ALK lung cancers are characterized by rare other mutations, a TTF-1 cell lineage, an acinar histology, and young onset.
      ,
      • Shaw AT
      • Yeap BY
      • Solomon BJ
      • et al.
      Effect of crizotinib on overall survival in patients with advanced non-small-cell lung cancer harbouring ALK gene rearrangement: a retrospective analysis.
      ,
      • Sun Y
      • Ren Y
      • Fang Z
      • et al.
      Lung adenocarcinoma from East Asian never-smokers is a disease largely defined by targetable oncogenic mutant kinases.
      ,
      • Zhang X
      • Zhang S
      • Yang X
      • et al.
      Fusion of EML4 and ALK is associated with development of lung adenocarcinomas lacking EGFR and KRAS mutations and is correlated with ALK expression.
      However, while these clinical characteristics may have value for population studies, they are insufficiently specific to be used to select individual patients for treatment with a targeted inhibitor. Similarly, these characteristics are insufficiently sensitive to be used as prerequisites for testing an individual patient for EGFR mutation or ALK fusion, as significant numbers of patients who might benefit from EGFR- or ALK-targeted therapy might be inappropriately excluded (TABLE 4, TABLE 5, TABLE 6, TABLE 7). Prediction models combining several of these variables to define patients who have a very low probability of EGFR mutations (eg, <1%) have been developed but will require further evaluation.
      • Girard N
      • Sima CS
      • Jackman DM
      • et al.
      Nomogram to predict the presence of EGFR activating mutation in lung adenocarcinoma.
      ,
      • Dogan S
      • Shen R
      • Ang DC
      • et al.
      Molecular epidemiology of EGFR and KRAS mutations in 3,026 lung adenocarcinomas: higher susceptibility of women to smoking-related KRAS-mutant cancers.
      TABLE 4EGFR Mutation Prevalence in Different Lung Adenocarcinoma Patient Populations
      Data for other populations were absent or too limited for analysis.
      EGFR Mutation Prevalence, %EGFR Mutation PositiveEGFR Mutation Negativen (N)
      Asian/Pacific
      References 7, 17, 32, 35, 48, 49, 55, 133, 184, 185, 191, 257, 264, 269, 270, 272–276, 278, 283–292.
      451547190531 (3452)
      White
      References 17, 32, 191, 192, 253, 273, 288, 292–294.
      24853268110 (3534)
      African American
      References 256, 288, 293.
      2019783 (97)
      Hispanic
      References 128, 254, 267, 288.
      17653074 (372)
      Asian/Indian
      Reference 295.
      521141061 (220)
      Abbreviations: n, number of studies; N, number of patients.
      a Data for other populations were absent or too limited for analysis.
      b References
      • Mok TS
      • Wu YL
      • Thongprasert S
      • et al.
      Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma.
      • Shaw AT
      • Yeap BY
      • Mino-Kenudson M
      • et al.
      Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK.
      • Girard N
      • Sima CS
      • Jackman DM
      • et al.
      Nomogram to predict the presence of EGFR activating mutation in lung adenocarcinoma.
      • Liu Y
      • Xu ML
      • Zhong HH
      • Heng WJ
      • Wu BQ
      EGFR mutations are more frequent in well-differentiated than in poor-differentiated lung adenocarcinomas.
      • Huang SF
      • Liu HP
      • Li LH
      • et al.
      High frequency of epidermal growth factor receptor mutations with complex patterns in non-small cell lung cancers related to gefitinib responsiveness in Taiwan.
      • Sonobe M
      • Manabe T
      • Wada H
      • Tanaka F
      Mutations in the epidermal growth factor receptor gene are linked to smoking-independent, lung adenocarcinoma.
      • Wu CC
      • Hsu HY
      • Liu HP
      • et al.
      Reversed mutation rates of KRAS and EGFR genes in adenocarcinoma of the lung in Taiwan and their implications.
      • Kawada I
      • Soejima K
      • Watanabe H
      • et al.
      An alternative method for screening EGFR mutation using RFLP in non-small cell lung cancer patients.
      • Lee YJ
      • Park IK
      • Park MS
      • et al.
      Activating mutations within the EGFR kinase domain: a molecular predictor of disease-free survival in resected pulmonary adenocarcinoma.
      • Soh J
      • Toyooka S
      • Ichihara S
      • et al.
      Impact of HER2 and EGFR gene status on gefitinib-treated patients with nonsmall-cell lung cancer.
      • Massarelli E
      • Varella-Garcia M
      • Tang X
      • et al.
      KRAS mutation is an important predictor of resistance to therapy with epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer.
      • Hotta K
      • Kiura K
      • Toyooka S
      • et al.
      Clinical significance of epidermal growth factor receptor gene mutations on treatment outcome after first-line cytotoxic chemotherapy in Japanese patients with non-small cell lung cancer.
      • Kondo M
      • Yokoyama T
      • Fukui T
      • et al.
      Mutations of epidermal growth factor receptor of non-small cell lung cancer were associated with sensitivity to gefitinib in recurrence after surgery.
      • Sasaki H
      • Endo K
      • Okuda K
      • et al.
      Epidermal growth factor receptor gene amplification and gefitinib sensitivity in patients with recurrent lung cancer.
      • Satouchi M
      • Negoro S
      • Funada Y
      • et al.
      Predictive factors associated with prolonged survival in patients with advanced non-small-cell lung cancer (NSCLC) treated with gefitinib.
      • Sutani A
      • Nagai Y
      • Udagawa K
      • et al.
      Gefitinib for non-small-cell lung cancer patients with epidermal growth factor receptor gene mutations screened by peptide nucleic acid-locked nucleic acid PCR clamp.
      • Taron M
      • Ichinose Y
      • Rosell R
      • et al.
      Activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor are associated with improved survival in gefitinib-treated chemorefractory lung adenocarcinomas.
      • Uramoto H
      • Sugio K
      • Oyama T
      • et al.
      Epidermal growth factor receptor mutations are associated with gefitinib sensitivity in non-small cell lung cancer in Japanese.
      • Wang Z
      • Wu YL
      • Zhang GC
      • Zhou Q
      • Xu CR
      • Guo AL
      EGFR/KRAS mutations and gefitinib therapy in Chinese NSCLC patients.
      • Xu JM
      • Han Y
      • Duan HQ
      • et al.
      EGFR mutations and HER2/3 protein expression and clinical outcome in Chinese advanced non-small cell lung cancer patients treated with gefitinib.
      • Wu J-Y
      • Shih J-Y
      • Chen K-Y
      • Yang C-H
      • Yu C-J
      • Yang P-C
      Gefitinib therapy in patients with advanced non-small cell lung cancer with or without testing for epidermal growth factor receptor (EGFR) mutations.
      • Asahina H
      • Yamazaki K
      • Kinoshita I
      • et al.
      A phase II trial of gefitinib as first-line therapy for advanced non-small cell lung cancer with epidermal growth factor receptor mutations.
      • Bae NC
      • Chae MH
      • Lee MH
      • et al.
      EGFR, ERBB2, and KRAS mutations in Korean non-small cell lung cancer patients.
      • Haneda H
      • Sasaki H
      • Lindeman N
      • et al.
      A correlation between EGFR gene mutation status and bronchioloalveolar carcinoma features in Japanese patients with adenocarcinoma.
      • Inoue A
      • Suzuki T
      • Fukuhara T
      • et al.
      Prospective phase II study of gefitinib for chemotherapy-naive patients with advanced non-small-cell lung cancer with epidermal growth factor receptor gene mutations.
      • Kang SM
      • Kang HJ
      • Shin JH
      • et al.
      Identical epidermal growth factor receptor mutations in adenocarcinomatous and squamous cell carcinomatous components of adenosquamous carcinoma of the lung.
      • Riely GJ
      • Pao W
      • Pham D
      • et al.
      Clinical course of patients with non-small cell lung cancer and epidermal growth factor receptor exon 19 and exon 21 mutations treated with gefitinib or erlotinib.
      • Soung YH
      • Lee JW
      • Kim SY
      • et al.
      Mutational analysis of EGFR and K-RAS genes in lung adenocarcinomas.
      • Sugio K
      • Uramoto H
      • Onitsuka T
      • et al.
      Prospective phase II study of gefitinib in non-small cell lung cancer with epidermal growth factor receptor gene mutations.
      • Sunaga N
      • Tomizawa Y
      • Yanagitani N
      • et al.
      Phase II prospective study of the efficacy of gefitinib for the treatment of stage III/IV non-small cell lung cancer with EGFR mutations, irrespective of previous chemotherapy.
      • Tang X
      • Varella-Garcia M
      • Xavier AC
      • et al.
      Epidermal growth factor receptor abnormalities in the pathogenesis and progression of lung adenocarcinomas.
      .
      c References
      • Shaw AT
      • Yeap BY
      • Mino-Kenudson M
      • et al.
      Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK.
      • Girard N
      • Sima CS
      • Jackman DM
      • et al.
      Nomogram to predict the presence of EGFR activating mutation in lung adenocarcinoma.
      • Massarelli E
      • Varella-Garcia M
      • Tang X
      • et al.
      KRAS mutation is an important predictor of resistance to therapy with epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer.
      • Ludovini V
      • Bianconi F
      • Pistola L
      • et al.
      Phosphoinositide-3-kinase catalytic alpha and KRAS mutations are important predictors of resistance to therapy with epidermal growth factor receptor tyrosine kinase inhibitors in patients with advanced non-small cell lung cancer.
      • Cohen V
      • Agulnik JS
      • Ang C
      • et al.
      Epidermal growth factor receptor mutations detected by denaturing high-performance liquid chromatography in nonsmall cell lung cancer: impact on response to therapy with epidermal growth factor receptor-tyrosine kinase inhibitors.
      • Taron M
      • Ichinose Y
      • Rosell R
      • et al.
      Activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor are associated with improved survival in gefitinib-treated chemorefractory lung adenocarcinomas.
      • Riely GJ
      • Pao W
      • Pham D
      • et al.
      Clinical course of patients with non-small cell lung cancer and epidermal growth factor receptor exon 19 and exon 21 mutations treated with gefitinib or erlotinib.
      • Tang X
      • Varella-Garcia M
      • Xavier AC
      • et al.
      Epidermal growth factor receptor abnormalities in the pathogenesis and progression of lung adenocarcinomas.
      • Cote ML
      • Haddad R
      • Edwards DJ
      • et al.
      Frequency and type of epidermal growth factor receptor mutations in African Americans with non-small cell lung cancer.
      • Moiseyenko VM
      • Procenko SA
      • Levchenko EV
      • et al.
      High efficacy of first-line gefitinib in non-Asian patients with EGFR-mutated lung adenocarcinoma.
      .
      d References
      • Harada T
      • Lopez-Chavez A
      • Xi L
      • Raffeld M
      • Wang Y
      • Giaccone G
      Characterization of epidermal growth factor receptor mutations in non-small-cell lung cancer patients of African-American ancestry.
      • Riely GJ
      • Pao W
      • Pham D
      • et al.
      Clinical course of patients with non-small cell lung cancer and epidermal growth factor receptor exon 19 and exon 21 mutations treated with gefitinib or erlotinib.
      • Cote ML
      • Haddad R
      • Edwards DJ
      • et al.
      Frequency and type of epidermal growth factor receptor mutations in African Americans with non-small cell lung cancer.
      .
      e References
      • Molina-Vila MA
      • Bertran-Alamillo J
      • Reguart N
      • et al.
      A sensitive method for detecting EGFR mutations in non-small cell lung cancer samples with few tumor cells.
      • Cortes-Funes H
      • Gomez C
      • Rosell R
      • et al.
      Epidermal growth factor receptor activating mutations in Spanish gefitinib-treated non-small-cell lung cancer patients.
      • Porta R
      • Sanchez-Torres JM
      • Paz-Ares L
      • et al.
      Brain metastases from lung cancer responding to erlotinib: the importance of EGFR mutation.
      • Riely GJ
      • Pao W
      • Pham D
      • et al.
      Clinical course of patients with non-small cell lung cancer and epidermal growth factor receptor exon 19 and exon 21 mutations treated with gefitinib or erlotinib.
      .
      f Reference
      • Sahoo R
      • Harini VV
      • Babu VC
      • et al.
      Screening for EGFR mutations in lung cancer, a report from India.
      .
      TABLE 5Clinicopathologic Characteristics in Relation to EGFR Mutation Status in Studies Containing Primarily Asian Patients
      Most studies contained primarily patients with adenocarcinoma.
      EGFR Mutation Prevalence, %EGFR Mutation PositiveEGFR Mutation Negativen (N)
      Age with cutoff, y
       <65
      References 7, 55, 184, 272, 276, 290.
      463704336 (803)
       ≥65
      References 55, 184, 272, 276, 290.
      384327095 (1141)
      Sex
       Female
      References 7, 32, 35, 48, 49, 55, 133, 184, 185, 257, 264, 269, 270, 272, 274–276, 278, 283–287, 289–291, 296.
      58102773327 (1760)
       Male
      References 32, 35, 48, 49, 55, 133, 184, 185, 257, 264, 269, 270, 272, 274–276, 278, 283–287, 289–291, 296
      3245696226 (1418)
      Smoking
       Never
      References 32, 35, 55, 133, 184, 185, 257, 264, 269, 270, 274–276, 278, 283–285, 287, 289–291, 296.
      5884359922 (1442)
       Ever
      References 32, 35, 55, 133, 184, 185, 257, 264, 269, 270, 274–276, 278, 283–285, 287, 289–291, 296.
      2626576722 (1032)
      History of smoking, pack-years
       0–10
      Reference 290.
      671051 (15)
       11–40
      Reference 290.
      45561 (11)
       >40
      Reference 290.
      235171 (22)
       >20
      Reference 272.
      2513401 (53)
      Histology
       Adenocarcinoma
      References 32, 35, 48, 49, 55, 133, 184, 185, 257, 264, 269, 270, 272, 274–276, 278, 283–286, 289–291, 296.
      501278125625 (2534)
       Squamous
      References 55, 257, 264, 272, 274, 275, 289, 296.
      581608 (168)
       Adenosquamous
      References 272, 289.
      67422 (6)
       Large cell
      References 48, 272, 275, 289.
      71144 (15)
      Differentiation
       Two grades
        Well
      References 119, 184, 202.
      37621073 (169)
        Moderate to poor
      References 119, 184, 202.
      14271623 (189)
       Three grades
        Well
      References 49, 184.
      6528152 (43)
        Moderate
      References 49, 184.
      4859632 (122)
        Poor
      References 49, 184.
      3417332 (50)
      Abbreviations: n, number of studies; N, number of patients.
      a Most studies contained primarily patients with adenocarcinoma.
      b References
      • Mok TS
      • Wu YL
      • Thongprasert S
      • et al.
      Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma.
      • Wu CC
      • Hsu HY
      • Liu HP
      • et al.
      Reversed mutation rates of KRAS and EGFR genes in adenocarcinoma of the lung in Taiwan and their implications.
      • Lee YJ
      • Park IK
      • Park MS
      • et al.
      Activating mutations within the EGFR kinase domain: a molecular predictor of disease-free survival in resected pulmonary adenocarcinoma.
      • Sutani A
      • Nagai Y
      • Udagawa K
      • et al.
      Gefitinib for non-small-cell lung cancer patients with epidermal growth factor receptor gene mutations screened by peptide nucleic acid-locked nucleic acid PCR clamp.
      • Xu JM
      • Han Y
      • Duan HQ
      • et al.
      EGFR mutations and HER2/3 protein expression and clinical outcome in Chinese advanced non-small cell lung cancer patients treated with gefitinib.
      • Sugio K
      • Uramoto H
      • Onitsuka T
      • et al.
      Prospective phase II study of gefitinib in non-small cell lung cancer with epidermal growth factor receptor gene mutations.
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      Identical epidermal growth factor receptor mutations in adenocarcinomatous and squamous cell carcinomatous components of adenosquamous carcinoma of the lung.
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      • Kim SY
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      Mutational analysis of EGFR and K-RAS genes in lung adenocarcinomas.
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      Prospective phase II study of gefitinib in non-small cell lung cancer with epidermal growth factor receptor gene mutations.
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      Phase II prospective study of the efficacy of gefitinib for the treatment of stage III/IV non-small cell lung cancer with EGFR mutations, irrespective of previous chemotherapy.
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      • Soh J
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      Detection of EGFR gene mutations using the wash fluid of CT-guided biopsy needle in NSCLC patients.
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      e References
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      • Sima CS
      • Jackman DM
      • et al.
      Nomogram to predict the presence of EGFR activating mutation in lung adenocarcinoma.
      • Liu Y
      • Xu ML
      • Zhong HH
      • Heng WJ
      • Wu BQ
      EGFR mutations are more frequent in well-differentiated than in poor-differentiated lung adenocarcinomas.
      • Huang SF
      • Liu HP
      • Li LH
      • et al.
      High frequency of epidermal growth factor receptor mutations with complex patterns in non-small cell lung cancers related to gefitinib responsiveness in Taiwan.
      • Sonobe M
      • Manabe T
      • Wada H
      • Tanaka F
      Mutations in the epidermal growth factor receptor gene are linked to smoking-independent, lung adenocarcinoma.
      • Wu CC
      • Hsu HY
      • Liu HP
      • et al.
      Reversed mutation rates of KRAS and EGFR genes in adenocarcinoma of the lung in Taiwan and their implications.
      • Kawada I
      • Soejima K
      • Watanabe H
      • et al.
      An alternative method for screening EGFR mutation using RFLP in non-small cell lung cancer patients.
      • Lee YJ
      • Park IK
      • Park MS
      • et al.
      Activating mutations within the EGFR kinase domain: a molecular predictor of disease-free survival in resected pulmonary adenocarcinoma.
      • Soh J
      • Toyooka S
      • Ichihara S
      • et al.
      Impact of HER2 and EGFR gene status on gefitinib-treated patients with nonsmall-cell lung cancer.
      • Hotta K
      • Kiura K
      • Toyooka S
      • et al.
      Clinical significance of epidermal growth factor receptor gene mutations on treatment outcome after first-line cytotoxic chemotherapy in Japanese patients with non-small cell lung cancer.
      • Kondo M
      • Yokoyama T
      • Fukui T
      • et al.
      Mutations of epidermal growth factor receptor of non-small cell lung cancer were associated with sensitivity to gefitinib in recurrence after surgery.
      • Sasaki H
      • Endo K
      • Okuda K
      • et al.
      Epidermal growth factor receptor gene amplification and gefitinib sensitivity in patients with recurrent lung cancer.
      • Satouchi M
      • Negoro S
      • Funada Y
      • et al.
      Predictive factors associated with prolonged survival in patients with advanced non-small-cell lung cancer (NSCLC) treated with gefitinib.
      • Sutani A
      • Nagai Y
      • Udagawa K
      • et al.
      Gefitinib for non-small-cell lung cancer patients with epidermal growth factor receptor gene mutations screened by peptide nucleic acid-locked nucleic acid PCR clamp.
      • Uramoto H
      • Sugio K
      • Oyama T
      • et al.
      Epidermal growth factor receptor mutations are associated with gefitinib sensitivity in non-small cell lung cancer in Japanese.
      • Wang Z
      • Wu YL
      • Zhang GC
      • Zhou Q
      • Xu CR
      • Guo AL
      EGFR/KRAS mutations and gefitinib therapy in Chinese NSCLC patients.
      • Xu JM
      • Han Y
      • Duan HQ
      • et al.
      EGFR mutations and HER2/3 protein expression and clinical outcome in Chinese advanced non-small cell lung cancer patients treated with gefitinib.
      • Wu J-Y
      • Shih J-Y
      • Chen K-Y
      • Yang C-H
      • Yu C-J
      • Yang P-C
      Gefitinib therapy in patients with advanced non-small cell lung cancer with or without testing for epidermal growth factor receptor (EGFR) mutations.
      • Asahina H
      • Yamazaki K
      • Kinoshita I
      • et al.
      A phase II trial of gefitinib as first-line therapy for advanced non-small cell lung cancer with epidermal growth factor receptor mutations.
      • Bae NC
      • Chae MH
      • Lee MH
      • et al.
      EGFR, ERBB2, and KRAS mutations in Korean non-small cell lung cancer patients.
      • Haneda H
      • Sasaki H
      • Lindeman N
      • et al.
      A correlation between EGFR gene mutation status and bronchioloalveolar carcinoma features in Japanese patients with adenocarcinoma.
      • Inoue A
      • Suzuki T
      • Fukuhara T
      • et al.
      Prospective phase II study of gefitinib for chemotherapy-naive patients with advanced non-small-cell lung cancer with epidermal growth factor receptor gene mutations.
      • Kang SM
      • Kang HJ
      • Shin JH
      • et al.
      Identical epidermal growth factor receptor mutations in adenocarcinomatous and squamous cell carcinomatous components of adenosquamous carcinoma of the lung.
      • Soung YH
      • Lee JW
      • Kim SY
      • et al.
      Mutational analysis of EGFR and K-RAS genes in lung adenocarcinomas.
      • Sugio K
      • Uramoto H
      • Onitsuka T
      • et al.
      Prospective phase II study of gefitinib in non-small cell lung cancer with epidermal growth factor receptor gene mutations.
      • Sunaga N
      • Tomizawa Y
      • Yanagitani N
      • et al.
      Phase II prospective study of the efficacy of gefitinib for the treatment of stage III/IV non-small cell lung cancer with EGFR mutations, irrespective of previous chemotherapy.
      • Otani H
      • Toyooka S
      • Soh J
      • et al.
      Detection of EGFR gene mutations using the wash fluid of CT-guided biopsy needle in NSCLC patients.
      f References