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The echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) gene fusion occurs in 2–7% of NSCLC cases[1,2] and is typically identified by Vysis ALK Break-Apart fluorescent in situ hybridization (FISH) assay. Tumors expressing this fusion respond to treatment crizotinib.
A 43 year-old never-smoker male presented with pericardial tamponade. Pericardio/thoracocentesis yielded 2.5 liters of fluid; light microscopy was consistent with lung adenocarcinoma. No epidermal growth factor receptor (EGFR) mutation or EML4-ALK rearrangement was detected.
Following talc pleurodesis, he was treated with 4 cycles of cisplatin/pemetrexed, however experienced a progression.
A specimen from the tumor was sent for comprehensive genomic profiling using a Next Generation Sequencing (NGS) assay in a CLIA laboratory (Foundation Medicine), identifying a complex ALK rearrangement. Several steps were then undertaken to better characterize 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 5 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 that multiple transcript products not readily identifiable existed.
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, explaining 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 39-fold over-expression of exons 20–29 of ALK relative to exons 1–19 (Figure 2).
ALK immunohistochemistry (IHC) was positive as displayed in Figure 1. Based on 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 CT-PET scans and after 4 months, the PET was negative and the chest CT scan showed further shrinkage of the primary lesion (RECIST 75%; Figure 3).
Many ALK rearrangements have been described in NSCLC. Most of these fusion variants are comprised of 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.
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.
Conflict of Interest
NP, MI – nothing to declare.
GAO, GP, VM, DL, PJS, and MTC are employees and equity holders of Foundation Medicine
FRH: Consultant (advisory board) for Pfizer (compensated) and research agreement through University of Colorado with Ventana/Roche.
LSG: Employee of Oncotest-Teva Pharmaceutical Industries, Petach Tikva, Israel