bs_bs_banner

INVITED REVIEW SERIES: UPDATE IN INTERVENTIONAL PULMONOLOGY SERIES EDITORS: FABIEN MALDONADO; ERIC S. EDELL; PATRICK J. BARRON AND REX C. YUNG

Molecular alterations in non-small-cell lung cancer: Perspective for targeted therapy and specimen management for the bronchoscopist KASIA CZARNECKA-KUJAWA1,2 AND KAZUHIRO YASUFUKU2 1

Division of Respirology and 2Thoracic Surgery, Toronto General Hospital, University Health Network, University of Toronto, Toronto, Canada

ABSTRACT Major advances have occurred over the past decade in our understanding of lung cancer pathobiology. Increasing knowledge of molecular aberrations in lung cancer, specifically the discovery of two driver genes in pharmacologically targetable tyrosine kinases involved in growth factor receptor signalling, epidermal growth factor receptor and anaplastic lymphoma kinase, has been of major therapeutic and prognostic importance. This discovery has allowed for new, personalized approach to the management of lung cancer. Recognizing the importance of molecular signatures of lung cancer, the College of American Pathologists, International Association for the Study of Lung Cancer and Association for Molecular Pathology released the first guidelines for molecular testing in lung cancer. The introduction of minimally invasive needle techniques for the evaluation of lung cancer patients, such as endobronchial ultrasound transbronchial needle aspiration and oesophageal ultrasound–fine-needle aspiration, has revolutionized the way lung cancer patients are assessed. Samples obtained using the minimally invasive needle approaches have been Correspondence: Kazuhiro Yasufuku, Interventional Thoracic Surgery Program, Associate Professor, Division of Thoracic Surgery, Toronto General Hospital, University Health Network, University of Toronto, 200 Elizabeth Street, 9N-957, Toronto, ON, Canada M5G2C4. Email: [email protected] The Authors: Dr Kasia Czarnecka-Kujawa is an Interventional Pulmonologist working with the Divisions of Respirology and Thoracic Surgery at the University Health Network in Toronto, Canada. Her research interests include comparative effectiveness research and health technology assessment in relation to the field of Interventional Pulmonology. Dr Kazuhiro Yasufuku is an internationally known thoracic surgeon working within the Division of Thoracic Surgery at the University Health Network, Toronto, Canada. His expertise and research interests include minimal invasive thoracic surgery and diagnostic procedures. In addition, Dr Yasufuku is working on exploring the utility of minimally invasive techniques for molecular staging and therapies in thoracic oncology. Conflict of interest/funding: Dr Yasufuku has received educational and research grants from Olympus Medical Systems Corp. Received 2 July 2014; invited to revise 10 July 2014; revised 15 July 2014; accepted 15 July 2014. © 2014 Asian Pacific Society of Respirology

shown to be sufficient not only for routine molecular testing but also for multigenic analysis. This allows bronchoscopist to assume an increasingly important role in the diagnostic workup of patients with lung cancer at all stages of the disease and contribute to personalizing the care of lung cancer patients. Key words: anaplastic lymphoma kinase, endobronchial ultrasound transbronchial needle aspiration, epidermal growth factor receptor, oesophageal ultrasound–fine-needle aspiration, lung cancer. Abbreviations: AE, adverse effect; ALK, anaplastic lymphoma kinase; AMP, American Association for Molecular Pathology; AR, acquired resistance; BAL, bronchoalveolar lavage; CAP, College of American Pathologists; CI, confidence interval; CT, computed tomography; EBUS-TBNA, endobronchial ultrasound transbronchial needle aspiration; EGFR, epidermal growth factor receptor; EML4, echinoderm microtubule-associated protein-like 4; EMT, epithelial mesenchymal transition; EUS-FNA, oesophageal ultrasound–fine-needle aspiration; FNA, fine-needle aspiration; HR, hazard ratio; IASLC, International Association for the Study of Lung Cancer; IHC, immunohistochemistry; KRAS, Kirsten rat sarcoma; LN, lymph node; NSCLC, non-small-cell lung cancer; OR, objective response; ORR, overall response rate; OS, overall survival; p53, protein 53; PCR, polymerase chain reaction; PFS, progression-free survival; PIK3, phosphatidylinositol-3kinase; RET, rearranged during transfection; ROS1, c-ros oncogene 1; SCLC, small-cell lung cancer; TBNA, transbronchial needle aspiration; TKI, tyrosine kinase inhibitor; uMS, ultra microsample; WT, wild-type.

INTRODUCTION Despite advances in our understanding of lung cancer molecular genetics, improvement in diagnostic assessment and management of patients with earlystage lung cancer, lung cancer is still the leading cause of cancer-related mortality, accounting for 1.4 million deaths worldwide and approximately 160 000 deaths annually in the United States. This represents more than 25% of all cancer-related deaths in the US and more than the colon, prostate and breast cancers combined, with a reported 5-year survival of approximately 15%.1,2 Respirology (2014) 19, 1117–1125 doi: 10.1111/resp.12377

1118 Low overall survival (OS) stems from the fact that on average 50% of patients with lung cancer present with advanced stage (stage IIIB or IV) where curative surgical or medical therapy is not possible.3 In the past decade, extensive research into molecular genetics of lung cancer has led to key discoveries that have revolutionized the management of advanced non-small-cell lung cancer (NSCLC). Different genetic drivers have been identified and entire molecular pathways have been deciphered, demonstrating that lung cancer is not a single entity with a common genetic driver, but a complex and heterogeneous disease with multiple pathways. Recognition of different genetic subtypes of NSCLC has led to the rapid evolution of targeted therapy altering the course of disease for many patients with advanced-stage NSCLC. In 2004, several independent groups discovered a mutation in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) in patients with dramatic radiographic and clinical response to the tyrosine kinase inhibitor (TKI) gefitinib.4,5 Superior response to targeted therapy with EGFR receptor antagonists in patients with activating EGFR mutations was confirmed in subsequent studies.6 In 2007, the echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) fusion gene was discovered, which encodes another type of tyrosine kinase.7 Dramatic responses to therapy with Crizotinib (ALK inhibitor) have been observed, which led to Food and Drug Administration approval of the drug for use in patients with advanced-stage NSCLC carrying ALK mutations.8,9 Identifying patients who would benefit from targeted therapy with EGFR or ALK inhibitors added another dimension to the assessment of patients with lung cancer. In light of these groundbreaking discoveries, the role of the bronchoscopist involved in planning invasive diagnostic and staging procedures in patients with lung cancer has acquired a new level of complexity. Histological confirmation of lung cancer is no longer acceptable as the sole goal of a diagnostic procedure. Bronchoscopists must ensure that adequate tissue has been provided for molecular diagnostic assessment and that it is done so in a minimally invasive and cost-effective way. To ensure the prompt and adequate assessment of patients with lung cancer, bronchoscopists are expected to work closely with cytopathologists at their institutions to ensure appropriate patient selection for correct molecular marker testing and confirm that sample collection and processing occur according to validated standards and in a timely fashion to facilitate early initiation of personalized therapy. To address these and other important issues in the management of NSCLC, to standardize the diagnostic approach and provide guidance for institutions involved in diagnosing and managing patients with lung cancer, three major institutions—the College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC) and the American Association for Molecular Pathology Respirology (2014) 19, 1117–1125

K Czarnecka-Kujawa and K Yasufuku

(AMP)—have developed in collaboration the first guideline statement providing an evidence-based framework for molecular testing in patients with NSCLC. This review article discusses the historical evolution of molecular diagnosis in lung cancer, summarizes the recently developed guidelines on molecular testing in NSCLC, provides a practical guide to tissue processing to ensure the highest yield from collected specimens and addresses the evolving role of bronchoscopists in the new, molecular landscape of lung cancer.

HISTORICAL PERSPECTIVE ON EVOLUTION OF LUNG CANCER MOLECULAR DIAGNOSTIC ASSESSMENT Up until the last decade, conventional therapy for stage IV NSCLC consisted of a platinum-based chemotherapy. Second line chemotherapy would be used in the event of first line chemotherapy failure. Palliative radiation may have been offered. However, all patients would eventually succumb to their cancer with an average of 5-year survival of 7 and 2% for Stage IIIB and IV cancer, respectively.10 However, a subset of patients with advanced NSCLC was shown to respond preferably to the TKI gefitinib, with improved progression-free survival (PFS) as compared with conventional chemotherapy.8 These patients were discovered to carry the activating mutation in the EGFR tyrosine kinase. Specific phenotypic features have been described in patients who are more likely to harbour the EGFR mutations including gender, smoking status and ethnicity, with the mutation being more often encountered in females, light or non-smokers and in patients of EastAsian origin.11,12 Histologically, these mutations have been described more commonly in low-grade adenocarcinomas than in the poorly differentiated mucinous or solid adenocarcinomas. However, they have been found in adenocarcinomas of all grades, patients with more extensive smoking status and in other ethnic groups; therefore, testing for the EGFR mutation should not be exclusively based on the gender, ethnicity, smoking status of the patient or on the grade of the tumour. Multiple clinical trials have explored the utility of targeted therapy with TKIs for the treatment of NSCLC. Several phase I, followed by phase II, clinical trials were conducted in the early 2000s with use of gefitinib and erlotinib—a synthetic EGFR-TKI.13–18 The TKI was administered to patients who progressed on conventional cytotoxic chemotherapy. Approximately 10% of this unselected cohort demonstrated symptomatic improvement and partial response in the phase I trials, with median time to progression of approximately 3 months.18 Phase II trials showed radiographic regressions in 14% of patients (95% confidence interval (CI), 11–18) and symptomatic improvement in 39% (95% CI, 34–43). Responders were found to be more often women, non-smokers © 2014 Asian Pacific Society of Respirology

Molecular alterations in NSCLC

and with adenocarcinoma subtype cancer. These observations led to the simultaneous discovery of recurring mutations in the tyrosine kinase domain of the EGFR gene in patients with significant response to gefitinib and erlotinib.19,20 In the select group of patients with activating EGFR receptor mutations, as high as 68% response rate to the TKI and PFS of 12 months have been observed.21–23 In 2009, the Iressa Pan-Asia Study in East Asian, never or only light smokers with stage IIIB or IV adenocarcinoma showed that the use of TKI as an upfront therapy in patients with activating mutations in the EGFR resulted in improved PFS as compared to treatment with conventional chemotherapy (disease progression hazard ratio (HR) or death of 0.48, P < 0.001).24 Based on these study results, in 2011, the American Society of Clinical Oncology suggested routine testing for EGFR mutations in patients with advanced lung adenocarcinomas who might benefit from targeted therapy.25 A recent meta-analysis assessed the effect of EGFR inhibitor therapy on PFS and OS in a total of 1450 patients studied in 23 trials (13 trials used the EGFR therapy as first line, 7 as second line and 3 as maintenance). Overall, EGFR-TKI treatment prolonged PFS in EGFR-positive patients in all settings, with HR for EGFR positive and negative patients as follows: 0.43 (95% CI: 0.38–0.49; P < 0.001), and 1.06 (95% CI: 0.94– 1.19; P = 0.35; P < 0.001; P interaction < 0.001); 0.34 (95% CI: 0.20–0.60; P < 0.001) and 1.23 (95% CI: 1.05–1.46; P = 0.01; P < 0.001; P interaction < 0.001); 0.15 (95% CI: 0.08–0.27; P < 0.001) and 0.81 (95% CI: 0.68–0.97; P = 0.02; P interaction < 0.001) when used as first, second line and as maintenance therapy, respectively. The treatment, however, did not impact OS between the groups, likely due to cross-over effect at the end of the studies.6 TKI treatment is now standard of care as first line therapy for lung cancer patients with advanced disease who carry the EGFR driver mutations. EML4/ALK is another important driver gene in lung cancer that was discovered by Soda et al. in 2007.7 Besides NSCLC, activating translocations of the ALK gene have been identified in other cancers, including neuroblastoma, anaplastic large-cell lymphoma and inflammatory myofibroblastic tumours. In NSCLC, EML4-ALK is an aberrant fusion gene that encodes a cytoplasmic chimeric protein with constitutive kinase activity. Multiple distinct EML4-ALK chimeric proteins have been identified.8 The incidence of EML4/ ALK fusion in cohorts of patients with NSCLC ranges from 1.6% to as high as 19.3%. The wide variation in rates is most likely attributable to few factors including diverse cohorts of patients either with no or preferential selection of patients with adenocarcinoma, use of different sensitivity detection assays and varying sample size of the cohorts. Patients with EML4/ALK fusions have been shown to be on average younger than patients with the wild-type (WT) ALK genes and patients with EGFR mutation and are more likely to be men and never-smokers or light smokers with adenocarcinomas and with WT EGFR gene.26 In the United States, the EML4/ALK aberration occurs in approximately 2–7% of NSCLC patients with © 2014 Asian Pacific Society of Respirology

1119 increased incidence in light or non-smokers with adenocarcinomas.8,9 Crizotinib (Xalkori, Pfizer, New York, NY, USA) is an oral adenosine triphosphate competitive selective inhibitor of the ALK and mesenchymal-epithelial transfer tyrosine kinase, inhibiting tyrosine phosphorylation of activated ALK. Its use has been tested in patients with advanced-stage lung carcinomas harbouring ALK gene rearrangements.27,28 Recently, Kwak et al. demonstrated an impressive response to Crizotinib in a cohort of 82 patients with EML/ALK mutation who were predominantly non-smokers or light smokers and with advanced lung adenocarcinomas. The overall response rate (ORR) reported was 57% with PFS rate of 72% at 6 months.8 In another phase 1 study of 149 ALK-positive never or former smokers (99%) predominantly with primary lung adenocarcinomas (97%), crizotinib showed marked efficacy with more than 60% of patients having an objective and rapid response, with median time to first documented objective response (OR) of 7.9 weeks. Response was durable (median duration of response was 49.1 weeks). Patients who derived the greatest OR were more likely to be treatment naive, have the lowest performance status score and were Asian.29 Median PFS was 9.7 months (95% CI: 7.7– 12.8). OS at 6 and 12 months was 87.9% (95% CI: 81.3– 92.3) and 74.8% (95% CI: 66.4–81.5), respectively. These findings have been confirmed by phase II and phase III clinical trials with ORR of 59.8%, occurring within the first 8 weeks of treatment in 71% of patients, with a median time to response of 6.1 weeks and median PFS of 8.1 months (95% CI: 6.8–9.7) in a preliminary cohort of 261 patients of ALK-positive adenocarcinoma.30 In the first phase III, randomized, open-label study, crizotinib was compared with standard single-agent chemotherapy (pemetrexed or docetaxel) in 347 EML4/ALK-positive, advanced NSCLC-pretreated patients. Crizotinib was superior to standard single-agent chemotherapy in response (ORR 65 vs 20%, P < 0.0001; ORR treatment-related data—65.7% with crizotinib vs 29.3% with pemetrexed and 6.9% with docetaxel) and PFS (median 7.7 vs 3 months, P < 0.0001; PFS treatmentrelated data—7.7 months with crizotinib vs 4.2 and 2.6 months with pemetrexed and docetaxel, respectively) in ALK mutation-positive patients who had been treated with first-line, platinum-based chemotherapy.31 Crizotinib has a good adverse-effect (AE) profile with 97% of patients reporting only minor treatmentrelated AEs.29–31 Clinical trials have been on the way to address the question of crizotinib resistance in patients with EML/ALK mutations with promising results.32 Other oncogene mutations have been described in lung adenocarcinoma. The most common one is in the Kirsten rat sarcoma (KRAS) oncogene, occurring in approximately 30% of adenocarcinomas predominantly in patients with smoking history. Ethnic variations have been described: Asians (5–10%), white Americans and Europeans (25–35%) and patients of African ancestry (15–25%).33–36 Respirology (2014) 19, 1117–1125

1120 Mutations in EGFR and ALK TK are mutually exclusive. Shaw et al. demonstrated that 84% of the preselected cohort of Asian, never or light smokers with adenocarcinoma had response to erlotinib or gefitinib and carried an activating EGFR mutation, whereas none harboured EML4/ALK fusion gene. Conversely, among the patients refractory to EGFRTKIs, 29% were positive for EML4/ALK. Similarly, none of the patients with the KRAS (another driver oncogene) mutation had response to EGFR-TKI. This suggests that in general, EGFR, EML4/ALK and the KRAS mutations are mutually exclusive rather than overlapping, which can have implication on molecular testing strategy.26 Overall reported response of patients with KRAS mutation to TKI therapy ranges from 0 to 26%, with higher response rates in patients with WT KRAS. However, the presence of the KRAS mutations should not guide decisions regarding initiation of TKI therapy, since KRAS WT tumours can have WT EGFR status and those tumours have been shown to respond less favourably to the TKI therapy than to the conventional chemotherapy. For that reason, if discovered up front, KRAS mutations status should not be used to guide patient selection for TKI therapy and in addition, EGFR testing should be performed.37–39 Other driver mutations associated with potential targetable pathways described in lung cancer include: phosphatidylinositol-3-kinase (PIK3)–protein kinase B, (AKT)–mammalian target of rapamycin located downstream of EGFR; epithelial mesenchymal transition (EMT) pathway, involving morphological change in cellular tight junctions, yielding cells more migratory and invasive;40 Tumour protein 53 (p53) oncogene found in 41.6% of NSCLC in multigenic analysis of lymph node (LN) material obtained by endobronchial ultrasound transbronchial needle aspiration (EBUSTBNA);41 v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), a protein affecting cell differentiation and division, occurring in 2–3% of NSCLC;42,43 c-ros oncogene 1 (ROS1) a receptor tyrosine kinase of the insulin family, found in ∼2% NSCLC;44 rearranged during transfection (RET) fusion gene, the kinesin family member 5B—ret proto-oncogene leading to aberrant activation of the RET kinase and cell proliferation, present in 1–2% of adenocarcinomas.

GUIDELINES? FRAMEWORK FOR MOLECULAR TESTING Given the vast amount of literature that has accumulated on the molecular genetics of lung cancer, in 2013 the CAP, the AMP and the IASLC have officially released molecular testing guidelines for the population of patients with lung cancer that harbour genetic driver mutations and who might benefit from targeted therapy with the EGFR and ALK TKIs. The guideline addressed 5 principal and 14 corollary questions regarding molecular testing in the NSCLC (9). The principal questions included: (i) When should molecular testing be performed?; (ii) How should molecular testing be performed?; (iii) How Respirology (2014) 19, 1117–1125

K Czarnecka-Kujawa and K Yasufuku

should ALK testing be performed?; (iv) Should other genes be routinely tested in lung cancer?; and (v) How should molecular testing of lung adenocarcinomas be implemented and operationalized? Fourteen subjects have been addressed in 15 grade A/B recommendations. The guidelines suggest testing all patients with advanced-stage tumours (stage IV according to the 7th edition on TNM staging) containing pure or mixed adenocarcinoma component, regardless of tumour grade, for the EGFR and ALK mutation status at the time of original diagnosis or tumour recurrence if the receptor status has not been determined previously. Tumours should be tested regardless of the patient’s age, gender, ethnicity and smoking status. Even though tumours expressing the EGFR mutation tend to occur more often in females, younger patients who are non- or light smokers, and the EML4/ALK-positive tumours occur more often in younger non- or light smokers, these clinical characteristics are insufficient as selection criteria for targeted therapy or molecular testing. Small cell and squamous cell carcinomas do not need to be subjected to molecular testing for EGFR and ALK mutations unless they contain adenocarcinoma component. High concordance with respect to EGFR mutation status has been reported between primary tumours and metastatic foci (>95%), therefore, either of the two sites can be used for molecular testing.45–47 The choice of site should be guided by the quality of the sample. Patients with stage I, II and III tumours may be tested for EGFR and ALK mutation status but this should occur at the discretion of local laboratory and in consultation with the local oncology teams. The rationale for testing early tumours is that in case of disease recurrence in patients presenting initially with less-advanced lesions, testing might have to be performed on old or difficult to track down specimen, or require subjecting patients to invasive procedures to obtain tissue for testing which can be of limited quality and quantity depending on local expertise. All this may cause delays in patient management. Molecular testing of early lesions must be balanced against costs and with priority given to patients with stage IV lesions. Two weeks (10 working days) have been agreed upon as the maximum turnaround time for EGFR and ALK testing. As new potential treatment targets are discovered in patients with lung cancer, standard molecular testing using multigenic assays may become the standard of care. However, given that many of the newly discovered targets like PIK3 ROS1, RET oncogenes are under experimental investigation with no approved personalized therapy for patients carrying these mutations, robust evidence to recommend routine molecular testing for these markers is lacking. Therefore, routine testing for these driver mutations is not recommended, except in setting of a clinical trial.9 If tissue is abundant and cost is not a concern, testing for both EGFR and ALK mutations may be performed. However, use of an algorithm with sequential, rather than simultaneous testing may be more cost-effective. The guidelines propose the use of three similar algorithms based on the observation that the © 2014 Asian Pacific Society of Respirology

Molecular alterations in NSCLC

three most common mutations encountered in lung cancer—EGFR, ALK and KRAS are mutually exclusive. (i) EGFR testing first followed by ALK testing (fluorescence in situ hybridization (FISH) method) if EGFR test is WT; (ii) EGFR testing using a more sensitive but less specific method (for example high-performance liquid chromatography) that may discover but fail to characterize in detail the EGFR mutation. If negative, ALK FISH testing may be performed. This would add an additional step but prevent a number of costly, definitive EGFR and ALK tests. (iii) Testing for KRAS (the most common mutation encountered in lung cancer). Patients with negative results would enter one of the two above algorithms. However, if tissue is sparse and sample might be exhausted on the KRAS testing, EGFR and ALK testing should be prioritized. Formalin-fixed paraffin-embedded fresh, frozen or alcohol-fixed specimens can all be used for polymerase chain reaction (PCR)-based EGFR mutation testing. Other tissue treatments should be avoided. Immunohistochemistry (IHC) can be used as a screening test for ALK if a test with high-performance characteristics is available, with confirmatory FISH assay as a definitive test. In all instances, bronchoscopist should work closely with cytopathologists and the cytopathology technicians who should guide them through the process of tissue collection, offering feedback on tissue quality, quantity and adequacy for effective molecular resting.

BRONCHOSCOPIST ROLE IN THE ERA OF MOLECULAR TESTING IN LUNG CANCER In the era where molecular genetics of lung cancer not only guides therapy but also predicts treatment response and prognosis, the bronchoscopist’s role in the process of diagnostic work-up, including lung cancer staging and restaging, has acquired a new and important dimension. Acquisition of tissue solely for diagnostic purposes is no longer sufficient. Planning an invasive procedure on a patient with suspected primary lung cancer, the bronchoscopist must think strategically to maximize procedure yield, optimally achieving both diagnosis and staging with one procedure while providing adequate tissue quantity and quality not only for histological cancer characterization but also for ancillary—molecular testing. With the introduction of ultrasound-based techniques (EBUS-TBNA, the oesophageal ultrasound– fine-needle aspiration (EUS-FNA) and radial probe-EBUS (rp-EBUS)) into the armamentarium of techniques at the bronchoscopist’s disposal, diagnostic and staging process of lung cancer in centres where these technologies and expertise are available, has become minimally invasive, has proven to be cost-effective and associated with high patient satisfaction.48,49 Historically, samples used for histopathological diagnosis in lung cancer patients came either from core needle biopsy specimens or from surgical specimens. However, a vast amount of literature has accumulated over the recent decade proving that cytology © 2014 Asian Pacific Society of Respirology

1121 specimens are not only sufficient for histological assessment of lung tumours but also for molecular testing.50–59 Bronchoscopists can utilize a variety of cytological specimens for diagnostic purposes including those coming from bronchoalveolar lavage (BAL), bronchial brushing, transbronchial needle aspiration (TBNA), whether via EBUS-TBNA or EUS-FNA procedures. Understanding limitations and advantages associated with each of the sampling methods is key to maximizing diagnostic yield, while minimizing the number of procedures per patient and degree of invasiveness. In 2007, Nakajima et al. demonstrated that EBUSTBNA samples are adequate for IHC and DNA mutational analysis in osteosarcoma metastatic to the mediastinum.50 Since then, a large body of evidence has accumulated proving that needle-based techniques, both EBUS-TBNA and EUS-FNA, provide sufficient quantity and quality material for molecular testing of EGFR, ALK, KRAS and less commonly encountered molecular markers and pathways like EMT pathway, from both fresh and fixed specimens. Reported adequacy of EBUS-TBNA and EUS-FNA samples for molecular diagnosis ranges from 77% to 98%.50–55 EBUS-TBNA samples have one of the lowest insufficiency rates (4%) for EGFR and KRAS mutational analysis (compared with computed tomography (CT)–fine-needle aspiration (FNA)—7.5% insufficiency rate; ultrasound-guided/superficial FNA—10%).52 With this paradigm change, needle-based minimally invasive techniques are becoming more frequently used for mediastinal staging and becoming the only way tissue diagnosis is obtained in many cases. In patients with biopsy-proven or suspected primary lung cancer and either mediastinal lymphadenopathy by size criteria or PET-positive mediastinal LNs, use of needle-based technique for mediastinal staging as the first invasive test has been shown to result in fewer invasive tests than approach following a different staging protocol (1.3 + 0.5 vs 2.3 + 0.5 tests/ patient respectively, P < 0.0001) and have fewer complications (0 of 30, 0% vs 18 of 108, 17%; P = 0.01).48 EBUS-TBNA was sufficient to guide treatment decisions without any other invasive tests in 88 (64%) patients. Simply changing the test sequence (use of EBUS-TBNA before a CT-guided biopsy) can eliminate up to two thirds of the complications and reduce costs related to CT-guided biopsies.48 Other cytology specimens—bronchial brushing in cases of endobronchial lesions, conventional TBNA or pleural fluid cytology—may provide sufficient material for molecular testing.59 However, close collaboration with the cytopathologist must take place at the time of sample collection or shortly after to ensure that sample cellularity is adequate. One study showed that while sometimes molecular profiling may be successful when the sample contains ∼100 tumour cells, it is more likely to be successful if >1000 cells have been provided.59 For centres where rapid on-site cytopathology evaluation is not available routinely, sample adequacy may be accurately assessed by the bronchoscopist after short-term training under supervision of a qualified Respirology (2014) 19, 1117–1125

1122 cytopathologist. One study showed that there was an 81% overall substantial agreement between observers (cytopathologist and a bronchoscopist evaluating the same sample) with the κ of 0.73 (95% CI: 0.61–0.86; P = 0.001), which became excellent in cases of malignant disease (κ, 0.81; 95% CI: 0.70–0.90; P = 0.001).60 Even though diagnostic yield of flexible bronchoscopy in lung cancer can be as high as 88% in central masses and airway invasion, overall sensitivity of flexible bronchoscopy with brushing and/or biopsy can be as low as 34% in lesions less than 2 cm. For that reason, careful patient selection for appropriate diagnostic procedure is crucial to maximize diagnostic yield.61 However, BAL fluid has been shown to be amenable to molecular testing, increasing sensitivity of cancer detection.62 Recent study by Li et al., evaluated yield of BAL for detection of KRAS and p35 mutations in 48 patients with peripheral lung cancer. KRAS and p53 detection rate in the primary lesion were 52% and 58%, respectively; in BAL fluid collected from the airway closest to the lesion, 38% and 44%, respectively. The combined detection of both KRAS and p53 mutations yielded a sensitivity of 66% for the diagnosis of peripheral NSCLC, which is markedly higher than that of cytology plus histology by first bronchoscopy (38%, P = 0.008). In each patient with the two gene mutations in BAL fluid, mutation type and location were the same as those of the primary tumour.62 This study showed that detection of the KRAS and p53 mutations in BAL fluid could be a helpful addition to cytology and histology examination for the diagnosis of peripheral NSCLC. However, it should not be relied on exclusively for diagnostic and molecular evaluation of lung lesions suspicious for lung cancer. Bronchoscopists also play an important role in patient evaluation in the setting of disease recurrence and acquired resistance (AR) to targeted therapy, where obtaining adequate tissue sample may have significant therapeutic implications. In one study, 25% of patients with enlarging mediastinal LNs following curative surgery for NSCLC whose mediastinal lymphadenopathy was re-evaluated with EBUSTBNA were shown to have a possible second primary lung cancer.63 Some of the patients were diagnosed with small-cell lung cancer (SCLC), which warrants different treatment approach than the NCSLC. Patients diagnosed with early second primary NSCLC underwent second curative surgery. Reported diagnostic accuracy of EBUS-TBNA in restaging is 95.1%.63 Approximately 70% of patients with sensitizing EGFR mutations experience initial clinical and radiographic response to TKI. However, AR to TKI develops almost invariably within 8–16 months, resulting in disease progression.64–67 Even though clinical management implications in such setting are under investigation, confirming disease progression may be of importance. The most common mechanism of AR in already mutated EGFR is the emergence of an additional EGFR tyrosine kinase mutation—T790M.64 Emergence of this AR is, however, not uniform but clonal and has been shown in approximately 50% of tumour cells at the time of treatment failure,64,67 which may be Respirology (2014) 19, 1117–1125

K Czarnecka-Kujawa and K Yasufuku

responsible for ongoing derived benefit from TKI therapy in some patients with documented AR.68,69 Alternatively, discovery of SCLC in patients with AR may warrant another therapeutic approach, again, stressing the importance of tissue confirmation in cases of suspected AR.69

FUTURE DIRECTIONS As the molecular complexity of lung cancer becomes elucidated, multiple potential therapeutic targets will be identified. Synchronous intervention with more than one compound (each aiming at inhibition of a different target) may become the new standard of care for some tumours. Diagnostic tissue samples will therefore have to be sufficient to test for multigenic aberrations. Recently, whole genome sequencing has come to attention as a tool with the potential to truly personalize the management of lung cancer. Whole genome sequencing of 178 squamous cell carcinomas and 183 adenocarcinomas showed that beside the more commonly found mutations in EGFR, ALK and KRAS proteins in adenocarcinomas, additional mutations were found in BRAF, U2AF1 (U2 auxiliary factor1), RBM10 (RNA-binding protein10), ARID1A (AT rich interactive domain1A). Strikingly, overall, 25 genes were shown to have statistically significant number of mutations in lung adenocarcinomas, each representing a possible therapeutic target. A smaller number of mutations was found in squamous cell carcinomas (11 genes with recurrent mutations).56 Ultrasound-guided techniques provide sufficient amount and quality samples for multigenetic analysis including p53 mutation, BRAF and PIK3CA, BRAF, RET and ROS1.53,57,58 Recently, Sakairi et al. demonstrated that even the ultra microsample (uMS) consisting of needle rinse product produced after EBUSTBNA sample collection (commonly considered a waste) can provide useful material for multigenetic analysis. DNA and RNA were extracted from the samples, and PCR-based techniques were used to detect target genetic aberrations. Even though the yield from uMS was only 34% (45 out of 134 already confirmed cancer cases were verified by uMS). This proves that useful material is present in even what may be considered a waste. Collecting uMS may become useful in cases of sparse tissue collection, increasing sensitivity to detect cancer when the cytology samples may be insufficient.58 In the future, as more AR mechanisms and targeted therapies are discovered, molecular analysis of tissue using multigenic approach may become the standard of care allowing the implementation of a prompt and accurate personalized approach to management of primary and recurred lung cancer.

REFERENCES 1 World Health Organization. Published 2012: Cancer fact sheet No. 297. [Accessed 2 Feb 2014.] Available from URL: http:// www.who.int/mediacentre/factsheets/fs297/en/ © 2014 Asian Pacific Society of Respirology

1123

Molecular alterations in NSCLC 2 Dela Cruz C, Tanoue L, Matthay R. Lung cancer: epidemiology, Etiology and Prevention. In: Tanoue L, Matthay R (eds) Clinics in Chest Medicine. Lung Cancer, Vol. 32. Elsevier Inc., Philadelphia, PA, 2011; 605–44. 3 Midthun D. Lung cancer: screening for lung cancer. In: Tanoue L, Matthay R (eds) Clinics in Chest Medicine. Lung Cancer, Vol. 32. Elsevier Inc., Philadelphia, PA, 2011; 659–68. 4 Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska FG et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N. Engl. J. Med. 2004; 350: 2129–39. 5 Paez JG, Jänne PA, Lee JC, Tracy S, Greulich H, Gabriel S, Herman P, Kaye FJ, Lindeman N, Boggon TJ et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004; 304: 1497–500. 6 Lee CK, Brown C, Gralla RJ, Hirsh V, Thongprasert S, Tsai CM, Tan EH, Ho JC, da Chu T, Zaatar A et al. Impact of EGFR inhibitor in non-small cell lung cancer on progression-free and overall survival: a meta-analysis. J. Natl Cancer Inst. 2013; 105: 595–605. 7 Soda M, Choi YL, Enomoto M, Takada S, Yamashita Y, Ishikawa S, Fujiwara S, Watanabe H, Kurashina K, Hatanaka H et al. Identification of the transforming EML4-ALK fusion gene in nonsmall-cell lung cancer. Nature 2007; 448: 561–6. 8 Kwak EL, Bang YJ, Camidge DR, Shaw AT, Solomon B, Maki RG, Ou SH, Dezube BJ, Jänne PA, Costa DB et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N. Engl. J. Med. 2010; 363: 1693–703. 9 Lindeman N, Cagle PT, Beasley MB, Chitale DA, Dacic S, Giaccone G, Jenkins RB, Kwiatkowski DJ, Saldivar JS, Squire J et al. Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. J. Mol. Diagn. 2013; 15: 415–53. 10 Goldstraw P, Crowley J, Chansky KK, Giroux DJ, Groome PA, Rami-Porta R, Postmus PE, Rusch V, Sobin L. The IASLC Lung Cancer Staging Project: proposals for the revision of the TNM stage groupings in the forthcoming (seventh) edition of the TNM classification of malignant tumours. J Thorac. Oncol. 2007; 2: 706–14. 11 Shigematsu H, Lin L, Takahashi T, Nomura M, Suzuki M, Wistuba II, Fong KM, Lee H, Toyooka S, Shimizu N et al. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J. Natl. Cancer Inst. 2005; 97: 339–46. 12 Rosell R, Moran T, Queralt C, Porta R, Cardenal F, Camps C, Majem M, Lopez-Vivanco G, Isla D, Provencio M et al. Screening for epidermal growth factor receptor mutations in lung cancer. N. Engl. J. Med. 2009; 361: 958–67. 13 Ranson M, Hammond LA, Ferry D, Kris M, Tullo A, Murray PI, Miller V, Averbuch S, Ochs J, Morris C et al. ZD1839, a selective oral epidermal growth factor receptor-tyrosine kinase inhibitor, is well tolerated and active in patients with solid, malignant tumors: results of a phase I trial. J. Clin. Oncol. 2002; 20: 2240–50. 14 Baselga J, Rischin D, Ranson MCalvert H, Raymond E, Kieback DG, Kaye SB, Gianni L, Harris A, Bjork T, Averbuch SD et al. Phase I safety, pharmacokinetic, and pharmacodynamics trial of ZD1839, a selective oral epidermal growth factor receptor tyrosine kinase inhibitor, in patients with five selected solid tumor types. J. Clin. Oncol. 2002; 20: 4292–302. 15 Herbst RS, Maddox A-M, Rothenberg ML, Small EJ, Rubin EH, Baselga J, Rojo F, Hong WK, Swaisland H, Averbuch SD et al. Selective oral epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 is generally well-tolerated and has activity in non- small-cell lung cancer and other solid tumors: results of a phase I trial. J. Clin. Oncol. 2002; 20: 3815–25. 16 Fukuoka M, Yano S, Giaccone G, Tamura T, Nakagawa K, Douillard JY, Nishiwaki Y, Vansteenkiste J, Kudoh S, Rischin D et al. Multi-institutional randomized phase II trial of gefitinib for © 2014 Asian Pacific Society of Respirology

17

18

19

20

21

22

23

24

25

26

27

28

29

30

previously treated patients with advanced non-small-cell lung cancer. J. Clin. Oncol. 2003; 21: 2237–46. Kris MG, Natale RB, Herbst RS, Lynch TJ Jr, Prager D, Belani CP, Schiller JH, Kelly K, Spiridonidis H, Sandler A et al. Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer: a randomized trial. JAMA 2003; 290: 2149–58. Miller VA, Kris MG, Shah N, Patel J, Azzoli C, Gomez J, Krug LM, Pao W, Rizvi N, Pizzo B et al. Bronchioloalveolar pathologic subtype and smoking history predict sensitivity to gefitinib in advanced non-small-cell lung cancer. J. Clin. Oncol. 2004; 22: 1103–9. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska FG et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N. Engl. J. Med. 2004; 350: 2129–39. Paez JG, Jänne PA, Lee JC, Tracy S, Greulich H, Gabriel S, Herman P, Kaye FJ, Lindeman N, Boggon TJ et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004; 304: 1497–500. Shepherd FA, Rodrigues Pereira J, Ciuleanu T, Tan EH, Hirsh V, Thongprasert S, Campos D, Maoleekoonpiroj S, Smylie M, Martins R et al. National Cancer Institute of Canada Clinical Trials Group: erlotinib in previously treated non-small-cell lung cancer. N. Engl. J. Med. 2005; 53: 123–32. Stinchcombe TE, Socinski MA. Gefitinib in advanced non-small cell lung cancer: does it deserve a second chance? Oncologist 2008; 13: 933–44. Thatcher N, Chang A, Parikh P, Rodrigues Pereira J, Ciuleanu T, von Pawel J, Thongprasert S, Tan EH, Pemberton K, Archer V et al. 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). Lancet 2005; 366: 1527–37. Mok TS, Wu YL, Thongprasert S, Yang CH, Chu DT, Saijo N, Sunpaweravong P, Han B, Margono B, Ichinose Y et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N. Engl. J. Med. 2009; 361: 947–57. Keedy VL, Temin S, Somerfield MR, Beasley MB, Johnson DH, McShane LM, Milton DT, Strawn JR, Wakelee HA, Giaccone G. American Society of Clinical Oncology provisional clinical opinion: epidermal growth factor receptor (EGFR) mutation testing for patients with advanced non-small-cell lung cancer considering first-line EGFR tyrosine kinase inhibitor therapy. J. Clin. Oncol. 2011; 29: 2121–7. Shaw AT, Yeap BY, Mino-Kenudson M, Digumarthy SR, Costa DB, Heist RS, Solomon B, Stubbs H, Admane S, McDermott U et al. Clinical features and outcome of patients with non-smallcell lung cancer who harbor EML4-ALK. J. Clin. Oncol. 2009; 27: 4247–53. McDermott U, Iafrate AJ, Gray NS, Shioda T, Classon M, Maheswaran S, Zhou W, Choi HG, Smith SL, Dowell L et al. Genomic alterations of anaplastic lymphoma kinase may sensitize tumors to anaplastic lymphoma kinase inhibitors. Cancer Res. 2008; 68: 3389–95. Christensen JG, Zou HY, Arango ME, Arango ME, Li Q, Lee JH, McDonnell SR, Yamazaki S, Alton GR, Mroczkowski B et al. Cytoreductive antitumor activity of PF-2341066, a novel inhibitor of anaplastic lymphoma kinase and c-Met, in experimental models of anaplastic large-cell lymphoma. Mol. Cancer Ther. 2007; 6: 3314–22. Camidge DR, Bang Y, Kwak EL. Progression-free survival (PFS) from a phase I study of crizotinib (PF-02341066) in patients with ALK-positive non-small cell lung cancer (NSCLC). J. Clin. Onc. 2011; 29: 2501. ASCO Annual Meeting Proceedings (PostMeeting Edition) (May 20 Supplement). Kim DW, Ahn M-J, Shi Y. Updated results of a global phase II study with crizotinib in advanced ALK-positive non-small cell Respirology (2014) 19, 1117–1125

1124

31

32

33

34

35

36

37

38

39

40 41

42 43

44

45

46

lung cancer (NSCLC). Presented at the 2012 ESMO; abstract 1230. Shaw AT. Phase III trial shows crizotinib superior to single-agent chemotherapy for ALK-positive advanced NSCLC. Presented at ESMO 2012; abstract 2862. Choi YL, Soda M, Yamashita Y, Ueno T, Takashima J, Nakajima T, Yatabe Y, Takeuchi K, Hamada T, Haruta H et al. Cancer Study Group. EML4-ALK mutations in lung cancer that confer resistance to ALK inhibitors. N. Engl. J. Med. 2010; 363: 1734–9. Broet P, Dalmasso C, Tan EH, Alifano M, Zhang S, Wu J, Lee MH, Régnard JF, Lim D, Koong HN et al. Genomic profiles specific to patient ethnicity in lung adenocarcinoma. Clin. Cancer Res. 2011; 17: 3542–50. Leidner RS, Fu P, Clifford B, Hamdan A, Jin C, Eisenberg R, Boggon TJ, Skokan M, Franklin WA, Cappuzzo F et al. Genetic abnormalities of the EGFR pathway in African American patients with non-small-cell lung cancer. J. Clin. Oncol. 2009; 27: 5620–6. Reinersman JM, Johnson ML, Riely GJ, Riely GJ, Chitale DA, Nicastri AD, Soff GA, Schwartz AG, Sima CS, Ayalew G et al. Frequency of EGFR and KRAS mutations in lung adenocarcinomas in African Americans. J. Thorac. Oncol. 2011; 6: 28–31. Shigematsu H, Lin L, Takahashi T, Nomura M, Suzuki M, Wistuba II, Fong KM, Lee H, Toyooka S, Shimizu N et al. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J. Natl. Cancer Inst. 2005; 97: 339–46. Mitsudomi T, Morita S, Yatabe YY, Negoro S, Okamoto I, Tsurutani J, Seto T, Satouchi M, Tada H, Hirashima T et al. West Japan Oncology Group: 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. Lancet Oncol. 2010; 11: 121–8. Fukuoka M, Wu YL, Thongprasert S, Sunpaweravong P, Leong SS, Sriuranpong V, Chao TY, Nakagawa K, Chu DT, Saijo N 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). J. Clin. Oncol. 2011; 29: 2866–74. Maemondo M, Inoue A, Kobayashi K, Sugawara S, Oizumi S, Isobe H, Gemma A, Harada M, Yoshizawa H, Kinoshita I et al. Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. N. Engl. J. Med. 2010; 362: 2380–8. Lee MY, Shen MR. Epithelial-mesenchymal transition in cervical carcinoma. Am. J. Transl. Res. 2012; 4: 1–13. Mohamed S, Yasufuku K, Nakajima T, Hiroshima K, Kubo R, Iyoda A, Yoshida S, Suzuki M, Sekine Y, Shibuya K et al. Analysis of cell cycle-related proteins in mediastinal lymph nodes of patients with N2-NSCLC obtained by EBUSTBNA: relevance to chemotherapy response. Thorax 2008; 63: 642–7. Bunn PA Jr, Doebele RC. Genetic testing for lung cancer: reflex versus clinical selection. J. Clin. Oncol. 2011; 29: 1943–5. Paik PK, Arcila ME, Fara M, Choe JY, Sima CS, Miller VA, Kris MG, Ladanyi M, Riely GJ. Clinical characteristics of patients with lung adenocarcinomas harboring BRAF mutations. J. Clin. Oncol. 2011; 2: 2046–51. Bergethon K, Shaw AT, Ou SH, Katayama R, Lovly CM, McDonald NT, Massion PP, Siwak-Tapp C, Gonzalez A, Fang R et al. ROS1 rearrangements define a unique molecular class of lung cancers. J. Clin. Oncol. 2012; 30: 863–70. Park S, Holmes-Tisch AJ, Cho EY, Shim YM, Kim J, Kim HS, Lee J, Park YH, Ahn JS, Park K et al. Discordance of molecular biomarkers associated with epidermal growth factor receptor pathway between primary tumors and lymph node metastasis in non-small cell lung cancer. J. Thorac. Oncol. 2009; 4: 809–15. Yatabe Y, Matsuo K, Mitsudomi T. Heterogeneous distribution of EGFR mutations is extremely rare in lung adenocarcinoma. J. Clin. Oncol. 2011; 29: 2972–7.

Respirology (2014) 19, 1117–1125

K Czarnecka-Kujawa and K Yasufuku 47 Sun L, Zhang Q, Luan H, Zhan Z, Wang C, Sun B. Comparison of KRAS and EGFR gene status between primary non-small cell lung cancer and local lymph node metastases: implications for clinical practice. J. Exp. Clin. Cancer Res. 2011; 30: 30. 48 Almeida FA, Casal RF, Jimenez CA, Eapen GA, Uzbeck M, Sarkiss M, Rice D, Morice RC, Ost DE. Quality gaps and comparative effectiveness in lung cancer staging: the impact of test sequencing on outcomes. Chest 2013; 144: 1776–82. 49 Yarmus LB, Akulian JA, Mathai SC, Sathiyamoorthy S, Sahetya S, Harris K, Gillespie C, Haas A, Feller-Kopman D, Sterman D et al. Comparison of moderate versus deep sedation for endobronchial ultrasound transbronchial needle aspiration. Ann. Am. Thorac. Soc. 2013; 10: 121–6. 50 Nakajima T, Yasufuku K, Suzuki M, Sekine Y, Shibuya K, Hiroshima K, Fujisawa T. Histological diagnosis of spinal chondrosarcoma by endobronchial ultrasound guided transbronchial needle aspiration: a case report. Respirology 2007; 12: 308–10. 51 Nakajima T, Yasufuku K, Suzuki M, Kimura H, Yoshino I. Assessment of epidermal growth factor receptor mutation by endobronchial ultrasound-guided transbronchial needle aspiration. Chest 2007; 132: 597–602. 52 Billah S, Stewart J, Staerkel G, Chen S, Gong Y, Guo M. EGFR and KRAS mutations in lung carcinoma: molecular testing by using cytology specimens. Cancer Cytopathol. 2011; 119: 111–17. 53 van Eijk R, Licht J, Schrumpf M, Talebian Yazdi M, Ruano D, Forte GI, Nederlof PM, Veselic M, Rabe KF, Annema JT et al. Rapid KRAS, EGFR, BRAF and PIK3CA mutation analysis of fine needle aspirates from non-small-cell lung cancer using allele-specific qPCR. PLoS ONE 2011; 6: 17791. 54 Santis G, Angell R, Nickless G, Quinn A, Herbert A, Cane P, Spicer J, Breen R, McLean E, Tobal K. Screening for EGFR and KRAS mutations in endobronchial ultrasound derived transbronchial needle aspirates in non-small cell lung cancer using COLD-PCR. PLoS ONE 2011; 6: 25191. 55 Tanner N, Watson P, Boylan A, Memoli JS, Pastis N, Taylor K, Garrett-Mayer E, Silvestri GA. Utilizing endobronchial ultrasound with fine-needle aspiration to obtain tissue for molecular analysis. A single-center experience. J. Bronchol., Intervent. Pulmonol. 2011; 18: 317–21. 56 Cagle P, Allen T, Olsen R. Lung cancer biomarkers. Present status and future developments. Arch. Pathol. Lab. Med. 2013; 137: 1191–8. 57 Nakajima T, Yasufuku K, Nakagawara A, Kimura H, Yoshino I. Multi-gene mutation analysis of metastatic lymph nodes in nonsmall cell lung cancer diagnosed by EBUS-TBNA. Chest 2011; 140: 1319–24. 58 Sakairi Y, Sato K, Itoga S, Saegusa F, Matsushita K, Nakajima T, Yoshida S, Takiguchi Y, Nomura F, Yoshino I. Transbronchial biopsy needle rinse solution used for comprehensive biomarker testing in patients with lung cancer. J. Thor. Oncol. 2014; 9: 26–32. 59 Lewandowska M, Jozwicki W, Jochymski C, Kowalewski J. Application of PCR methods to evaluate EGFR, KRAS and BRAF mutations in a small number of tumor cells in cytological material from lung cancer patients. Oncol Rep 2013; 30: 45–52. 60 Bonifazi M, Sediari M, Ferretti M, Tang XP, Shi WL, Du YJ. The role of the pulmonologist in rapid on-site cytologic evaluation of transbronchial needle aspiration: a prospective study. Chest 2014; 145: 60–5. 61 Rivera P, Mehta A, Wahidi M. Establishing the diagnosis of lung cancer: diagnosis and management of lung cancer, 3rd ed: american college of chest physicians evidence-based clinical practice guidelines. Chest 2013; 143(Suppl. 5): 142–65. 62 Li J, Hu YM, Wang Y, Tang XP, Shi WL, Du YJ. Gene mutation analysis in non-small cell lung cancer patients using bronchoalveolar lavage fluid and tumor tissue as diagnostic markers. Int. J. Biol. Markers 2014; doi: 10.5301/jbm.5000075. 63 Anraku M, Pierre AF, Nakajima T, de Perrot M, Darling GE, Waddell TK, Keshavjee S, Yasufuku K. Endobronchial ultrasound-guided transbronchial needle aspiration in the © 2014 Asian Pacific Society of Respirology

Molecular alterations in NSCLC management of previously treated lung cancer. Ann. Thorac. Surg. 2011; 92: 251–5. 64 Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, Zakowski MF, Kris MG, Varmus H. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med. 2005; 2: 73. 65 Bean J, Brennan C, Shih JY, Riely G, Viale A, Wang L, Chitale D, Motoi N, Szoke J, Broderick S et al. MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 20932–7. 66 Engelman JA, Janne PA. Mechanisms of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer. Clin. Cancer Res. 2008; 14: 2895–9.

© 2014 Asian Pacific Society of Respirology

1125 67 Jackman D, Pao W, Riely GJ, Engelman JA, Kris MG, Jänne PA, Lynch T, Johnson BE, Miller VA. Clinical definition of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer. J. Clin. Oncol. 2010; 28: 357–60. 68 Chmielecki J, Foo J, Oxnard GR, Hutchinson K, Ohashi K, Somwar R, Wang L, Amato KR, Arcila M, Sos ML et al. Optimization of dosing for EGFR-mutant non-small cell lung cancer with evolutionary cancer modeling. Sci. Transl. Med. 2011; 3: 90ra59. 69 Sequist LV, Waltman BA, Dias-Santagata D, Digumarthy S, Turke AB, Fidias P, Bergethon K, Shaw AT, Gettinger S, Cosper AK et al. Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors. Sci. Transl. Med. 2011; 3: 75ra26.

Respirology (2014) 19, 1117–1125

Molecular alterations in non-small-cell lung cancer: perspective for targeted therapy and specimen management for the bronchoscopist.

Major advances have occurred over the past decade in our understanding of lung cancer pathobiology. Increasing knowledge of molecular aberrations in l...
163KB Sizes 0 Downloads 4 Views