Higher RET Gene Expression Levels Do Not Represent anAlternative RET Activation Mechanism in Medullary Thyroid Carcinoma

Abstract

This study was designed to investigate whether RET (rearranged during transfection) mRNA over-expression could be considered an alternative driver event for the development of medullary thyroid carcinoma (MTC), and if different RET isoforms could play a role in MTC tumorigenesis. Eighty-three MTC patients, whose mutational profile was previously identified by next-generation sequencing (NGS) IONS5, were included in this study. Expression analysis was performed by the quantitative reverse transcription-polymerase chain reaction technique. RET expression levels were found to be significantly higher in cases with RET somatic mutations than in cases that were negative for RET somatic mutations (p = 0.003) as well as in cases with a somatic mutation, either in RET or RAS than in cases negative for both these mutations (p = 0.01). All cases were positive for the RET51 isoform expression while only 72/83 (86.7%) were positive for RET9 isoform expression. A statistically significant higher expression of the RET51 isoform was found in cases positive for RET somatic mutation than in cases either positive for RAS mutation (p = 0.0006) or negative for both mutations (p = 0.001). According to our data, RET gene over-expression does not play a role in MTC tumorigenesis, neither as an entire gene or as an isoform. At variance, the RET gene, and in particular the RET51 isoform, is expressed higher in RET mutated cases. On the basis of these results we can hypothesize that the overexpression of RET, and in particular of RET51, could potentiate the transforming activity of mutated RET, making these cases more aggressive.

Keywords: RAS; RET; mRNA expression; medullary thyroid carcinoma.

Figure 1

RET gene expression levels according to the somatic mutation profile. A statistically significant higher expression level was observed in RET+. Gene expression levels are reported as ΔCt (ΔCt = Ct RET − Ct G6PD) where Ct is the threshold cycle for qRT-PCR. The lowest is the ΔCt value, and the highest is the mRNA expression level.

Figure 2

(A): RET gene expression levels according to the somatic mutation profile. A statistically significant higher RET gene expression level was observed in RET+ cases with respect to RET− and RAS+ cases. (B): gene expression levels according to the somatic mutation profile. A statistically significant higher RET gene expression level was observed in RET+ or RAS+ cases with respect to cases negative for both gene alterations (RET− and RAS−).

Figure 3

RET51 isoform expression levels according to the somatic mutation profile. A statistically significant higher expression level was observed in RET+ cases than in RAS+ cases and RET− and RAS− cases. Gene expression levels are reported as ΔCt (ΔCt = Ct RET – Ct G6PD) where Ct is the threshold cycle for qRT-PCR. The lowest is the ΔCt value, and the highest is the mRNA expression level.

Figure 4

(A): RET51 isoform expression levels according to the somatic mutation profile. A statistically significant higher RET51 expression level was observed in RET-positive cases with respect to cases negative for a RET mutation. (B): RET51 isoform expression levels according to the somatic mutation profile. A statistically significant higher RET gene expression level was observed in positive cases with respect to cases negative for both genes. Expression levels are reported as ΔCt (ΔCt = Ct RET – Ct G6PD) where Ct is the threshold cycle for qRT-PCR. The lowest is the ΔCt value, and the highest are the gene expression levels.