Identification of new ALK and RET gene fusions from colorectal and lung cancer biopsies

Abstract

Applying a next-generation sequencing assay targeting 145 cancer-relevant genes in 40 colorectal cancer and 24 non-small cell lung cancer formalin-fixed paraffin-embedded tissue specimens identified at least one clinically relevant genomic alteration in 59% of the samples and revealed two gene fusions, C2orf44-ALK in a colorectal cancer sample and KIF5B-RET in a lung adenocarcinoma. Further screening of 561 lung adenocarcinomas identified 11 additional tumors with KIF5B-RET gene fusions (2.0%; 95% CI 0.8-3.1%). Cells expressing oncogenic KIF5B-RET are sensitive to multi-kinase inhibitors that inhibit RET.

Figure 1

DNA alterations identified in 40 CRC FFPE specimens. (a) The columns in the table denote samples, and the rows denote genes. A number inside the cell indicates the number of alterations of a specific type identified here. (b) A 5,194,955-bp tandem duplication generates an in-frame C2orf44-ALK gene fusion. (c) The RNA sequence of the C2orf44-ALK gene fusion shows aberrant splicing. UTR, untranslated region. (d) RNA sequencing shows an 89.8-fold increase in expression of ALK beginning at exon 20 relative to exons 1–19.

Figure 2

DNA alterations identified in 24 NSCLC FFPE specimens. (a) The columns in the table denote samples, and the rows denote genes. (b) An 11,294,741-bp inversion generates an in-frame KIF5B-RET gene fusion and the reciprocal RET-KIF5B fusion. (c) Protein domain structure of the RET-KIF5B and KIF5B-RET fusions. (d) RNA sequencing shows a 7.3-fold increase in expression of RET beginning at exon 12 relative to exons 1–11. (e) Focal moderate cytoplasmic immunoreactivity for RET protein expression (using avidin-biotin peroxidase). Scale bar, 100 μm; inset, 10 μm. (f) Ba/F3 cells with the KIF5B-REF fusion were treated with different drugs at the indicated concentrations, and viable cells were measured after 72 h of treatment and plotted relative to untreated controls. (g) Cells from e were treated with increasing concentrations of sunitinib or gefitinib for 6 h, and immunoblotting was used to detect the indicated proteins. Conc, concentration; pRET, phosphorylated RET.