Next-Generation Sequencing Identifies and Immunohistochemistry Confirms a Novel Crizotinib-Sensitive ALK Rearrangement in a Patient with Metastatic Non–Small-Cell Lung Cancer
Authors: Nir Peled, Gary Palmer, Fred R. Hirsch, Murry W. Wynes, Maya Ilouze, Marileila Varella-Garcia, Lior Soussan-Gutman, Geoff A. Otto, Philip J. Stephens, Jeffrey S. Ross, Maureen T. Cronin, Doron Lipson, Vincent A. Miller
Journal: Journal of Thoracic Oncology (2012)

The echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) gene fusion occurs in 2% to 7% of non–small-cell lung cancer (NSCLC) cases1Kwak EL Bang YJ Camidge DR et al.Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer.N Engl J Med. 2010; 363: 1693-1703Crossref PubMed Scopus (3812) Google Scholar,2Shaw AT Yeap BY Mino-Kenudson M et al.Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK.J Clin Oncol. 2009; 27: 4247-4253Crossref PubMed Scopus (1632) Google Scholar and is typically identified by Vysis ALK Break-Apart fluorescent in situ hybridization (FISH) assay. Tumors expressing this fusion respond to treatment using crizotinib.1Kwak EL Bang YJ Camidge DR et al.Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer.N Engl J Med. 2010; 363: 1693-1703Crossref PubMed Scopus (3812) Google Scholar A 43-year-old male never-smoker presented with pericardial tamponade. Pericardio/thoracocentesis yielded 2.5 liters of fluid; light microscopy was consistent with lung adenocarcinoma. No epidermal growth factor receptor mutation or EML4-ALK rearrangement was detected. After talc pleurodesis, he was treated with four cycles of cisplatin/pemetrexed. However, he experienced a progression. A specimen from the tumor was sent for comprehensive genomic profiling using a next-generation sequencing (NGS) assay in a Clinical Laboratory Improvement Amendments laboratory (Foundation Medicine), identifying a complex ALK rearrangement. Several steps were then undertaken to characterize better the genomic abnormality. FISH testing was negative using standard criteria (Figure 1). NGS analysis of genomic DNA revealed a complex rearrangement of ALK, involving breakpoints in at least five different genomic loci. One of the breakpoints was in ALK intron 19, the canonical breakpoint for EML4-ALK fusions. No clear fusion product was predicted; we suspected the existence of multiple transcript products that were not readily identifiable. We thus sequenced cDNA from the same sample to study, both, the expression level of ALK and the structure of any potential fusion product. The cDNA sequence demonstrated that the expressed product of the complex rearrangement was the canonical EML4-ALK fusion gene (EML4 exons 1–13, ALK exons 20–29). We hypothesized that at the genomic level the fused EML4 and ALK genes were separated by other small genomic shards; this explained why the EML4-ALK fusion was not detected by FISH. This connective region is likely to be removed during splicing and results in a functional EML4-ALK transcript. We next showed a 39-fold overexpression of exons 20–29 of ALK relative to exons 1–19 (Figure 2). ALK immunohistochemistry was positive, as displayed in Figure 1. On the basis of these results, crizotinib was begun. Within 2 weeks, the patient reported an improvement in pain in the pubic area and in exercise tolerance. Significant improvement was seen on initial computed tomography and positron emission tomography scans, and after 4 months, the positron emission tomography scan was negative and the chest computed tomography scan showed further shrinkage of the primary lesion (Response Evaluation Criteria In Solid Tumors 75%; Figure 3). Approximately 3% to 7% of lung tumors harbor ALK fusions 1Kwak EL Bang YJ Camidge DR et al.Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer.N Engl J Med. 2010; 363: 1693-1703Crossref PubMed Scopus (3812) Google Scholar, 2Shaw AT Yeap BY Mino-Kenudson M et al.Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK.J Clin Oncol. 2009; 27: 4247-4253Crossref PubMed Scopus (1632) Google Scholar, 3Rikova K Guo A Zeng Q et al.Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer.Cell. 2007; 131: 1190-1203Abstract Full Text Full Text PDF PubMed Scopus (1915) Google Scholar, 4Soda M Choi YL Enomoto M et al.Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer.Nature. 2007; 448: 561-566Crossref PubMed Scopus (4304) Google Scholar, 5Lipson D Capelletti M Yelensky R et al.Identification of new ALK and RET gene fusions from colorectal and lung cancer biopsies.Nat Med. 2012; 18: 382-386Crossref PubMed Scopus (714) Google Scholar, 6Wong DW Leung EL So KK et al.The EML4-ALK fusion gene is involved in various histologic types of lung cancers from nonsmokers with wild-type EGFR and KRAS.Cancer. 2009; 115: 1723-1733Crossref PubMed Scopus (640) Google Scholar ALK fusions are more commonly found in light smokers (<10 pack-years) and/or never smokers.1Kwak EL Bang YJ Camidge DR et al.Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer.N Engl J Med. 2010; 363: 1693-1703Crossref PubMed Scopus (3812) Google Scholar,2Shaw AT Yeap BY Mino-Kenudson M et al.Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK.J Clin Oncol. 2009; 27: 4247-4253Crossref PubMed Scopus (1632) Google Scholar,4Soda M Choi YL Enomoto M et al.Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer.Nature. 2007; 448: 561-566Crossref PubMed Scopus (4304) Google Scholar,6Wong DW Leung EL So KK et al.The EML4-ALK fusion gene is involved in various histologic types of lung cancers from nonsmokers with wild-type EGFR and KRAS.Cancer. 2009; 115: 1723-1733Crossref PubMed Scopus (640) Google Scholar,7Inamura K Takeuchi K Togashi Y et al.EML4-ALK lung cancers are characterized by rare other mutations, a TTF-1 cell lineage, an acinar histology, and young onset.Mod Pathol. 2009; 22: 508-515Crossref PubMed Scopus (401) Google Scholar ALK fusions are also associated with younger age 2Shaw AT Yeap BY Mino-Kenudson M et al.Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK.J Clin Oncol. 2009; 27: 4247-4253Crossref PubMed Scopus (1632) Google Scholar,6Wong DW Leung EL So KK et al.The EML4-ALK fusion gene is involved in various histologic types of lung cancers from nonsmokers with wild-type EGFR and KRAS.Cancer. 2009; 115: 1723-1733Crossref PubMed Scopus (640) Google Scholar,7Inamura K Takeuchi K Togashi Y et al.EML4-ALK lung cancers are characterized by rare other mutations, a TTF-1 cell lineage, an acinar histology, and young onset.Mod Pathol. 2009; 22: 508-515Crossref PubMed Scopus (401) Google Scholar and adenocarcinomas with acinar histology6Wong DW Leung EL So KK et al.The EML4-ALK fusion gene is involved in various histologic types of lung cancers from nonsmokers with wild-type EGFR and KRAS.Cancer. 2009; 115: 1723-1733Crossref PubMed Scopus (640) Google Scholar,7Inamura K Takeuchi K Togashi Y et al.EML4-ALK lung cancers are characterized by rare other mutations, a TTF-1 cell lineage, an acinar histology, and young onset.Mod Pathol. 2009; 22: 508-515Crossref PubMed Scopus (401) Google Scholar or signet-ring cells.2Shaw AT Yeap BY Mino-Kenudson M et al.Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK.J Clin Oncol. 2009; 27: 4247-4253Crossref PubMed Scopus (1632) Google Scholar Recent studies demonstrate that the frequency EML4-ALK fusion transcript is detected in 2% to 7% of lung adenocarcinomas1Kwak EL Bang YJ Camidge DR et al.Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer.N Engl J Med. 2010; 363: 1693-1703Crossref PubMed Scopus (3812) Google Scholar,2Shaw AT Yeap BY Mino-Kenudson M et al.Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK.J Clin Oncol. 2009; 27: 4247-4253Crossref PubMed Scopus (1632) Google Scholar; these tumors are sensitive to crizotinib. Many ALK rearrangements have been described in NSCLC. Most of these fusion variants comprised various portions of the EML4 gene fused with a consistent portion of the ALK gene. In addition, non-EML4 fusion partners have also been identified, including KIF5B and TFG.3Rikova K Guo A Zeng Q et al.Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer.Cell. 2007; 131: 1190-1203Abstract Full Text Full Text PDF PubMed Scopus (1915) Google Scholar The novel rearrangement identified in this case is complex and was not detected by the Break-Apart FISH assay. NGS showed that the patient's tumor harbored a complex EML4-ALK rearrangement at the genomic level. Clinical and radiographic evidence confirmed a rapid response to crizotinib. NGS should be considered in NSCLC patients with high likelihood of a driver kinase alteration when none is identified by other methods.