Differentiating Alternative Splice Variant Patterns of Human Telomerase Reverse Transcriptase in Thyroid Neoplasms

Abstract

Background: Although fine-needle aspiration (FNA) biopsy of thyroid nodules is very sensitive in detecting thyroid malignancy, it remains ambiguous in 20-30% of cases. Current biomarkers for thyroid cancer lack either the sensitivity or specificity to substantially address this clinical problem. The aim of this study was to investigate the gene expression patterns of human telomerase reverse transcriptase (hTERT) alternative splice variants in benign and malignant thyroid tumors in an attempt to find a more reliable biomarker in the differential diagnosis of thyroid nodules. Methods: One hundred and thirty-three thyroid tumors from eight histopathological tumor types were collected from patients undergoing thyroid surgery at Johns Hopkins Hospital. Gene expression patterns of hTERT alternative splice variants were investigated in the tumors by nested reverse transcriptase-PCR. Telomerase enzyme activity was evaluated in a subset of 16 samples associated with the different hTERT patterns. Association of c-myc expression and hTERT patterns was also examined. Results: Malignant thyroid tumors exhibited a greater proportion of the active full-length hTERT transcript (0.57 AE 0.15) than inactive splice variants, a À (0.13 AE 0.02), or b À =a À b À deletion transcripts (0.30 AE 0.11; p < 0.001). The opposite was observed in benign tumors, which exhibited greater proportions of b À =a À b À deletion transcripts (0.64 AE 0.08) than either the full-length (0.19 AE 0.06) or a À deletion transcripts (0.17 AE 0.02; p < 0.001). Similar results were observed among a diagnostically challenging subset of 50 thyroid tumors that were suspicious for malignancy on FNA. Further, increased telomerase enzymatic activity was only associated with expression of the full-length hTERT isoform. In contrast, c-myc expression, which has been implicated in hTERT regulation, correlated with overall hTERT transcription without specificity for expression of the full-length isoform. Conclusions: These differences in gene expression patterns of hTERT alternative splice variants may provide a useful adjunct to FNA diagnosis of suspicious thyroid tumors.

Telomere Length Is Related to Alternative Splice Patterns of Telomerase in Thyroid Tumors

“…The relative gene expression levels of hTERT alternative splice variants were analyzed by nested PCR, as previously described. 29 The first round of PCR was maintained in the exponential phase. Amplified products were separated in 2% agarose gels with nucleic acid gel stain (Cambrex, Rockland, ME) and were visualized under UV light.…”

“…29 The splice variant assay was repeated for the subset of samples in this study to have a side-by-side comparison with the quantitative assessment of total hTERT transcript expression levels. Figure 2A is a representative agarose gel image showing the splice variant patterns of four of the most common thyroid tumor types.…”

“…We recently identified four hTERT splice transcript variants in thyroid tumors: full-length, ␣-deletion, ␤-deletion, and ␣-␤-deletion transcripts. 29 We also looked for the other possible hTERT splice variants in thyroid tumors but did not find any evidence of the other species, including the ␥-deletion, which was identified in hepatocellular carcinoma cell lines. Telomerase enzyme activity was shown by several groups to depend on the presence of full-length hTERT gene expression.…”

“…33,35 A previous study on hTERT splice variants in thyroid tumors confirmed that it was the fraction or percentage of full-length transcript, rather than the overall level of hTERT expression, that correlated directly with enzymatic telomerase activity. 29 No study to date, however, has examined whether telomere length correlates with hTERT alternative splice variant patterns in thyroid tumors.…”

“…29 This study included 34 malignant tumors: 15 papillary thyroid carcinomas (PTCs), 12 follicular variant of PTCs (FVPTCs), 4 follicular carcinomas (FCs), and 3 Hürthle cell carcinomas (HCs); and 27 benign lesions: 12 follicular adenomas (FAs), 6 Hürthle cell adenomas (HAs), and 9 adenomatoid nodules (ANs). Because FCs and HCs are relatively infrequent, resulting in limited sample numbers, we grouped follicular neoplasms with or without Hürthle cell morphologic features together for the statistical analyses.…”