Figure 2

Detection of ALK fusion gene and MET exon 14 skipping events using the NanoString technology. (A) Detection of gene fusions, e.g. involving ALK, by the NanoString assay is based on expression (counts) of the 3′, 5′ part of the gene, and fusion specific probes as described elsewhere (see e.g.¹¹). The actual count values (left panel) for ALK related probes in sample S_0003 reveals the, likely, exact ALK fusion (EML4-ALK_E13:A20) and demonstrates the differential expression of the 3′ and 5′ probes of the ALK gene when a fusion occurs. Combining a 3′/5′ probe ratio with fusion specific probe expression identifies five ALK fusion positive samples (red samples) in the upper right quadrant of a scatter plot of the 3′/5′ expression ratio versus the expression of ALK fusion specific probes (left panel) (see¹¹ for further details) for cases subjected to fusion gene analysis (see Table 1). (B) Identification of three patients harboring MET exon 14 skipping events (red samples). Detection is based on high expression of a specific junction probe spanning exon 13–15 (excluding exon 14) (y-axis), versus a ratio of the mean expression for probes representing exons 3–4 and 20–21 divided by exon 14 specific expression (x-axis). Samples harboring MET exon 14 skipping events are visualized in the upper right quadrant as these report high junction probe counts and differential expression of exon 14 and exons 3–4 and 20–21 probe counts. (C) Raw NanoString count data for the S_0297_1 sample, which was tested clinically ALK positive by IHC, but was called FISH negative. NanoString analysis identifies a likely EML4-ALK_E13:A20 fusion. One 5′ probe demonstrates a high background count compared to remaining 5′ probes.