Nuclear-specific accumulation oftelomerase reverse transcriptase(TERT) mRNA inTERTpromoter mutated follicular thyroid tumours visualised by in situ hybridisation: a possible clinical screening tool?

Abstract

AimsUpregulation of the telomerase reverse transcriptase (TERT) gene is a frequent finding in follicular thyroid carcinomas (FTCs) with metastatic features. The augmented expression is usually caused by TERT promoter mutations. As TERT protein immunohistochemistry might not correlate to TERT mRNA levels in follicular thyroid tumours, we therefore sought to determine if visualisation of TERT mRNA through in situ hybridisation could highlight high-risk cases.MethodsWe collected formalin-fixated paraffin-embedded tissues from 26 follicular thyroid tumours; 7 FTCs, 2 follicular thyroid tumours of uncertain malignant potential (FT-UMPs) and a single Hürthle cell carcinoma with established TERT promoter mutations and gene expression, as well as 16 FTCs with no TERT gene aberrancy or gene expression, and assessed them using RNA Scope in situ hybridisation (ISH) and TERT probes targeting the two main TERT transcripts (TERT1 and TERT2).ResultsTERT 1 and/or 2 mRNA was found by ISH in 8/10 cases with established promoter mutations and mRNA expression, whereas all 16 cases without TERT gene aberrancies or gene expression were negative (Fisher’s exact p<0.001). Strikingly, TERT mRNA was visualised in the nuclear compartment only, thereby corroborating earlier studies suggesting a non-conventional role for TERT in tumour biology. Moreover, TERT mRNA expression was scattered across the tissue sections and only found in a few percentages of tumour nuclei.ConclusionsTERT mRNA seems to be focally expressed and localised exclusively to the nucleus in TERT promoter mutated follicular thyroid tumours, possibly reflecting a true biological and unorthodox phenomenon worthy of further investigations.

5hmC immunohistochemistry: a predictor of TERT promoter mutational status in follicular thyroid carcinoma?

“…TERT aberrancies may exhibit spatial heterogeneity in follicular thyroid tumors, which has been shown for TERT promoter mutations [17,18] as well as for TERT mRNA expression visualized by ISH [10]. It was therefore expected that some heterogeneity could be observed regarding the expression of 5hmC; it is however interesting that as many as half of all cases showed heterogeneous expression patterns -a nding that lacks a credible explanation.…”

“…TERT promoter mutations and TERT gene expression are not unequivocally needed for the global loss of 5hmC in thyroid cancer. There is much to be revealed regarding the biology of TERT promoter mutations in follicular thyroid tumors, as TERT protein expression itself may not be correlated to TERT promoter mutations and TERT mRNA expression [9] as well as the recent observation of nuclear-speci c TERT mRNA expression [10].…”

“…A cohort of differentiated follicular cell-derived and oncocytic thyroid tumors which has previously been evaluated for TERT promoter mutations and TERT expression using qRT-PCR and ISH [10] was chosen for 5hmC IHC analyses. The cohort consisted of 26 FTCs, 2 FT-UMPs, and one oncocytic thyroid carcinoma (OTC), of which 10 (7 FTCs, 2 FT-UMPs, and the single OTC) showed TERT promoter mutations and TERT gene expression, while the remaining cases were TERT promoter wildtype and displayed absent TERT mRNA levels.…”

“…As of this, the 2022 WHO classi cation of endocrine and neuroendocrine tumors acknowledges the role of TERT promoter mutations in highlighting cases at risk of disease dissemination [8], and more focus is currently diverted to developing robust screening tools to detect TERT-expressing tumors in clinical routine. Speci cally, while TERT immunohistochemistry (IHC) is not yet considered a reliable method, studies using TERT mRNA in situ hybridization (ISH) have shown promising results [9,10].…”

5hmC Immunohistochemistry: A Predictor of TERT Promoter Mutational Status in Follicular Thyroid Carcinoma?

“…Specifically, whereas TERT immunohistochemistry (IHC) is not yet considered a reliable method, studies using TERT mRNA in situ hybridization (ISH) have shown promising results. 9,10 5-Hydroxymethylcytosine (5hmC) immunoreactivity is decreased in various malignant tumors, such as melanoma 11 and glioma 12 as well as in papillary thyroid carcinoma (PTC). 13,14 5hmC is produced when 5-methylcytosine is demethylated via ten-eleven translocation (TET) enzymes, and 5hmC may thus serve as an epigenetic marker of global de-methylation events across the genome.…”