Detection of Circulating Thyroid Cancer Cells by Reverse Transcription-PCR for Thyroid-stimulating Hormone Receptor and Thyroglobulin: The Importance of Primer Selection

Abstract

Monitoring for thyroid cancer recurrence is routinely done through measurement of serum thyroglobulin (Tg) and 131I whole-body scanning (WBS) after total thyroidectomy and radioactive iodine ablation (1). Serum Tg has been a useful marker to detect residual or metastatic disease, but its limitations include interassay variability, insufficient sensitivity of some commercial assays, and the frequent presence of interfering anti-Tg antibodies in patient serum (2)(3). Although the ability of serum Tg to detect metastatic disease improves greatly after thyroid hormone withdrawal, hormone withdrawal produces symptomatic hypothyroidism and significant morbidity in many patients. Sensitive detection of circulating cancer cells by reverse transcription-PCR (RT-PCR) of tumor-specific mRNA appears to be a useful adjunct in monitoring of some other malignancies (4)(5). RT-PCR has been used to detect thyroid cells in circulation by amplifying transcripts of the thyroid tissue-specific Tg gene (6)(7)(8), but Tg mRNA can be found normally in circulation (7)(8). Recently, real-time quantitative RT-PCR has been reported to detect small amounts of Tg mRNA in the blood of healthy individuals and to identify thyroid cancer patients with recurrent and residual disease (9)(10)(11). Furthermore, Savagner et al. (11) reported that the amount of a Tg mRNA alternative splicing variant closely correlated with the thyroid volume and thyroid-stimulating hormone (TSH) concentration. Thyroid carcinomas contain functional TSH receptor (TSHR) (12)(13), and differentiated thyroid cancer micrometastases have been detected by RT-PCR of TSHR and Tg mRNAs (14). TSHR has not been exploited to detect circulating cancer cells, and smaller TSHR transcripts have been detected in human lymphocytes (15). We investigated the specificity of different PCR primer pairs in the amplification of TSHR and Tg mRNA signals in blood samples from healthy individuals and in thyroid cancer tissue. Selected primer …