Anaplastic lymphoma kinase (ALK) gene rearrangement in non-small cell lung cancer (NSCLC): Results of a multi-centre ALK-testing
Introduction
Lung cancer is the leading cause of death by cancer worldwide [1]. As less than 15% of the patients present with a resectable tumor at the time of diagnosis, prognosis remains dramatically poor [2], [3]. In the last years, strategies to improve personalized medicine focused on the analysis of the epidermal growth factor receptor (EGFR), which is associated with activating mutations of the tyrosine kinase (TK) domain in about 15% of the adenocarcinomas of the lung. In order to block this TK activity, inhibitors (gefitinib, erlotinib) have been developed and successfully applied in patients with activating TK mutations [4], [5]. More recently, Soda et al. [6] showed that genomic breaks in the gene of anaplastic lymphoma kinase (ALK) are detectable in a subset of ∼4% (range: 0.9–13%) [6], [7], [8], [9], [10], [11] of non-small cell lung cancer (NSCLC). In these tumors, a small paracentric inversion within the short arm of chromosome 2 leads to a fusion gene comprising EML4 and ALK resulting in a consecutive expression of the ALK kinase domain [6]. Whereas the fused ALK gene region remains constant (exon 20), different EML4 exons can be affected. In addition, other fusion partners than EML4 have been described [8], [12], [13], pointing out to a new molecular subgroup [14] of NSCLC (ALK-rearranged) which consists almost exclusively of adenocarcinomas without mutations of EGFR or KRAS, and preferentially arises in non-smokers [8], [15], [16]. Crizotinib, a small orally applicable ALK inhibitor has shown promising disease control rates in NSCLC patients with rearranged ALK-genes and significantly improved survival rates compared to crizotinib naive controls [17], [18]. Therefore this inhibitor has received approval by the American Food and Drug Administration (FDA) in August 2011 and the European Medicines Agency (EMA) in October 2012 for the treatment of patients with locally advanced or metastatic ALK-positive NSCLC. Remarkably, in America a FDA-approved ALK-FISH assay is required whereas the EMA refers to ALK-positivity without stipulation of a certain ALK test. Therefore, the reliability of any kind of ALK testing in Europe has to be approved prior to therapy with ALK inhibitors. To ensure this reliability, round robin tests are indispensable to identify ALK testing most suitable for this purpose. To this end, we selected 10 ALK pre-tested NSCLC-cases, which were subsequently re-evaluated by an expert panel (8 institutes of pathology) prior to the roll-out of an open round robin test. All 10 cases were tested for ALK-breaks by FISH and for ALK-expression by immunohistochemistry (IHC) by all participants: IHC produced almost concordant results in ALK-break negative cases, whereas tumors with ALK-breaks revealed very inhomogeneous results by means of IHC. ALK-FISH testing led to very consistent data – irrespective of the FISH probe applied – with the exception of one case with a low number of affected tumor cells. This discrepant FISH case was omitted from the nation (German)-wide round robin test. ALK-IHC, which revealed heterogeneous results in ALK-break positive cases was included in the round robin test only as an optional test and not used for validation.
Section snippets
Case selection
A pre-selection was performed by the Institute of Pathology (Charité) to identify cases with ALK-breaks involving tumor samples from the Institutes of Pathology Charité Berlin, University Hospital Heidelberg, Berlin-Buch, Vivantes Hospital Berlin, Hamburg-West and Minden (all from Germany). Finally 10 NSCLC cases were selected, 5 of them displayed ALK-breaks (4 with predominantly split signals [SS] and 1 with predominantly single red signals [SRS]) based on the institutional pre-screening. All
Data of quality control
The aim of our study was to allow the evaluation of at least three tissue cores per case for each participant. Therefore all participants received 4 consecutive TMA-derived tissue sections (total number 32 sections) in order to allow potential repetition of FISH or IHC analysis. Five participants performed IHC once whereas three participants repeated ALK IHC-detection (11 slides). Reported IHC-results were finally based on 234/240 evaluable tissue cores. FISH was performed more than once by all
Discussion
ALK tyrosine kinase inhibitors (TKI) as crizotinib show promising therapeutic effects in ALK-gene rearranged NSCLCs [17], [18]. With its approval in Europe (October 2012), the reliable identification of NSCLC-patients with ALK-alterations is of urgent priority. Currently, the method of choice for the detection of ALK breaks in NSCLC is FISH. Immunohistochemistry for the detection of ALK protein expression is – in contrast to anaplastic large cell lymphoma (ALCL) – not sufficiently reliable most
Conclusion
In order to identify cases with unequivocal ALK-FISH data, an internal quality control round involving 8 experienced diagnostic centers was performed prior to the initiation of a German-wide round robin test in the frame of the QuiP initiative. Based on the results of this internal quality control round, the following criteria for the definition of an ALK-positive cases have been established for the German-wide test: (i) a distance of significantly more than one FISH signal between a given 5′
Conflict of interest statement
One expert panel meeting and the preparation of the study was supported by PFIZER within the framework of the FALKE-project (Fusion of EML4-ALK epidemiol-ogy Evaluation).
Acknowledgement
One expert panel meeting and the preparation of the study was supported by PFIZER within the framework of the FALKE-project (Fusion of EML4-ALK epidemiology Evaluation).
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2017, European Journal of Medicinal ChemistryCitation Excerpt :In 2007, echinoderm microtubule-associated protein-like-4 (EML4)-ALK, found in over 6% of non-small cell lung cancers (NSCLC), was recognized as a promising oncogenic drug target [2]. Although the specific physiological function of ALK in cancer remains unclear, ALK fusion protein has been found in various human cancers [3], such as breast, colorectal, inflammatory myofibroblastic tumors (IMT), diffuse large B-cell lymphoma (DLBCL), and most notably in 2–7% of NSCLC and 70% of ALCL [4]. Moreover, it has been reported that the amplification or point mutations of ALK is involved in the development of neuroblastoma (NB), anaplastic thyroid cancer, and ovarian cancer [5].