RET fusion as a novel driver of medullary thyroid carcinoma

Abstract

Introduction: Oncogenic RET tyrosine kinase gene fusions and activating mutations have recently been identified in lung cancers, prompting initiation of targeted therapy trials in this disease. Although RET point mutation has been identified as a driver of tumorigenesis in medullary thyroid carcinoma (MTC), no fusions have been described to date.

Objective: We evaluated the role of RET fusion as an oncogenic driver in MTC.

Methods: We describe a patient who died from aggressive sporadic MTC < 10 months after diagnosis. Her tumor was evaluated by means of next-generation sequencing, including an intronic capture strategy.

Results: A reciprocal translocation involving RET intron 12 was identified. The fusion was validated using a targeted break apart fluorescence in situ hybridization probe, and RNA sequencing confirmed the existence of an in-frame fusion transcript joining MYH13 exon 35 with RET exon 12. Ectopic expression of fusion product in a murine Ba/F3 cell reporter model established strong oncogenicity. Three tyrosine kinase inhibitors currently used to treat MTC in clinical practice blocked tumorigenic cell growth.

Conclusion: This finding represents the report of a novel RET fusion, the first of its kind described in MTC. The finding of this potential novel oncogenic mechanism has clear implications for sporadic MTC, which in the majority of cases has no driver mutation identified. The presence of a RET fusion also provides a plausible target for RET tyrosine kinase inhibitor therapies.

Figure 1.

The MHY13-RET kinase fusion is caused by a translocation of chromosome 17 and chromosome 10 joining the first 35 exons of MHY13 with exon 12–20 of RET. The in-frame junction of the fusion transcription was confirmed with Sanger sequencing of cDNA generated from patient tumor.

Figure 2.

RET rearrangement detected by fluorescence in situ hybridization in the sporadic MTC of a patient who died of disease at age 46 years. The RET (10q11.21) break apart probe was from Cymogen DX, with the centromeric side labeled in orange and the telomeric side labeled in green. Cells without rearrangement show overlap with the generation of a yellow signal, whereas cells with rearrangement (arrows) show isolated red and green signals.

Figure 3.

A, MYH13-RET fusion transcript supports IL-3-independent Ba/F3 cell growth. Control and fusion constructs were transfected into Ba/F3 cells to assess the rescue cell viability in the absence of IL-3. Full-length reference sequences (WT) for RET and MYH13, along with the nonspecific protein mCherry were included as negative controls. To establish oncogenicity, constructs encoding both predicted fusion constructs, RET-MYH13 and MYH13-RET, were compared to the established positive control PIK3CA (H1047R). Data display the cell viability determined at 1.5 weeks for three independent transfections ± SD. Asterisks indicate significant difference from mCherry control (P < .001). B-D, Tyrosine kinase inhibitors targeting RET block MYH13-RET fusion-driven Ba/F3 cell growth. Cells were seeded in 96-well format at 1 × 10⁴ cells per well in medium with IL-3 or without IL-3. After overnight incubation, sunitinib (B), vandetanib (C), and cabozantinib (D) were added at indicated concentrations (0–10 μm; DMSO as vehicle). A final concentration of 0.1% DMSO was used in all wells. Data display the cell viability at 72 hours as mean ± SD normalized to 0 μm control (100%). Asterisks indicate significant difference from +IL-3 control (P < .005). Cell viability was measured using CellTiter-Glo assay (Promega) (A) or Prestoblue Cell viability Reagent (Life Technologies, Inc) (B–D) according to manufacturers' instruction.