Saudi Journal of Ophthalmology (2013) 27, 187–192
Ocular Oncology Update
Ocular metastasis of lung adenocarcinoma with ELM4-ALK translocation: A case report with a review of the literature Kailun Jiang, MD a,b; Seymour Brownstein, MD a,b,⇑; Harmanjatinder S. Sekhon, MD, PhD b; Scott A. Laurie, MD c; Kay Lam, MD a,b; Steven Gilberg, MD a; William Britton, MD a
Abstract Choroidal metastasis is the most common intraocular neoplasm and is associated with significant morbidity. In a small percentage of patients, ocular manifestation may be the initial presentation of a systemic malignancy and can be diagnostically difficult to distinguish from ocular primary malignancies. Herein, we present a case of a never-smoker whose ocular pathology was integral to the diagnosis and management of a lung adenocarcinoma harboring a rare oncogene. Through this case, we have explored important diagnostic and therapeutic considerations of pulmonary metastases to the choroid. Keywords: Choroidal metastasis, Non-small cell lung carcinoma, Adenocarcinoma, EML4-ALK translocation, Targeted therapy, Crizotinib Ó 2013 Production and hosting by Elsevier B.V. on behalf of Saudi Ophthalmological Society, King Saud University. http://dx.doi.org/10.1016/j.sjopt.2013.06.011
Introduction Metastasis is the most common intraocular neoplasm mostly localizing to the choroid.1 Ocular metastasis occurs in 2–9% of all malignancies, of which 47–81% are from breast primaries and 9–23% are from lung primaries.2 69–88% of intraocular metastases localize to the choroid, with the majority of lung metastatic lesions being unifocal.1,3 Metastatic ocular lesions may be clinically silent, with only an estimated 12% involving the macula.3 At the time of ocular diagnosis, 34% have an unknown primary.3 The World Health Organization estimated that 12.7 million new cases of cancer occurred worldwide in 2008.4 Lung cancer is the most frequent malignant neoplasm, accounting for 16.5% of all new malignancy diagnoses in men and 8.5% in
women worldwide.4 The incidence of symptomatic ocular lung metastasis is between 1% and 7%.1 Due to the reduction in smoking amongst the population, relatively more individuals are developing non-small cell lung carcinoma (NSCLC), particularly adenocarcinoma.5 Lung adenocarcinoma can arise in never-smokers, and such patients are more likely to have an activating mutation of the epidermal growth factor receptor (EGFR) pathway or echinoderm microtubule associated protein and anaplastic lymphoma kinase (EML4-ALK) gene translocation.5 Much progress has been made in the understanding and identification of the cellular mechanism underlying tumorigenesis and progression. These mechanisms primarily involve a multitude of pathways including the well recognized EGFR and K-ras anti-apoptotic pathways.5,6 Herein, we describe a case of lung adenocarcinoma
Available online 5 July 2013 a
Department of Ophthalmology, University of Ottawa, The Ottawa Hospital, The Ottawa Hospital Research Institute, Ottawa, Ontario, Canada Department of Pathology and Laboratory Medicine, University of Ottawa, The Ottawa Hospital, The Ottawa Hospital Research Institute, Ottawa, Ontario, Canada c Department of Medical Oncology, University of Ottawa, The Ottawa Hospital, The Ottawa Hospital Research Institute, Ottawa, Ontario, Canada b
⇑ Corresponding author. Address: University of Ottawa Eye Institute, 501 Smyth Road, Suite W6213, Ottawa, Ontario K1H 8L6, Canada. Tel.: +1 (613) 737 8824; fax: +1 (613) 737 8826. e-mail address: [email protected]
(S. Brownstein). Peer review under responsibility of Saudi Ophthalmological Society, King Saud University
Production and hosting by Elsevier
Access this article online: www.saudiophthaljournal.com www.sciencedirect.com
188 with a novel gene rearrangement of EML4-ALK that presented initially as a symptomatic ocular metastasis. We provide an in-depth discussion of ocular pulmonary metastases and management options.
Case report A 44-year-old, never-smoker, otherwise healthy man presented with 3-days of a superior visual field defect in his left eye (OS) with a visual acuity of 20/25. Dilated funduscopic examination disclosed an 11 10 mm amelanotic choroidal mass near the macula OS (Fig. 1A). Ultrasonographic examination showed a dome-shaped lesion measuring 3.9 mm in height with high internal reflectivity. Diagnostic imaging revealed a 55 40 mm left lower lobe (LLL) lung lesion (Fig. 1B), which on fine needle aspirate biopsy (FNAB) was
K. Jiang et al. identified as an adenocarcinoma. Genetic testing was negative for EGFR mutation. Biopsy specimens of the mediastinal lymph nodes were positive for metastatic disease and he was staged at T2, N2, and M1.7 A magnetic resonance imaging (MRI) scan of the brain detected an intraocular lesion measuring 10 4 mm (Fig. 1C). During the 8 weeks’ investigative process, the ocular lesion continued to enlarge and the patient experienced increasing pain and decreasing vision. Nine weeks after his initial presentation, he received targeted radiation therapy to the posterior pole OS (30 Gy in 10 fractions). While the ocular pain and tumor size diminished, the patient’s visual acuity did not improve (Fig. 1D). He also was treated aggressively with chemotherapy (cisplatin and vinorelbine) and radiotherapy (60 Gy in 20 fractions over the course of 7 weeks) targeted to the primary lesion 10 weeks following his initial presentation. He responded well with a marked reduction in the LLL tumor size
Figure 1. (A) Left fundus of a choroidal metastasis showed a large dome-shaped amelanotic tumor with retinal pigment epithelium clumping. (B) Left lower lobe lesion was revealed by lateral chest X-ray. (C–E) Using serial MRI studies, it was noted that combined radiotherapy and chemotherapy were initially effective at reducing tumor size (arrows) (C, pre-treatment; D, post-treatment). However, over the subsequent 1.5 years, tumor size slowly enlarged (E, arrow) causing significant ocular pain. (F) Cross sections of the enucleated left globe revealed an amelanotic tumor mass (CA) in the posterior aspect of the globe measuring 20 mm in base and 9 mm in height. (G) The tumor cells invaded through the lamina cribrosa (arrow) to the margin of the surgical transection (hematoxylin-eosin, 25).
Ocular metastasis of lung adenocarcinoma with ELM4-ALK translocation: and no evidence of any new lesions upon computed tomographic (CT) restaging. While the ocular tumor mass was stable, the patient’s visual deficits continued to worsen. Eight months after his initial presentation, he had no light perception with worsening ocular pain. Over the next year, followup studies showed progressive metastatic disease with new lesions identified in the liver, cerebellum, and multiple small nodules in the left lung base. Also, the mediastinal nodes enlarged and his chronic cough progressed to hemoptysis. The patient began to experience pain on extraocular movement and a repeated MRI showed progression of the OS lesion (Fig. 1E). He underwent enucleation for palliative pain management 14 months after being diagnosed with non-small cell lung carcinoma (Fig. 1F). Post-operatively, ocular pain levels improved. His systemic disease continued to progress, with an enlargement of the hepatic metastasis and the multiple lung nodules. Due to disease progression, he was treated with a second course of chemotherapy on a clinical trial consisting of docetaxel. He tolerated this treatment well and no diseaserelated symptoms aside from slight worsening orbital discomfort were observed.
Gross pathological examination (Fig. 1F) disclosed a posterior choroidal tumor measuring 20 mm in maximal base and 9 mm in maximal height. On histopathological examination, the tumor formed nests, cords, and islands surrounded by fibrovascular stroma and displayed a perivascular growth pattern with numerous cholesterol clefts. The tumor cells invaded the anterior-posterior section of the optic nerve extending to its cut edge (Fig. 1G). The tumor cells had pleomorphic nuclei, prominent nucleoli, abundant cytoplasm with well-demarcated borders and neoplastic atypical mitoses were noted. The tumor formed occasional gland-like structures (Fig. 2A) and contained cells with abundant vacuolated cytoplasm with well demarcated cell boarders imparting a signet ring appearance (Fig. 2B), which stained strongly positive for mucus production (Fig. 2C–F). The prior FNAB and mediastinal lymph node biopsies showed similar cytomorphological features (Fig. 2G and H). The tumor extended into and destroyed the overlying degenerated, necrotic retina. It also invaded the adjacent sclera to 25% of its depth, but there was no evidence of extrascleral extension or invasion into the subarachnoid space.
Figure 2. (A–E) The ocular tumor formed gland-like structure (A, hematoxylin-eosin, 100) with numerous signet ring cells with a vacuolated cytoplasm and a displaced crescent shaped nucleus (B, arrows, hematoxylin-eosin, 400) and stained strongly positive for mucus production (C, arrows, periodic acid-Schiff, 400; D, arrows, colloidal iron, 400; E, arrow, mucicarmine, 400). (F) Transmission electron microscopy of the ocular specimen identified tumor cells with the cytoplasm filled with a predominantly radiolucent vacuolated fibrillo-granular material with displacement of the nucleus into a crescent cap (arrow) in a signet ring configuration (original magnification 2500). (G and H) Reassessment of the lung nodule FNAB (G, hematoxylineosin, 400) and mediastinal lymph node (H, hematoxylin-eosin, 400) demonstrated similar signet ring cells (arrows).
K. Jiang et al.
Figure 3. (A–C) Immunohistochemical staining for ALK gene rearrangement using anti-ALK antibody (5A4) showed extensive positivity in all three biopsy specimens (A, lung nodule FNAB, 100; B, mediastinal lymph node, 100; C, choroidal metastasis, 100). (D) FISH analysis of the choroidal metastatic lesion is positive for ALK translocation as indicated by the separation of the red and green fluorescent marks. In the absence of gene translocation, the red and green fluorophores overlap (arrows).
Thereafter, the patient’s neoplasm was screened for a new target, ALK expression by immunohistochemistry, which showed diffuse, strong staining in the FNAB, lymph nodes, and globe (Fig. 3A–C). Florescent in situ hybridization (FISH) of the ocular metastatic lesion demonstrated that he was positive for EML4-ALK gene translocation (Fig. 3D). Because the results from a randomized trial suggest that ALK inhibitor, crizotinib, was superior to other chemotherapies in patients with ALK rearrangement, his therapy was switched to crizotinib.8 He has remained on this therapy for over 6 months and remained in stable clinical condition with no evidence of disease progression at the time of this report.
The choroid is the most common location for metastatic seeding, due in large part to its rich vascular supply.3 These lesions frequently arise from a silent primary.3 Rarely, a symptomatic choroidal metastasis may be the initial presentation of a lung neoplasm. One recent meta-analysis approximated the number of cases presenting in such fashion was 55 cases. Of these 55 cases, 42% were adenocarcinoma and 22% of the patients were never-smokers.9
important to obtain a comprehensive history as 66–97% of patients evaluated for a metastatic choroidal lesion already would have been diagnosed with a primary neoplasm elsewhere.2 Metastases to the choroid are usually nonpigmented with the exception of metastatic spread from cutaneous melanoma primaries.3,11 Metastatic choroidal lesions are generally plateau or dome shaped and are often associated with retinal detachment.3 The presence of serous detachment and multiple choroidal lesions is more suggestive of metastases.10,11 Several methods are available for direct characterization of choroidal lesion, including ultrasonography, fundus autofluorescence (FAF) and FNAB. Ultrasonography differentiates between choroidal melanoma, metastasis, and hemangioma with 90% accuracy.12 Metastatic lesions are usually hyperechogenic as compared to uveal melanoma on A-mode.12 FAF may be used to aid in the evaluation of the tumor margin.13 In 17% of patients with uveal metastatic lesions, a primary neoplastic source may never be found.3 In this population it would be beneficial to obtain a definitive diagnosis through FNAB, with the preservation of visual function and generally has not been shown to cause seeding along the needle tract.3
It can be diagnostically challenging to distinguish a metastatic lesion from a primary ocular neoplasm and from spaceoccupying lesions. Uveal melanoma is the most common primary intraocular malignancy.10 Six to 10% of patients with uveal melanoma also have another primary neoplasm.10 It is
Lung primaries are categorized into small cell and nonsmall cell lung carcinoma; approximately 80% of all cases are non-small cell carcinoma.7,14 Non-small cell carcinomas are primarily adenocarcinoma, squamous cell carcinoma, or large cell carcinoma.14 Due to the declining smoking inci-
Ocular metastasis of lung adenocarcinoma with ELM4-ALK translocation: dence, 55% of non-small cell lung carcinomas now are adenocarcinoma.5,7,15 Adenocarcinoma can be seen in non-smokers and is the most common type of lung cancer in women.7,16 Adenocarcinoma usually begins in the periphery of the lung; it is a slow growing lesion, leading to prolonged latency prior to diagnosis.16–18 Consequently, these patients are often diagnosed at an advanced stage with metastatic disease and a median survival duration of 6–10 months.7 On the other hand, squamous cells carcinoma accounts for 25–31% of NSCLC and is more prevalent in smokers.7 These lesions are usually central, originate and involve the main conducting airway, and are symptomatic early into the disease process.7,18 Large cell carcinoma is a diagnosis of exclusion with no glandular or squamous differentiation making up approximately 13% of NSCLC and similar to adenocarcinoma typically occurring in the peripheral regions of the lung.7,18 Small cell carcinoma comprises 20% of all lung cancers7 Although, this type of neoplasm is associated with a rapid growth rate and early metastasis, small cell carcinoma is generally highly responsive to chemotherapy and radiotherapy.7,18 Similar to squamous cell carcinoma, small cell carcinomas are usually found centrally.7 Smoking is a substantial predisposing risk factor in developing small cell carcinoma.7
Therapy Early treatment offers the best hope for the preservation of vision in metastatic uveal cancer. Several approaches exist to treat these lesions; the utility of each depends upon the presenting characteristics of the lesion. In general, choroidal metastases respond favorably to systemic therapy targeted toward the primary neoplasm.2 However, local therapy is warranted when there is an enlargement of the lesion, pain, or when the ocular lesion is the sole metastatic finding. In instances where residual visual function remains, radiotherapy (brachytherapy or external beam), laser photocoagulation, and more recently intraocular delivery of bevacizumab may be offered.2,19 Brachytherapy is most suitable for discrete small to medium sized lesions, whereas larger lesions close to the optic nerve and macula are more amenable to external beam delivery.20 The treatment goal for ocular metastasis is palliative with the preservation of function and quality of life. Surgical interventions, such as enucleation, are reserved for salvage procedures in the treatment of break-through disease following initial radiation and/or chemotherapy treatment, as occurred with our patient.2,9
Special therapeutic considerations Modern biochemical and genetic characterization of NSCLC has yielded an insight into the oncogenes involved in the pathogenesis of these neoplasms. The oncogenes most commonly associated with NSCLC are EGFR and Kras.5,6 Both of these proteins are involved in cellular survival and proliferation pathways.5,21 Identification of mutant variants in these genes has stimulated research for the development of genetically tailored targeted therapy. An estimated 16.7% of patients with NSCLC have an activating mutation in EGFR; this rate is higher in patients with adenocarcinoma subtype (49%).22,23 In general, the mutations occur in exclusion of one another.5,21 Overexpression, dys-
regulation, and activated mutations of EGFR result in neoplastic proliferation.24 Strong associations exist between mutations in EGFR and a history of never-smokers, female, and adenocarcinoma.5,23 Chemotherapeutic drugs have been formulated to inhibit EGFR activated pathways. The response rate to these EGFR tyrosine kinase inhibitors (EGFR TKIs) is strongly dependent on the EGFR mutation variant. In sensitive mutations such as the exon-19 deletion and exon-21 L858R point mutation that make up 90% of all EGFR mutation patients, the response rate to the EGFR TKIs is approximately 67%.25,26 In randomized trials, these agents are superior to standard chemotherapy in patients with EGFR mutation.27 K-ras is a downstream effector of EGFR, and activating mutations in K-ras are found in 21–43% of individuals with NSCLC.28 Mutations in K-ras constitutively activated the downstream cascades leading to neoplastic proliferation.29 Similar to EGFR, K-ras mutations are more likely to be found in adenocarcinoma.5 However, there is a stronger association between the presence of K-ras mutation and a past history of smoking.5 Because K-ras is downstream to EGFR, inhibitors to EGFR are often insufficient to downregulate K-ras mediated pathways and thus individuals with a K-ras mutation have a poor prognosis for survival.21,30 Our patient was negative for an EGFR mutation by polymerase chain reaction (PCR) analysis. K-ras mutation assessment was not available to our institution. However, he experienced significant disease progression with formation of new lesions and an enlargement of existing lesions despite an initial positive response to cisplatin and vinorelbine. Based on our patient’s clinical history and histopathological findings, we felt that it was prudent to explore the presence of a mutation in another less frequent oncogene, ALK. ALK’s most frequent translocation partner is EML4 in NSCLC, forming an EML4-ALK complex.31 This rearrangement mutation is present in approximately 5% of NSCLC and is more likely to occur in adenocarcinoma.8 Lung adenocarcinomas with the signet ring component and mucinous subtype have been shown to be strongly associated with ALK translocation.32–34 An ALK gene rearrangement also is more frequent in younger individuals and in never-smokers.33 For our patient, the signet ring morphology was initially revealed in the choroidal lesion following enucleation. Reassessment of the original lung FNAB and mediastinal lymph nodes similarly found signet ring cells (Fig. 2G and H). Given that our patient fit into all these associative demographics, and that proven target therapy exists, the patient was tested and found to be positive for EML4-ALK gene recombination (Fig. 3). Based on this result, his treatment was switched to crizotinib, which is currently the only approved ALK tyrosine kinase inhibitor in North America. For patients with ALK gene rearrangement, 2-year survival rates improved from 12% to 55% with crizotinib treatment.8,35
Conclusion Our case highlights the importance of involving ophthalmologists in a collaborative fashion in the ongoing systemic care of cancer patients, as choroidal metastasis may be the first sign of a relapse of a known primary malignant neoplasm or the initial presentation of an unknown primary. For our patient, the histopathological findings assisted in the identifica-
192 tion of the mutation, which allowed him to receive targeted and tailored therapy. With the recent stride in personalized medicine specific histopathological diagnoses are of paramount importance to guide targeted therapies for better patient care management.
Conflict of interest The authors declared that there is no conflict of interest.
References 1. Su HT, Chen YM, Perng RP. Symptomatic ocular metastases in lung cancer. Respirology (Carlton, Vic) 2008;13:303–5. 2. Kanthan GL, Jayamohan J, Yip D, Conway RM. Management of metastatic carcinoma of the uveal tract: an evidence-based analysis. Clin Exp Ophthalmol 2007;35:553–65. 3. Shields CL, Shields JA, Gross NE, Schwartz GP, Lally SE. Survey of 520 eyes with uveal metastases. Ophthalmology 1997;104:1265–76. 4. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010;127:2893–917. 5. Subramanian J, Govindan R. Lung cancer in never smokers: a review. J Clin Oncol 2007;25:561–70. 6. Chowdhuri SR, Xi L, Pham TH, et al. EGFR and KRAS mutation analysis in cytologic samples of lung adenocarcinoma enabled by laser capture microdissection. Modern Pathol 2012;25:548–55. 7. Hoffman PC, Mauer AM, Vokes EE. Lung cancer. Lancet 2000;355:479–85. 8. Shaw AT, Kim DW, Nakagawa K, et al. Crizotinib versus chemotherapy in advanced ALK-positive lung cancer. New Eng J Med 2013;368:2385–94. 9. Singh N, Kulkarni P, Aggarwal AN, et al. Choroidal metastasis as a presenting manifestation of lung cancer: a report of 3 cases and systematic review of the literature. Medicine 2012;91:179–94. 10. Singh P, Singh A. Choroidal melanoma. Oman J Ophthalmol 2012;5:3–9. 11. Frenkel S, Pe’er J. Choroidal metastasis of adenocarcinoma of the lung presenting as pigmented choroidal tumor. Case Reports Ophthalmol 2012;3:311–6. 12. Verbeek AM. Differential diagnosis of intraocular neoplasms with ultrasonography. Ultrasound Med Biol 1985;11:163–70. 13. Natesh S, Chin KJ, Finger PT. Choroidal metastases fundus autofluorescence imaging: correlation to clinical, OCT, and fluorescein angiographic findings. Ophthalmic Surg Lasers & Imaging 2010;41:406–12. 14. Hou J, Aerts J, den Hamer B, et al. Gene expression-based classification of non-small cell lung carcinomas and survival prediction. PLoS ONE 2010;5:e10312. 15. Akunuru S, Palumbo J, Zhai QJ, Zheng Y. Rac1 targeting suppresses human non-small cell lung adenocarcinoma cancer stem cell activity. PLoS ONE 2011;6:e16951. 16. Charloux A, Quoix E, Wolkove N, Small D, Pauli G, Kreisman H. The increasing incidence of lung adenocarcinoma: reality or artefact? A review of the epidemiology of lung adenocarcinoma. Int J Epidemiol 1997;26:14–23. 17. Hayabuchi N, Russell WJ, Murakami J. Slow-growing lung cancer in a fixed population sample. Radiologic assessments. Cancer 1983;52:1098–104. 18. Travis WD, Brambilla E, Muller-Hermelink KH, Harris CC. World health organization classification of tumours. Pathology and genetics of tumours of the lung pleura thymus and heart. Lyon: IARC Press; 2004.
K. Jiang et al. 19. de la Barquera Cordero AS, Hidalgo RA. Intravitreal bevacizumab for choroidal metastasis of lung carcinoma; a case report. J Ophthalmic Vision Res 2010;5:265–8. 20. George B, Wirostko WJ, Connor TB, Choong NW. Complete and durable response of choroid metastasis from non-small cell lung cancer with systemic bevacizumab and chemotherapy. J Thorac Oncol 2009;4:661–2. 21. Schmid K, Oehl N, Wrba F, Pirker R, Pirker C, Filipits M. EGFR/KRAS/ BRAF mutations in primary lung adenocarcinomas and corresponding locoregional lymph node metastases. Clin Cancer Res 2009;15:4554–60. 22. Wu YL, Zhong WZ, Li LY, et al. Epidermal growth factor receptor mutations and their correlation with gefitinib therapy in patients with non-small cell lung cancer: a meta-analysis based on updated individual patient data from six medical centers in mainland China. J Thorac Oncol 2007;2:430–9. 23. Bell DW, Brannigan BW, Matsuo K, et al. Increased prevalence of EGFR-mutant lung cancer in women and in East Asian populations: analysis of estrogen-related polymorphisms. Clin Cancer Res 2008;14:4079–84. 24. Grunwald V, Hidalgo M. Developing inhibitors of the epidermal growth factor receptor for cancer treatment. J Natl Cancer Inst 2003;95:851–67. 25. Jackman DM, Miller VA, Cioffredi LA, et al. Impact of epidermal growth factor receptor and KRAS mutations on clinical outcomes in previously untreated non-small cell lung cancer patients: results of an online tumor registry of clinical trials. Clin Cancer Res 2009;15:5267–73. 26. 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;12:169–76. 27. Mok TS, Wu YL, Thongprasert S, et al. Gefitinib or carboplatinpaclitaxel in pulmonary adenocarcinoma. New Eng J Med 2009;361:947–57. 28. Zinsky R, Bolukbas S, Bartsch H, Schirren J, Fisseler-Eckhoff A. Analysis of KRAS mutations of Exon 2 Codons 12 and 13 by snapshot analysis in comparison to common DNA sequencing. Gastroenterol Res Pract 2010;2010:789363. 29. Marchetti A, Milella M, Felicioni L, et al. Clinical implications of KRAS mutations in lung cancer patients treated with tyrosine kinase inhibitors: an important role for mutations in minor clones. Neoplasia (New York, NY) 2009;11:1084–92. 30. Guan JL, Zhong WZ, An SJ, et al. KRAS mutation in patients with lung cancer: a predictor for poor prognosis but not for EGFR-TKIs or chemotherapy. Ann Surg Oncol 2013;20:1381–8. 31. Sasaki T, Rodig SJ, Chirieac LR, Janne PA. The biology and treatment of EML4-ALK non-small cell lung cancer. Eur J Cancer (Oxford, England: 1990) 2010;46:1773–80. 32. Ohba T, Sugio K, Kometani T, et al. Signet ring cell adenocarcinoma of the lung with an EML4-ALK fusion gene mimicking mucinous (colloid) adenocarcinoma: a case report. Lung Cancer (Amsterdam, Netherlands) 2011;73:375–8. 33. Popat S, Gonzalez D, Min T, et al. ALK translocation is associated with ALK immunoreactivity and extensive signet-ring morphology in primary lung adenocarcinoma. Lung Cancer (Amsterdam, Netherlands) 2012;75:300–5. 34. Thunnissen E, Bubendorf L, Dietel M, et al. EML4-ALK testing in nonsmall cell carcinomas of the lung: a review with recommendations. Virchows Arch 2012;461:245–57. 35. 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. Lancet Oncol 2011;12:1004–12.