Targeted therapy in lung cancer has had a major influence

Transcription

1 Review Article Lung Cancer in the Era of Targeted Therapy A Cytologist s Perspective Maureen F. Zakowski, MD Context. The diagnosis and treatment of non small cell lung cancer have changed dramatically in the past few years. The discovery of activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor and the use of drugs that successfully target those mutations are among the key advances that have led to a shift in the practice of oncology and pathology, with perhaps the greatest effect on the field of cytology. Objectives. To present the perspective of a practicing thoracic pathologist and cytopathologist on the developments that have changed practice and to place those changes in a broader context. Data Sources. Literature review, studies undertaken or participated in by the author, and personal experience. Conclusions. Cytologists are in an ideal position to influence appropriate testing and treatment in the era of targeted therapy. Lung pathology has led the way in the era of targeted therapy, in no small part due to cytology. (Arch Pathol Lab Med. 2013;137: ; doi: /arpa RA) Targeted therapy in lung cancer has had a major influence on the practice of cytology. Personalized medicine and targeted therapy have become commonplace terms and expectations for the patient with cancer, with attention given to this topic in all areas of media from the scientific literature to the evening news. 1 3 No cancer is more in need of a personalized or targeted approach than lung cancer. It remains the largest cancer killer in the westernized world, with approximately new cases, and more than deaths estimated for 2012 in the United States. 4 This is in contrast to breast cancer, for which, there are expected to be about new cases in 2012, with fewer than deaths. 4 Little progress has been made in the search for a lung cancer cure, at least in part because most patients present at a stage too late to be surgical candidates. Unfortunately, there is no cure for lung cancer without surgery, although surgery and chemotherapy may both be required. The diagnosis of lung cancer in most patients is made using limited cytology and small amounts of biopsy material, and the pathologist is now faced with the challenges of meeting the needs of a changing landscape in the evaluation and treatment of lung cancer with limited amounts of tissue. This article will review the major changes in the field of non small cell lung cancer (NSCLC) and share the perspective of the cytopathologist faced with a practice with increasing challenges. LUNG CANCER SCREENING AND DIAGNOSIS Unlike cervical carcinoma, which was greatly reduced in the United States because of cytology screening, 5 no effective screening techniques have yet been identified for the general population with lung cancer. The National Lung Screening Trial showed that participants who received lowdose helical computed tomography scans had a 20% lower risk of dying from lung cancer than did participants who received standard chest x-rays. That trial, however, was restricted to people aged 55 to 74 years with at least a 30 Accepted for publication February 8, pack-year history of smoking. 6 Published as an Early Online Release April 18, Because most patients with lung cancer are not surgical From the Pathology Fellowship Program, Memorial Sloan Kettering, New York, New York. candidates, most samples available for analysis will be based The authors have no relevant financial interest in the products or on cytology or small-biopsy materials. However, epidemiology and targeted therapy in lung cancer have changed companies described in this article. This work was supported by Boehringer Ingelheim Pharmaceuticals, Inc (Ridgefield, Connecticut). Editorial assistance was provided able from patients who go to surgery, it is questionable cytology practice. Although more-extensive tissue is avail- by Staci Heise, PhD, of MedErgy (Yardley, Pennsylvania), who was whether that obviates the limitations related to initial contracted by Boehringer Ingelheim for that service. The author meets criteria for authorship as recommended by the International diagnosis. In recent years, we have seen changes in Committee of Medical Journal Editors (ICMJE), fully accepts respiratory tract cytology from improvements in imaging responsibility for content and editorial decisions, and was involved and fine-needle aspiration (FNA) techniques, the use of at all stages of manuscript development. The author received no flexible bronchoscopy, and the use of endobronchial compensation related to the development of the manuscript. Reprints: Maureen Zakowski, MD, Pathology Fellowship Program, ultrasound (EBUS) as a real-time staging procedure. At the Memorial Sloan Kettering, 1275 York Ave, New York, NY ( zakowskm@mskcc.org). a central squamous cell tumor histology caused by smoking same time, primary neoplastic lung disease has shifted from 1816 Arch Pathol Lab Med Vol 137, December 2013 Lung Cancer in the Era of Targeted Therapy Zakowski

2 Table 1. Subtyping Non Small Cell Lung Cancer on Cytology Specimen Type Cytology Accuracy Compared With Histology, % Cases, No. Source, y FNA, BB, BW, BL with IHC 128 Rekhtman et al, FNA Nizzoli et al, FNA cell blocks with IHC 103 Righi et al, FNA, BB, BW, BL 93; IHC needed less frequently in cytology 101 Sigel et al, Abbreviations: BB, bronchial brush; BW, bronchial wash; BL, bronchial lavage; FNA, fine-needle aspiration; IHC, immunohistochemistry. to a peripheral adenocarcinoma, often in nonsmokers and with an almost equal incidence in men and women. The changes in oncology practice have resulted, in part, because of the recognition that histologic and cytologic subtyping of NSCLC is necessary before treatments can be chosen and that genotyping must be done for mutation analysis, which may also direct therapy. The small cell NSCLC division is no longer sufficient; cytologists make that distinction with great accuracy. 7 There may, however, be some reluctance on the part of the cytology community to commit to a NSCLC subtype when they have only limited material for analysis, in part, because, for most of the history of cytology practice, that distinction was not necessary all NSCLCs were treated the same. The classic criteria for those subtypes existed, but may not often have been applied. As a result, the skills necessary to make that distinction may have withered, and there is always a need and desire to reduce cytologic-histologic correlation discrepancies. To reintroduce the pathology community to the practice of distinguishing adenocarcinoma from squamous carcinoma, we must look at the reasons driving the need for histologic separation. MUTATIONS ASSOCIATED WITH LUNG CANCER The current state of lung cancer diagnosis and treatment rests on the discovery of activating mutations in adenocarcinoma. 8 These mutations are not believed to occur in nonadenocarcinoma, although they may occasionally be found in tumors of adenosquamous subtype. 9 The most common of these mutations are epidermal growth factor receptor (EGFR), Kirsten rat sarcoma viral oncogene homolog (KRAS), and rearrangement of the anaplastic lymphoma receptor tyrosine kinase (ALK), and to a lesser degree v-raf murine sarcoma viral oncogene homolog B1 (BRAF). 10 The techniques used to identify these mutations are readily applicable to pathology and cytology material. ALK rearrangements can be readily identified by fluorescence in situ hybridization (FISH) using cytology cell-block material, and more recently, immunohistochemical antibodies have become available to detect that rearrangement; how their use will be incorporated into pathology practice has not yet been defined. 11 Small-molecule inhibitor drugs targeted to some of these mutations have been evaluated clinically, such as tyrosine kinase inhibitors (TKIs) for EGFR and crizotinib for ALK. 12,13 The US Food and Drug Administration (FDA) requires a FISH demonstration of ALK rearrangement before the use of crizotinib. 13 Although the FDA does not require a demonstration of EGFR mutations for the use of TKIs, those patients with EGFR mutations are most likely to respond to TKIs. There is also the recognition that there are histology-specific indications and toxicities for certain chemotherapeutic and inhibitory agents. 14 This indication of therapy by histologic subtype is exemplified by the successful use of pemetrexed in nonsquamous carcinomas, and the contraindication for the use of bevacizumab in squamous carcinoma. Interestingly, that usage has coined the phrase nonsquamous, which has solidly entered the clinician s vocabulary and has necessitated a change in the way pathologists talk about NSCLC. The discovery of activating mutations in EGFR was reported almost simultaneously by 3 separate groups in ,15,16 EGFR mutations were found in better-differentiated adenocarcinomas, such as the bronchioloalveolar subtype, by most investigators, 17,18 although some reported an association with papillary subtype. 19 Although certain histology features may be associated with mutations, histology cannot be used as a surrogate for mutation testing. 20 The most appropriate way to predict for response to EGFR TKIs is to test for EGFR mutations with molecular methods, not by determining EGFR copy number with FISH or chromogenic in situ hybridization techniques. 20,21 THE EMERGING VISIBILITY OF THE PATHOLOGIST AND THE CYTOLOGIST IN LUNG CANCER THERAPY Histologic subtyping as a force behind treatment decisions is recognized by at least one drug manufacturer of chemotherapeutic agents. Lilly Oncology (Indianapolis, Indiana) has mounted an expansive campaign in medical journals called It s Histological to highlight the importance of pathology in the diagnosis and treatment of lung cancer. Pathologists are now the target of advertising campaigns. This is very different from when pathologists were unknown to patients and clinicians. Targeted therapy is changing the way pathology and pathologists are perceived. With the elevated importance of the pathologic workup of lung cancer samples, more pressure is being placed on pathologists to do additional and more-varied tests with less available tissue. This has had a direct effect on the practice of cytology, which has always dealt with minute amounts of diagnostic material. The use of cytology in diagnosing lung cancer is well documented. The ability of cytology to render a specific histologic subtype using conventional material, however, is still somewhat controversial. Several relatively large studies document the validity of cytology, relative to histology, in subtyping NSCLC. Although 4 large series demonstrate accuracies of 64% to 100% (Table 1), there remains a small but real discrepancy in the accuracy of small biopsy (needle core) material in contrast to the gold standard of large resection specimens. Immunohistochemistry (IHC) as an adjunct to hematoxylin-eosin morphology to refine pathologic diagnoses has been well documented and performs extremely well in cytology material, particularly in cell block and ThinPrep (Hologic, Inc, Bedford, Massachusetts) specimens. The preferred panel of IHC stains for the separation of NSCLC into adenocarcinoma and squamous cell carcinoma subtypes most often includes thyroid transcription factor 1 (TTF-1) and p63. TTF-1 positivity (outside of the setting of thyroid carcinoma) supports both lung primary and adenocarcinoma (Figure 1), whereas p63 positivity is a Arch Pathol Lab Med Vol 137, December 2013 Lung Cancer in the Era of Targeted Therapy Zakowski 1817

3 Figure 1. Thyroid transcription factor 1 (TTF1) stained cell block. Note the intense nuclear staining (original magnification 3200). Figure 2. Cytology cell block of adenocarcinoma showing squamoid features of generous, pink cytoplasm and angulated nuclei (hematoxylin-eosin, original magnification 3200). strong indicator of squamous differentiation. Although both of these stains can have a slight overlapping pattern, it is more common to have a TTF-1 þ squamous cell carcinoma than it is to have a p63 þ adenocarcinoma; it is rare for a squamous carcinoma to lack p63 positivity. 26 Both these stains have sharp, easy-to-read, nuclear signals and can be useful in cytology cell-block material, which often demonstrates a squamoid artifact (Figure 2) when the sample is adenocarcinoma. p40 is emerging as an extremely sensitive and specific marker of squamous differentiation in NSCLC. 26 Other useful IHC stains include napsin A and PE10 for adenocarcinoma and 34bE12 for squamous carcinoma. CK5/6 positivity can help support squamous differentiation but is considered second line in usefulness. CK7 and CK20 play little, if any, role in separating adenocarcinoma from squamous cell carcinoma. Although the ability of the cytology laboratory to accurately classify NSCLC subtypes is well documented, it is not well publicized. A recent article 14 in the Journal of Clinical Oncology states that FNA does not provide optimal specimens to enable histologic diagnosis based on architectural patterns. This is written by a clinician and a surgical pathologist and highlights the misconceptions in the literature. Fine-needle aspiration cytology does not, and never has, provided histologic architecture for anything. The American Society of Clinical Oncology guidelines of 2009 also state that, in the routine care of patients, reasonable efforts [should be taken] to obtain more tissue than what is contained in a routine cytology specimen, 27(p6252) and yet, most of these patients will never have more tissue available than that obtained as a cytology specimen. The National Comprehensive Cancer Network guidelines 28 recommend histologic subtyping followed by EGFR mutation testing (category 1) for adenocarcinoma in the metastatic and recurrent setting. That is tantamount to saying that cytology material must be used for such studies. As previously mentioned, EGFR, KRAS, ALK, and BRAF are the most frequently identified driver mutations in NSCLC. It is commonly assumed that cytology cannot provide enough material to identify these mutations. Yet studies 22,29 31 have demonstrated the utility of routine cytology specimens, such as brushes, washes, lavages, and FNA and EBUS material, as well as pleural and pericardial fluid in identifying mutations (Table 2). The success rate of cytology material, however, does depend somewhat on the type of specimen. There are other impediments to using cytology as the source for mutation analysis. For example, Clark 32 points out that few studies compare the cellularity of FNAs with the quality or quantity of the desired analyte or evaluate the effect of preanalytic handling or processing of cytology specimens on molecular tests. He also raises the issue of the need for standardized and optimized FNA processing devices for molecular testing. Nevertheless, molecular testing of cytology material can be very successful in identifying activating mutations. Cytology material can also be used outside of the strictly diagnostic setting. It is a fast, inexpensive, and nonin- Table 2. Success Rate of Molecular Testing for EGFR/KRAS Mutations Using Cytology Material Specimen Type % Suitable Cases, No. Source, y Routine cytology Rekhtman et al, EBUS TBNA Nakajima et al, EUS or EBUS FNA Schuurbiers et al, FNA, PL, BAL, BW Smouse et al, Abbreviations: BAL, bronchoalveolar lavage; BW, bronchial washing; EBUS, endobronchial ultrasound guided; EUS, transesophageal ultrasound guided; FNA, fine-needle aspiration; PL, pleural fluids; TBNA, transbronchial needle aspiration Arch Pathol Lab Med Vol 137, December 2013 Lung Cancer in the Era of Targeted Therapy Zakowski

4 Figure 3. Composite figure showing the immunoreactivity of mutation-specific, monoclonal antibody 2085 (Cell Signaling Technology, Inc, Danvers, Massachusetts) for the detection of the exon 19 (E746 A750)del. The photomicrographs on the left-hand side of the figure show a hematoxylin-eosin stained biopsy (A), cell block (C), and Papapanicolaou-stained ThinPrep (Hologic) specimens (E) matched on the right with positive staining with exon 19del monoclonal antibody (B, D, and F). These 3 different tumors were scored as 3 þ (original magnifications 3100 [A through D] and 3400 [E and F]. vasive way to gather information that may inform treatment or collect data for future studies in identifying molecular targets. In a trial conducted at Memorial Sloan- Kettering Cancer Center (New York, New York), cytology and small-biopsy material played a major role in rebiopsying patients with acquired resistance to EGFR TKIs. 33 The aims of this study were to determine the feasibility of rebiopsy in this setting (ie, would patients present Arch Pathol Lab Med Vol 137, December 2013 Lung Cancer in the Era of Targeted Therapy Zakowski 1819

5 themselves for rebiopsy after disease had been established to obtain tissue for studies that might not benefit the patient directly), to determine the frequency and spectrum of secondary EGFR mutations, and to collect material to search for novel mechanisms of acquired resistance to EGFR TKIs. One hundred fifty-three specimens were collected from 121 patients; 27 patients (22%) had multiple specimens studied. Approximately 50% of the specimens (n ¼ 76) consisted of minimally invasive samples of core biopsies or FNA material, 22% (n ¼ 34) were fluids that might otherwise have been discarded, and 22% were surgical specimens (n ¼ 34). Most surgical and cytology specimens (96 of 109; 88%) were adequate for analysis, and 71% (24 of 34) of fluid samples were adequate. Some samples may have been too degenerated or paucicellular to analyze, and bone specimens performed poorly because of the decalcification procedure effect on DNA. The FNAs performed better than the core biopsies did, which was most likely due to the greater sampling ability of fine needles. This kind of a trial is not just proof of principle but has also led to enhanced detection of the T790M mutation and helped to determine the natural history of patients with tumors harboring that mutation. 34 This is one example of the potential for maximizing the limited tissue that is often the only readily obtainable tumor in metastatic or recurrent lung cancer. Another area where cytology specimens can be exploited for maximal information in lung cancer is with the use of new, mutation-specific, immunohistochemical antibodies against exon 21 L858R and exon 19 (15 base pair) mutations (Figure 3, A through F). 35 In one study, 94 lung adenocarcinomas from cytology (cell block and ThinPrep) and small biopsy samples with known EGFR status on corresponding resection specimens were studied using these antibodies. 36 In these 94 cases there were 47 exon 19 deletions, 35 exon 21 L858R mutations, and 12 wild-type cases. These cases were stained and graded using a negative (0 1 þ ) and positive (2 þ 3 þ ) scoring system. The sensitivity for exon 19 deletions was 79% in cytology, and small biopsies compared with 85% in tissue. The sensitivity for exon 21 mutations was 66% compared with 76% in tissue. The specificity and positive predictive value of both antibodies were 100%. No wild-type cases were reactive. This application of IHC could eliminate the need for additional biopsies for mutation determination in positive cases and can be used with bone specimens, when the DNA is usually too degraded by decalcification procedures to be useful. The reports of the IHC staining could also become incorporated into the cytology report, saving time and resources. Immunohistochemistry staining for total protein EGFR in lung cancer does not correspond closely to mutation status 20 and currently has no effective role in the analysis of NSCLC. It is standard practice to analyze adenocarcinoma for the presence of activating mutations in EGFR, KRAS, and ALK. At my institution (Memorial Sloan-Kettering Cancer Center), that is done in a reflex setting, with the pathologist initiating the testing at diagnosis. All adenocarcinomas and adenosquamous carcinomas are analyzed using Sequenom, Inc (San Diego, California), technology without direct request from the clinician. As these mutations are mutually exclusive, after negative results in EGFR and KRAS studies, ALK is examined using a Vysis (Abbott Molecular, Des Plaines, Illinois) FISH break-apart probe. 37 Patients are not treated with a TKI unless a mutation in EGFR is identified. The Iressa Pan-Asia Study (IPASS) trial 20 has demonstrated that patients without such mutations do not benefit from TKI therapy, and we do not believe there are any histologic, demographic, clinical, or other surrogates for mutation testing. Standard chemotherapy is the treatment of choice for patients with KRAS-mutated adenocarcinomas. The FDA recently approved the use of crizotinib in patients with identified ALK mutations. 13 The Vysis ALK break-apart FISH probe is readily applied to material from cytology cell blocks and extends the usefulness of cytology in the management of patients with lung adenocarcinoma. CONCLUSIONS AND CLINICAL IMPLICATIONS In general, cytology material is underused in the current practice of medicine in the era of targeted therapy. A greater effort can be made by cytology laboratories to collect and store material for IHC and molecular studies. One practical approach is the routine use of cell blocks. Extra passes can be taken at the FNA, and material can be paraffin-embedded and stored for both diagnostic and research purposes. The presence of a cytotechnologist or pathologist during the procedure greatly facilitates the collection of adequate material. The minimum amount of material that is needed for molecular studies is as little as 700 tumor cells. 38 The tumor sample should not be diluted with nonmalignant cells, and using a hematoxylin-eosin stained slide as a guide, normal tissue can be trimmed out of a paraffin block to enrich the sample being analyzed. If a block or ThinPrep sample is not available, it is technically possible to scrape tumor cells directly off the stained cytology slides for analysis. The slides can be photographed or scanned before that is done to maintain a permanent record of the diagnosis if all the material is to be used for molecular studies. As pointed out by Clark, 32 cytopathologists must be engaged in clinical therapeutic aspects of cancer to be knowledgeable partners in health care. To maintain a central role in patient care, surgical pathologists and cytopathologists must incorporate molecular diagnoses for understanding and characterization of disease. If pathologists resist this change, the consequences are significant, as highlighted by Lauwers et al. 39 Allowing others (nonpathologists) to perform molecular testing moves the central role of tissue-based diagnosis out of pathology, compromises the strategic position of pathology (cytology) at the crossroads of clinical practice of medicine and scientific understanding of disease, and loses revenue. Molecular diagnostics is a billion dollar business, and a monetary share of that business should find its way back to anatomic pathology, where resources can be directed to better understanding and integration of molecular diagnostics into patient care. References 1. Kaiser J. Cancer research: looking for a target on every tumor. Science. 2009;326(5950): Hayden EC. Personalized cancer therapy gets closer. Nature. 2009; 458(7235): Varmus H. The new era in cancer research. Science. 2006;312(5777): Siegel R, Naishadham D, Jemal A. Cancer statistics, CA Cancer J Clin. 2012;62(1): American Cancer Society. Cancer Facts & Figures, Atlanta, GA: American Cancer Society; Aberle DR, Adams AM, Berg CD, et al; National Lung Screening Trial Research Team. Reduced lung-cancer mortality with low-dose computed tomographic screening. N Engl J Med. 2011;365(5): Arch Pathol Lab Med Vol 137, December 2013 Lung Cancer in the Era of Targeted Therapy Zakowski

6 7. Sakr L, Roll P, Payan MJ, et al. Cytology-based treatment decision in primary lung cancer: is it accurate enough? [published online ahead of print October 5, 2011]. Lung Cancer. 2012;75(3): Pao W, Miller V, Zakowski M, et al. EGF receptor gene mutations are common in lung cancers from never smokers and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci U S A. 2004; 101(36): Rekhtman N, Paik PK, Arcila ME, et al. Clarifying the spectrum of driver oncogene mutations in biomarker-verified squamous carcinoma of lung: lack of EGFR/KRAS and presence of PIK3CA/AKT1 mutations. Clin Cancer Res. 2012; 18(4): Pao W, Iafrate AJ, Su Z. Genetically informed lung cancer medicine. J Pathol. 2011;223(2): Murakami Y, Mitsudomi T, Yatabe Y. A screening method for the ALK fusion gene in NSCLC. Front Oncol. 2012;2:24. doi: /fonc Pao W, Miller VA, Politi KA, et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med. 2005;2(3):e73. doi: /journal. pmed XALKORI (crizotinib) capsules, oral [package insert]. New York, NY: Pfizer Laboratories; Langer CJ, Besse B, Gualberto A, Brambilla E, Soria JC. The evolving role of histology in the management of advanced non-small cell lung cancer. J Clin Oncol. 2010;28(36): Lynch TJ, Bell DW, Sordella R, 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(21): Paez JG, Janne PA, Lee JC, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science. 2004;304(5676): Zakowski MF, Hussain S, Pao W, et al. Morphologic features of adenocarcinoma of the lung predictive of response to the epidermal growth factor receptor kinase inhibitors erlotinib and gefitinib. Arch Pathol Lab Med. 2009;133(3): Miller VA, Riely GJ, Zakowski MF, et al. Molecular characteristics of bronchioloalveolar carcinoma and adenocarcinoma, bronchioloalveolar carcinoma subtype, predict response to erlotinib. J Clin Oncol. 2008;26(9): Motoi N, Szoke J, Riely GJ, et al. Lung adenocarcinoma: modification of the 2004 WHO mixed subtype to include the major histologic subtype suggests correlations between papillary and micropapillary adenocarcinoma subtypes, EGFR mutations and gene expression analysis. Am J Surg Pathol. 2008;32(6): 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). J Clin Oncol. 2011; 29(21): Sholl LM, Xiao Y, Joshi V, et al. EGFR mutation is a better predictor of response to tyrosine kinase inhibitors in non-small cell lung carcinoma than FISH, CISH, and immunohistochemistry. Am J Clin Pathol. 2010;133(6): Rekhtman N, Brandt SM, Sigel CS, et al. Suitability of thoracic cytology for new therapeutic paradigms in non-small cell lung carcinoma: high accuracy of tumor subtyping and feasibility of EGFR and KRAS molecular testing. J Thorac Oncol. 2011;6(3): Nizzoli R, Tiseo M, Gelsomino F, et al. Accuracy of fine needle aspiration cytology in the pathological typing of non-small cell lung cancer. J Thorac Oncol. 2011;6(3): Righi L, Graziano P, Fornari A, et al. Immunohistochemical subtyping of nonsmall cell lung cancer not otherwise specified in fine-needle aspiration cytology: a retrospective study of 103 cases with surgical correlation. Cancer. 2011;117(15): Sigel CS, Moreira AL, Travis WD, et al. Subtyping of non-small cell lung carcinoma: a comparison of small biopsy and cytology specimens. J Thorac Oncol. 2011;6(11): Pelosi G, Fabbri A, Bianchi F, et al. DNp63 (p40) and thyroid transcription factor-1 immunoreactivity on small biopsies or cellblocks for typing non-small cell lung cancer: a novel two-hit, sparing-material approach. J Thorac Oncol. 2012;7(2): Azzoli CG, Baker S Jr, Temin S, et al; American Society of Clinical Oncology. American Society of Clinical Oncology Clinical Practice Guideline update on chemotherapy for stage IV non small cell lung cancer. J Clin Oncol. 2009;27(36): National Comprehensive Cancer Network. Clinical Practice Guidelines in Oncology: non small cell lung cancer, version NCCN Guidelines Web site. Accessed November 28, Nakajima T, Yasufuku K, Suzuki M, et al. Assessment of epidermal growth factor receptor mutation by endobronchial ultrasound-guided transbronchial needle aspiration. Chest. 2007;132(2): Schuurbiers OC, Looijen-Salamon MG, Ligtenberg MJ, van der Heijden HF. A brief retrospective report on the feasibility of epidermal growth factor receptor and KRAS mutation analysis in transesophageal ultrasound- and endobronchial ultrasound-guided fine needle cytological aspirates. J Thorac Oncol. 2010;5(10): Smouse JH, Cibas ES, Janne PA, Joshi VA, Zou KH, Lindeman NI. EGFR mutations are detected comparably in cytologic and surgical pathology specimens of nonsmall cell lung cancer. Cancer. 2009;117(1): Clark DP. Seize the opportunity: underutilization of fine-needle aspiration biopsy to inform targeted cancer therapy decisions. Cancer. 2009;117(5): Arcila ME, Oxnard GR, Nafa K, et al. Rebiopsy of lung cancer patients with acquired resistance to EGFR inhibitors and enhanced detection of the T790M mutation using a locked nucleic acid-based assay. Clin Cancer Res. 2011;17(5): Oxnard GR, Arcila ME, Sima CS, et al. Acquired resistance to EGFR tyrosine kinase inhibitors in EGFR-mutant lung cancer: distinct natural history of patients with tumors harboring the T790M mutation. Clin Cancer Res. 2011; 17(6): Brevet M, Arcila M, Ladanyi M. Assessment of EGFR mutation status in lung adenocarcinoma by immunohistochemistry using antibodies specific to the two major forms of mutant EGFR. J Mol Diagn 2010; 12(2): Ang D, Brevet M, Ladanyi M, Zakowski MF. Assessment of EGFR mutation status in needle biopsies and cytology specimens of lung adenocarcinoma by immunohistochemistry using antibodies to two major forms of mutant EGFR. Lab Invest. 2010;90(suppl 1):87A. 37. Sequist LV, Heist RS, Shaw AT, et al. Implementing multiplexed genotyping of non small cell lung cancers into routine clinical practice. Ann Oncol. 2011; 22(12): Brandt SM, Tafe LJ, Arcila ME, et al. Performance of cytologic specimens in EGFR and KRAS molecular testing with a focus on minimal cellularity requirements. Mod Pathol. 2011;24(suppl 1):405A 406A. 39. Lauwers GY, Black-Schaffer S, Salto-Tellez M. Molecular pathology in contemporary diagnostic pathology laboratory: an opinion for the active role of surgical pathologists. Am J Surg Pathol. 2010;34(1): Arch Pathol Lab Med Vol 137, December 2013 Lung Cancer in the Era of Targeted Therapy Zakowski 1821