There are several challenges in lung cytology including equivocal cytological findings, lung cancer subclassification, and predictive marker analysis. Fluorescence in situ hybridization (FISH) detects chromosomal alterations that underlie the development and progression of cancer, and pinpoints targets of new anticancer drugs. Detection of such targets by FISH can be of diagnostic utility in morphologically difficult cases. Multitarget FISH with four different chromosomal probes improves the sensitivity of cytology in the diagnosis of lung cancer and clarifies equivocal cytological findings. FISH is becoming increasingly important in the field of predictive marker analysis. FISH is the gold standard to identify ALK rearrangements for treatment with the ALK inhibitor crizotinib (Xalkori). EGFR mutation analysis is the method of choice for selecting patients for therapy with EGFR tyrosine kinase inhibitors (TKIs), whereas the EGFR gene copy number has not been confirmed as a reliable predictive marker. MET amplification is an important mechanism of secondary resistance to EGFR TKIs and a candidate predictive marker for targeted second-line treatment of TKI-resistant non-squamous non-small cell lung cancer. FGFR1 amplification is a promising new marker in squamous cell carcinoma that may predict the response to FGFR1 inhibitors. In conclusion, there are an increasing number of clinically relevant FISH applications in lung cytology. We provide an overview on the current role of FISH in respiratory cytology.

Keywords:
Cytology, Cytoblocks, Lung cancer, Fluorescence in situ hybridization, ALK, EGFR
1.
Halling KC, Kipp BR: Fluorescence in situ hybridization in diagnostic cytology. Hum Pathol 2007;38:1137–1144.
2.
Bubendorf L, Grote HJ, Syriänen K: Molecular techniques; in Bibbo M, Wilbur D (eds): Comprehensive Cytopathology. London, Saunders Elsevier, 2008.
3.
Romeo MS, Sokolova IA, Morrison LE, Zeng C, Baron AE, Hirsch FR, Miller YE, Franklin WA, Varella-Garcia M: Chromosomal abnormalities in non-small cell lung carcinomas and in bronchial epithelia of high-risk smokers detected by multi-target interphase fluorescence in situ hybridization. J Mol Diagn 2003;5:103–112.
4.
Bubendorf L, Muller P, Joos L, Grilli B, Vogel S, Herzog M, Barascud A, Feichter G, Dalquen P, Tamm M: Multitarget FISH analysis in the diagnosis of lung cancer. Am J Clin Pathol 2005;123:516–523.
5.
Voss JS, Kipp BR, Halling KC, Henry MR, Jett JR, Clayton AC, Rickman OB: Fluorescence in situ hybridization testing algorithm improves lung cancer detection in bronchial brushing specimens. Am J Respir Crit Care Med 2010;181:478–485.
6.
Halling KC, Rickman OB, Kipp BR, Harwood AR, Doerr CH, Jett JR: A comparison of cytology and fluorescence in situ hybridization for the detection of lung cancer in bronchoscopic specimens. Chest 2006;130:694–701.
7.
Varella-Garcia M, Schulte AP, Wolf HJ, Feser WJ, Zeng C, Braudrick S, Yin X, Hirsch FR, Kennedy TC, Keith RL, Baron AE, Belinsky SA, Miller YE, Byers T, Franklin WA: The detection of chromosomal aneusomy by fluorescence in situ hybridization in sputum predicts lung cancer incidence. Cancer Prev Res (Phila) 2010;3:447–453.
8.
Gill RK, Vazquez MF, Kramer A, Hames M, Zhang L, Heselmeyer-Haddad K, Ried T, Shilo K, Henschke C, Yankelevitz D, Jen J: The use of genetic markers to identify lung cancer in fine needle aspiration samples. Clin Cancer Res 2008;14:7481–7487.
9.
Massion PP, Zou Y, Uner H, Kiatsimkul P, Wolf HJ, Baron AE, Byers T, Jonsson S, Lam S, Hirsch FR, Miller YE, Franklin WA, Varella-Garcia M: Recurrent genomic gains in preinvasive lesions as a biomarker of risk for lung cancer. PLoS One 2009;4:e5611.
10.
Glatz K, Savic S, Glatz D, Francz G, Barascud A, Grilli B, Herzog M, Dalquen P, Feichter G, Spieler P, Tamm M, Bubendorf L: An online quiz uncovers limitations of morphology in equivocal lung cytology. Cancer 2006;108:480–487.
11.
Savic S, Glatz K, Schoenegg R, Spieler P, Feichter G, Tamm M, Bubendorf L: Multitarget fluorescence in situ hybridization elucidates equivocal lung cytology. Chest 2006;129:1629–1635.
12.
Schramm M, Wrobel C, Born I, Kazimirek M, Pomjanski N, William M, Kappes R, Gerharz CD, Biesterfeld S, Bocking A: Equivocal cytology in lung cancer diagnosis: improvement of diagnostic accuracy using adjuvant multicolor fish, DNA-image cytometry, and quantitative promoter hypermethylation analysis. Cancer Cytopathol 2011;119:177–192.
13.
Guber A, Greif J, Rona R, Fireman E, Madi L, Kaplan T, Yemini Z, Gottfried M, Katz RL, Daniely M: Computerized analysis of cytology and fluorescence in situ hybridization (FISH) in induced sputum for lung cancer detection. Cancer Cytopathol 2010;118:269–277.
14.
Prindiville SA, Ried T: Interphase cytogenetics of sputum cells for the early detection of lung carcinogenesis. Cancer Prev Res (Phila) 2010;3:416–419.
15.
Field JK, Smith RA, Aberle DR, Oudkerk M, Baldwin DR, Yankelevitz D, Pedersen JH, Swanson SJ, Travis WD, Wisbuba II, Noguchi M, Mulshine JL: International Association for the Study of Lung Cancer Computed Tomography Screening Workshop 2011 report. J Thorac Oncol 2012;7:10–19.
16.
Eberhard DA, Giaccone G, Johnson BE: Biomarkers of response to epidermal growth factor receptor inhibitors in Non-Small-Cell Lung Cancer Working Group: standardization for use in the clinical trial setting. J Clin Oncol 2008;26:983–994.
17.
Popper H, Wrba F, Gruber-Mosenbacher U, Hulla W, Pirker R, Hilbe W, Studnicka M, Mohn-Staudner A, Ploner F: Histology-based algorithm in the molecular diagnosis of mutations of the epidermal growth factor receptor (EGFR) in non-small cell lung cancer (in German). Wien Klin Wochenschr 2011;123:316–321.
18.
Bruno P, Mariotta S, Ricci A, Duranti E, Scozzi D, Noto A, Mancini R, Giarnieri E, Giovagnoli MR: Reliability of direct sequencing of EGFR: comparison between cytological and histological samples from the same patient. Anticancer Res 2011;31:4207–4210.
19.
Thunnissen E, Kerr KM, Herth FJ, Lantuejoul S, Papotti M, Rintoul RC, Rossi G, Skov BG, Weynand B, Bubendorf L, Katrien G, Johansson L, Lopez-Rios F, Ninane V, Olszewski W, Popper H, Jaume S, Schnabel P, Thiberville L, Laenger F: The challenge of NSCLC diagnosis and predictive analysis on small samples: practical approach of a working group. Lung Cancer 2012;76:1–18.
20.
Travis WD, Brambilla E, Noguchi M, Nicholson AG, Geisinger KR, Yatabe Y, Beer DG, Powell CA, Riely GJ, Van Schil PE, Garg K, Austin JH, Asamura H, Rusch VW, Hirsch FR, Scagliotti G, Mitsudomi T, Huber RM, Ishikawa Y, Jett J, Sanchez-Cespedes M, Sculier JP, Takahashi T, Tsuboi M, Vansteenkiste J, Wistuba I, Yang PC, Aberle D, Brambilla C, Flieder D, Franklin W, Gazdar A, Gould M, Hasleton P, Henderson D, Johnson B, Johnson D, Kerr K, Kuriyama K, Lee JS, Miller VA, Petersen I, Roggli V, Rosell R, Saijo N, Thunnissen E, Tsao M, Yankelewitz D: International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society international multidisciplinary classification of lung adenocarcinoma. J Thorac Oncol 2011;6:244–285.
21.
Paik JH, Choi CM, Kim H, Jang SJ, Choe G, Kim DK, Kim HJ, Yoon H, Lee CT, Jheon S, Choe JY, Chung JH: Clinicopathologic implication of ALK rearrangement in surgically resected lung cancer a proposal of diagnostic algorithm for ALK-rearranged adenocarcinoma. Lung Cancer 2012;76:403–409.
22.
Fischer AH, Cibas ES, Howell LP, Kurian EM, Laucirica R, Moriarty AT, Renshaw AA, Zakowski MF, Young NA: Role of cytology in the management of non-small-cell lung cancer. J Clin Oncol 2011;29:3331–3332.
23.
Hasanovic A, Rekhtman N, Sigel CS, Moreira AL: Advances in fine needle aspiration cytology for the diagnosis of pulmonary carcinoma. Patholog Res Int 2011;2011:897292.
24.
Chowdhuri SR, Xi L, Pham TH, Hanson J, Rodriguez-Canales J, Berman A, Rajan A, Giaccone G, Emmert-Buck M, Raffeld M, Filie AC: EGFR and KRAS mutation analysis in cytologic samples of lung adenocarcinoma enabled by laser capture microdissection. Mod Pathol 2012;25:548–555.
25.
Savic S, Tapia C, Grilli B, Rufle A, Bihl MP, de Vito Barascud A, Herzog M, Terracciano L, Baty F, Bubendorf L: Comprehensive epidermal growth factor receptor gene analysis from cytological specimens of non-small-cell lung cancers. Br J Cancer 2008;98:154–160.
26.
Sasaki T, Janne PA: New strategies for treatment of ALK-rearranged non-small cell lung cancers. Clin Cancer Res 2011;17:7213–7218.
27.
Camidge DR, Kono SA, Flacco A, Tan AC, Doebele RC, Zhou Q, Crino L, Franklin WA, Varella-Garcia M: Optimizing the detection of lung cancer patients harboring anaplastic lymphoma kinase (ALK) gene rearrangements potentially suitable for ALK inhibitor treatment. Clin Cancer Res 2010;16:5581–5590.
28.
Bonanno L, Favaretto A, Rugge M, Taron M, Rosell R: Role of genotyping in non-small cell lung cancer treatment: current status. Drugs 2011;71:2231–2246.
29.
Sasaki T, Rodig SJ, Chirieac LR, Janne PA: The biology and treatment of EML4-ALK non-small cell lung cancer. Eur J Cancer 2010;46:1773–1780.
30.
Kwak EL, Bang YJ, Camidge DR, Shaw AT, Solomon B, Maki RG, Ou SH, Dezube BJ, Janne PA, Costa DB, Varella-Garcia M, Kim WH, Lynch TJ, Fidias P, Stubbs H, Engelman JA, Sequist LV, Tan W, Gandhi L, Mino-Kenudson M, Wei GC, Shreeve SM, Ratain MJ, Settleman J, Christensen JG, Haber DA, Wilner K, Salgia R, Shapiro GI, Clark JW, Iafrate AJ: Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med 2010;363:1693–1703.
31.
Shaw AT, Yasothan U, Kirkpatrick P: Crizotinib. Nat Rev Drug Discov 2011;10:897–898.
32.
Shaw AT, Yeap BY, Solomon BJ, Riely GJ, Gainor J, Engelman JA, Shapiro GI, Costa DB, Ou SH, Butaney M, Salgia R, Maki RG, Varella-Garcia M, Doebele RC, Bang YJ, Kulig K, Selaru P, Tang Y, Wilner KD, Kwak EL, Clark JW, Iafrate AJ, Camidge DR: 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;12:1004–1012.
33.
McLeer-Florin A, Moro-Sibilot D, Melis A, Salameire D, Lefebvre C, Ceccaldi F, de Fraipont F, Brambilla E, Lantuejoul S: Dual IHC and FISH testing for ALK gene rearrangement in lung adenocarcinomas in a routine practice: a French study. J Thorac Oncol 2012;7:348–354.
34.
Shaw AT, Solomon B, Kenudson MM: Crizotinib and testing for ALK. J Natl Compr Canc Netw 2011;9:1335–1341.
35.
Bergethon K, Shaw AT, Ou SH, Katayama R, Lovly CM, McDonald NT, Massion PP, Siwak-Tapp C, Gonzalez A, Fang R, Mark EJ, Batten JM, Chen H, Wilner KD, Kwak EL, Clark JW, Carbone DP, Ji H, Engelman JA, Mino-Kenudson M, Pao W, Iafrate AJ: ROS1 rearrangements define a unique molecular class of lung cancers. J Clin Oncol 2012;30:863–870.
36.
Fukuoka M, Wu YL, Thongprasert S, Sunpaweravong P, Leong SS, Sriuranpong V, Chao TY, Nakagawa K, Chu DT, Saijo N, Duffield EL, Rukazenkov Y, Speake G, Jiang H, Armour AA, To KF, Yang JC, Mok TS: 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;29:2866–2874.
37.
Cappuzzo F, Hirsch FR, Rossi E, Bartolini S, Ceresoli GL, Bemis L, Haney J, Witta S, Danenberg K, Domenichini I, Ludovini V, Magrini E, Gregorc V, Doglioni C, Sidoni A, Tonato M, Franklin WA, Crino L, Bunn PA Jr, Varella-Garcia M: Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-small-cell lung cancer. J Natl Cancer Inst 2005;97:643–655.
38.
Zhu CQ, da Cunha Santos G, Ding K, Sakurada A, Cutz JC, Liu N, Zhang T, Marrano P, Whitehead M, Squire JA, Kamel-Reid S, Seymour L, Shepherd FA, Tsao MS: Role of KRAS and EGFR as biomarkers of response to erlotinib in National Cancer Institute of Canada Clinical Trials Group Study BR.21. J Clin Oncol 2008;26:4268–4275.
39.
Varella-Garcia M, Diebold J, Eberhard DA, Geenen K, Hirschmann A, Kockx M, Nagelmeier I, Ruschoff J, Schmitt M, Arbogast S, Cappuzzo F: EGFR fluorescence in situ hybridisation assay: guidelines for application to non-small-cell lung cancer. J Clin Pathol 2009;62:970–977.
40.
Zlobec I, Raineri I, Schneider S, Schoenegg R, Grilli B, Herzog M, Savic S, Bubendorf L: Assessment of mean EGFR gene copy number is a highly reproducible method for evaluating FISH in histological and cytological cancer specimens. Lung Cancer 2010;68:192–197.
41.
Shepherd FA: Molecular selection trumps clinical selection. J Clin Oncol 2011;29:2843–2844.
42.
Khambata-Ford S, Harbison CT, Hart LL, Awad M, Xu LA, Horak CE, Dakhil S, Hermann RC, Lynch TJ, Weber MR: Analysis of potential predictive markers of cetuximab benefit in BMS099, a phase III study of cetuximab and first-line taxane/carboplatin in advanced non-small-cell lung cancer. J Clin Oncol 2010;28:918–927.
43.
Licitra L, Mesia R, Rivera F, Remenar E, Hitt R, Erfan J, Rottey S, Kawecki A, Zabolotnyy D, Benasso M, Storkel S, Senger S, Stroh C, Vermorken JB: Evaluation of EGFR gene copy number as a predictive biomarker for the efficacy of cetuximab in combination with chemotherapy in the first-line treatment of recurrent and/or metastatic squamous cell carcinoma of the head and neck: extreme study. Ann Oncol 2011;22:1078–1087.
44.
Janku F, Garrido-Laguna I, Petruzelka LB, Stewart DJ, Kurzrock R: Novel therapeutic targets in non-small cell lung cancer. J Thorac Oncol 2011;6:1601–1612.
45.
Cappuzzo F, Camidge DR, Varella-Garcia M: Is FISH floating or still swimming in the lung cancer ocean? Ann Oncol 2011;22:493–499.
46.
Pallis AG, Fennell DA, Szutowicz E, Leighl NB, Greillier L, Dziadziuszko R: Biomarkers of clinical benefit for anti-epidermal growth factor receptor agents in patients with non-small-cell lung cancer. Br J Cancer 2011;105:1–8.
47.
Ellis PM, Blais N, Soulieres D, Ionescu DN, Kashyap M, Liu G, Melosky B, Reiman T, Romeo P, Shepherd FA, Tsao MS, Leighl NB: A systematic review and canadian consensus recommendations on the use of biomarkers in the treatment of non-small cell lung cancer. J Thorac Oncol 2011;6:1379–1391.
48.
Li C, Sun Y, Fang R, Han X, Luo X, Wang R, Pan Y, Hu H, Zhang Y, Pao W, Shen L, Ji H, Chen H: Lung adenocarcinomas with HER2-activating mutations are associated with distinct clinical features and HER2/EGFR copy number gains. J Thorac Oncol 2012;7:85–89.
49.
Yap TA, Vidal L, Adam J, Stephens P, Spicer J, Shaw H, Ang J, Temple G, Bell S, Shahidi M, Uttenreuther-Fischer M, Stopfer P, Futreal A, Calvert H, de Bono JS, Plummer R: Phase I trial of the irreversible EGFR and HER2 kinase inhibitor BIBW 2992 in patients with advanced solid tumors. J Clin Oncol 2010;28:3965–3972.
50.
Ross HJ, Blumenschein GR Jr, Aisner J, Damjanov N, Dowlati A, Garst J, Rigas JR, Smylie M, Hassani H, Allen KE, Leopold L, Zaks TZ, Shepherd FA: Randomized phase II multicenter trial of two schedules of lapatinib as first- or second-line monotherapy in patients with advanced or metastatic non-small cell lung cancer. Clin Cancer Res 2010;16:1938–1949.
51.
Pao W, Chmielecki J: Rational, biologically based treatment of EGFR-mutant non-small-cell lung cancer. Nat Rev Cancer 2010;10:760–774.
52.
Spigel DR, Ervin TJ, Ramlau R, Daniel DB, Goldschmidt JH, Blumenschein GR, Krzakowski MJ, Robinet G, Clement-Duchene C, Barlesi F, Govindan R, Patel T, Orlov SV, Wertheim MS, Zha J, Pandita W, Yu W, Yauch RL, Patel PH, Peterson C: Final efficacy from OAM4558G, a randomized phase II study evaluating, MetMab or placebo in combination with erlotinib in advanced NSCLC (abstract). J Clin Oncol 2011;29:7505.
53.
Matsubara D, Ishikawa S, Oguni S, Aburatani H, Fukayama M, Niki T: Molecular predictors of sensitivity to the met inhibitor pha665752 in lung carcinoma cells. J Thorac Oncol 2010;5:1317–1324.
54.
Weiss J, Sos ML, Seidel D, Peifer M, Zander T, Heuckmann JM, Ullrich RT, Menon R, Maier S, Soltermann A, Moch H, Wagener P, Fischer F, Heynck S, Koker M, Schottle J, Leenders F, Gabler F, Dabow I, Querings S, Heukamp LC, Balke-Want H, Ansen S, Rauh D, Baessmann I, Altmuller J, Wainer Z, Conron M, Wright G, Russell P, Solomon B, Brambilla E, Brambilla C, Lorimier P, Sollberg S, Brustugun OT, Engel-Riedel W, Ludwig C, Petersen I, Sanger J, Clement J, Groen H, Timens W, Sietsma H, Thunnissen E, Smit E, Heideman D, Cappuzzo F, Ligorio C, Damiani S, Hallek M, Beroukhim R, Pao W, Klebl B, Baumann M, Buettner R, Ernestus K, Stoelben E, Wolf J, Nurnberg P, Perner S, Thomas RK: Frequent and focal FGFR1 amplification associates with therapeutically tractable FGFR1 dependency in squamous cell lung cancer. Sci Transl Med 2010;2:62ra93.
55.
Angulo B, Suarez-Gauthier A, Lopez-Rios F, Medina PP, Conde E, Tang M, Soler G, Lopez-Encuentra A, Cigudosa JC, Sanchez-Cespedes M: Expression signatures in lung cancer reveal a profile for EGFR-mutant tumours and identify selective PIK3CA overexpression by gene amplification. J Pathol 2008;214:347–356.
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