Please cite this article in press as: Just P-A, et al. Histologic subtypes, immunohistochemistry, FISH or molecular screening for the accurate diagnosis of ALK-rearrangement in lung cancer: A comprehensive study of Caucasian non-smokers. Lung Cancer (2011), doi:10.1016/j.lungcan.2011.11.004 ARTICLE IN PRESS G Model LUNG-3963; No. of Pages 7 Lung Cancer xxx (2011) xxx–xxx Contents lists available at SciVerse ScienceDirect Lung Cancer j our na l ho me p age: www.elsevier.com/locate/lungcan Histologic subtypes, immunohistochemistry, FISH or molecular screening for the accurate diagnosis of ALK-rearrangement in lung cancer: A comprehensive study of Caucasian non-smokers Pierre-Alexandre Just a,b , Aurélie Cazes c , Anne Audebourg a , Anatole Cessot d , Karine Pallier d , Claire Danel c , Marie-Cécile Vacher-Lavenu a , Pierre Laurent-Puig b,d , Benoît Terris a , Hélène Blons b,d,∗ a APHP, Université Paris Descartes, Groupe Hospitalier Cochin-Broca-Hôtel Dieu, Service d’anatomie et de cytologie pathologiques, Paris, F-75014, France b APHP, Hôpital européen Georges Pompidou, Service de biochimie, Paris, F-75015, France c APHP, Hôpital européen Georges Pompidou, Service d’anatomie et de cytologie pathologiques, Paris, F-75015, France d Université Paris Descartes, Sorbonne Paris Cité; INSERM UMR-S775 Molecular Basis of Response to Xenobiotics, Paris, F-75006, France a r t i c l e i n f o Article history: Received 28 September 2011 Received in revised form 3 November 2011 Accepted 5 November 2011 Keywords: Non-small cell lung carcinoma EML4-ALK fusion protein Reverse transcriptase polymerase chain reaction Quantitative RT-PCR Fluorescent in situ hybridization Immunohistochemistry Pathology a b s t r a c t EML4-ALK adenocarcinomas constitute a new molecular subgroup of lung tumours that respond very well to crizotinib, an ALK inhibitor. However, the diagnosis of ALK rearrangement in lung cancer is challenging. The aim of this study was to compare the diagnostic accuracy of five different methods in a series of 20 EGFR wt/wt lung adenocarcinomas from non- or light- smokers. Multiplex RT-PCR was considered as gold standard and identified four ALK-rearranged tumours among the 20 tested tumours. qRT-PCR got an interpretability rate of 100% and accurately typed all 20 tumours. qRT-PCR from corresponding formalin- fixed paraffin-embedded (FFPE) specimens got an interpretability rate of 65%. Out of the four previously identified ALK-rearranged cases, three were interpretable and two were retrieved using FFPE qRT-PCR. ALK break-apart FISH got an interpretability rate of 60% and accurately typed all of the twelve remaining cases. Anti-ALK immunohistochemistry (IHC) accurately typed all twenty tumours using a cut-off value of strong staining of 100% tumour cells. The 16 non ALK-rearranged tumours got no/light staining in 13 cases, and a moderate staining of 80–100% tumour cells in 3 cases. We then analysed four solid signet-ring lung adenocarcinomas. FFPE qRT-PCR, FISH and immunohistochemistry were concordant in three cases, with positive and negative results in respectively one and two cases. The fourth case, which was positive by FISH and immunohistochemistry but negative by RT-PCR, was shown to have a non-EML4-ALK ALK- rearrangement. As various factors such as RNA quality, fixation quality and type of ALK rearrangement may impede ALK screening, we propose a combined FISH/molecular biology diagnostic algorithm in which anti-ALK immunohistochemistry is used as a pre-screening step. © 2011 Elsevier Ireland Ltd. All rights reserved. 1. Introduction In 2007, anaplastic lymphoma kinase (ALK) was shown to be involved in the oncogenesis [1,2] of a small subset (∼4%) [3] of lung carcinomas. In these tumours, a small inversion within the short arm of chromosome 2 leads to an open reading frame that links the first exons of EML4 to the 3 ′ part of ALK (from exon 20) [2]. EML4-ALK tumours, which are now considered as a new molecular subgroup of lung tumours, are almost exclusively adenocarcinomas, without mutation of EGFR or KRAS, and preferentially arise in non-smokers [3]. Crizotinib, a small orally dispensable ALK inhibitor, is currently ∗ Corresponding author at: Hôpital européen Georges Pompidou, Service de biochimie, 20 rue Leblanc, F-75908 Paris cedex 15, France. Tel.: +33 1 56 09 38 82; fax: +33 1 56 09 33 93. E-mail address: helene.blons@egp.aphp.fr (H. Blons). tested in a phase III clinical trial and the preliminary results of the phase II trial evidenced a surprising 90% disease control rate in ALK- rearranged lung tumours [4]. Nevertheless, if crizotinib confirms its high therapeutic efficiency, it is likely that its use will be restricted to tumours with a proven ALK rearrangement. However, assessing ALK rearrangement in lung tumours remains a diagnostic challenge [3]. In the literature, reverse transcriptase polymerase chain reaction (RT-PCR) of the EML4-ALK transcripts [5] appeared to be a sensitive and specific tool. However, thirteen variants of EML4-ALK have been already described according to the break point on EML4 (from exon 2 to exon 20) [3]. Furthermore, beside EML4, TFG [1] and KIF5B [6,7] have also been described to be fused to ALK in rare cases. Thus, RT-PCR has to be multiplexed, i.e. several forward primers on EML4 (± on other fusion partners) are required in order to target all of the potential fusion vari- ants of ALK [5,6]. As some EML4-ALK RT-PCR amplicons size more than 1000 bp, the diagnostic relevance of RT-PCR directly relies 0169-5002/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2011.11.004