EML4-ALK fusion lung cancer: a rare acquired event

Abstract

A recurrent gene fusion between EML4 and ALK in 6.7% of non-small cell lung cancers (NSCLCs) and NKX2-1 (TTF1, TITF1) high-level amplifications in 12% of adenocarcinomas of the lung were independently reported recently. Because the EML4-ALK fusion was only shown by a reverse transcription-polymerase chain reaction approach, we developed fluorescent in situ hybridization assays to interrogate more than 600 NSCLCs using break-apart probes for EML4 and ALK. We found that EML4-ALK fusions occur in less than 3% of NSCLC samples and that EML4 and/or ALK amplifications also occur. We also observed that, in most cases in which an EML4/ALK alteration is detected, not all of the tumor cells harbor the lesion. By using a detailed multi-fluorescent in situ hybridization probe assay and reverse transcription-polymerase chain reaction, we have evidence that other, more common mechanisms besides gene inversion exist including the possibility of other fusion partners for ALK and EML4. Furthermore, we confirmed the NKX2-1 high-level amplification in a significant subset of NSCLC and found this amplification to be mutually exclusive to ALK and EML4 rearrangements.

Figure 1

Schematic design of the break-apart FISH assays and exemplary findings. (A) Schematic of the break-apart FISH assays for EML4 and ALK. (B) Loss of DNA on 2p outside the area encoding EML4 and ALK (exemplary for ALK: loss of the green-labeled probe telomeric of ALK). (C) Loss of interstitial DNA between EML4 and ALK (exemplary for ALK: loss of the red-labeled probe centromeric of ALK). (D) Break-apart of ALK (wild-type allele as one yellow, and one single red and green probe signal for the rearranged allele). (E) Cluster-like amplification of the ALK focus (multiple yellow signals).

Figure 2

RT-PCR results of selected cases that were found to be positive or negative for ELM4 and/or ALK rearrangement by FISH. Primers specific to the ELM4-ALK variant 1 fusion transcript (EML4-ALK, exon 6 of ELM4 and exon 20 of ALK1), downstream ALK (3′ ALK, exons 28 and 29), and GAPDH were used in combination with PCR of cDNA derived from RNA extracted from FFPE tissue of the indicated cases or of two cases (n1 and n2) that were negative for rearrangements. Results from FISH of ELM4 or ALK rearrangement are given below the case number. Positive indicators of rearrangement were attributed to cases if break-apart and/or deletion was observed for gene-specific FISH probes. Refer to Table 1 for specifics for each case shown here. Fifty-nucleotide fragment ladder (lane L) and sizes are shown to the left.