EVALUATION OF CIRCULATING THYROID-SPECIFIC TRANSCRIPTS AS MARKERS OF THYROID CANCER RELAPSE Luisa BARZON 1 , Marco BOSCARO 2 , Monia PACENTI 1 , Augusto TACCALITI 2 and Giorgio PAL ` U 1 * 1 Department of Histology, Microbiology and Medical Biotechnologies, University of Padova, Italy 2 Division of Endocrinology, University of Ancona, Italy Circulating thyroid-specific transcripts have been sug- gested as potential molecular markers of residual or recur- rent thyroid cancer. We assessed the accuracy of real-time RT-PCR-based detection of a panel of thyroid-specific mark- ers, including TG, TPO, TSHR, NIS and PDS, in comparison with serum TG measurements in a series of 55 patients operated for differentiated thyroid cancer (DTC). Serum TG levels were higher in patients with residual thyroid tissue or metastatic cancer than in disease-free patients during thy- roid hormone suppressive therapy (THST) and after stimu- lation with rhTSH (P < 0.05). Recombinant hTSH increased serum TG values in patients with tumor relapse (P < 0.05), but not in disease-free patients. This assay showed high spec- ificity and good sensitivity in detecting tumor relapse (accu- racy under THST  81.4%; after rhTSH stimulation  90.9%). TPO and TSHR mRNA, either under THST or after rhTSH, showed a significant correlation with disease status for mo- lecular assays. Qualitative analysis of baseline and stimulated TG, NIS and PDS mRNA showed high sensitivity but low specificity in the prediction of thyroid cancer recurrence or metastases (accuracy under THST  51%, 43% and 54%, respectively), whereas TPO and TSHR mRNA assays had higher specificity but low sensitivity, with accuracy under THST of 67% and 61%, respectively, that improved when these tests were combined. Our findings indicate that serum TG assay after TSH stimulation is the most accurate test for monitoring DTC. Combined measurements of TPO and TSHR mRNA levels during THST may represent a specific test for early detection of DTC relapse. © 2004 Wiley-Liss, Inc. Key words: real-time RT-PCR; thyroglobulin; recombinant human thyroid stimulating hormone; thyroid-specific genes; thyroid scintig- raphy; tumor marker Differentiated thyroid carcinoma (DTC), which comprises pap- illary and follicular thyroid cancer, is the most common thyroid malignancy and accounts for 1% of all human cancers. Its inci- dence is increasing in many countries, likely due to exposure of the population to thyroid radiation. Differentiated thyroid carcinoma generally has an excellent prognosis after treatment with total or near-total thyroidectomy followed by 131-iodine ( 131 I) ablation. However, about 20 – 40% of patients with DTC may develop recurrences or metastases even after successful initial therapy. A lifelong follow-up is therefore recommended for early discovery of residual or recurrent disease. Management protocols include periodic radioiodine whole body scanning (WBS) and measurements of serum thyroglobulin (TG) protein by immunoassay. 1 Thyroid hormone withdrawal or, more recently, administration of recombinant human thyroid-stimulating hormone (rhTSH) is required to produce the TSH stimulation needed to achieve maximal accuracy for these diagnostic tests. In comparison with thyroid hormone withdrawal, stimulation with rhTSH avoids side-effects associated with hypothyroidism. Serum TG measurement shows some advantages over WBS in the fol- low-up of DTC, having a higher sensitivity and no false positive results after TSH stimulation, 2,3 besides avoidance of exposure to radiation, lower costs and wide availability. A major disadvantage of this test, however, is the presence of interfering anti-TG anti- bodies in 10 –20% of patients. In the search of a more accurate test for diagnosis of residual or recurrent DTC, the possibility to detect transcripts from circulating thyroid carcinoma cells in the peripheral blood has been recently investigated, based on the experience achieved in detecting circu- lating tumor cells in patients with other malignancies, such as melanoma, breast cancer and prostate cancer. 4 Qualitative and quantitative RT-PCR assays for TG mRNA in peripheral blood have been evaluated as markers of metastatic or residual disease in patients with DTC. Measurement of TG mRNA in peripheral blood in the presence of anti-TG antibodies was found to be more sensitive than TG immunoassay, 5–11 but specificity of TG mRNA assay was low and results had poor correlation with clinical status and serum TG levels. 12–14 An explanation of false-positive results was accounted to the presence of circulating thyroid cells also in whole blood samples of healthy individuals 6 and to illegitimate TG transcription in lymphocytes and granulocytes, as demonstrated in patients without thyroid gland. 5,15 Besides TG mRNA, other mo- lecular markers have been employed for early detection of metas- tases including tissue- or tumor-specific mRNA transcripts, such as thyroid peroxidase (TPO), 5,16 sodium/iodide symporter (NIS), 8 on- cofetal fibronectin, 17 human kallikrein 2 18 and RET/PTC1 rear- rangement. 5 No comparative evaluations have been carried out so far, however, to define the relevance of these tests in the manage- ment of patients with DTC. We employed quantitative analysis in peripheral blood of sev- eral thyroid-specific transcripts of genes involved in the synthesis of thyroid hormones, i.e., TG, TPO, NIS, Pendred syndrome (PDS) and thyroid stimulating hormone receptor (TSHR), as markers of circulating thyroid cells for early diagnosis of recurrent/metastatic DTC. These genes are expressed selectively in thyroid follicular cells and in the vast majority of DTC, but are not expressed or are expressed at very low levels in peripheral blood cells. Because expression of these genes in the thyroid follicular cell is dependent on TSH stimulation, measurements were carried out during thyroid hormone suppressive therapy (THST) and after rhTSH stimulation. As demonstrated with serum TG, indeed, TSH stimulation could increase the production of thyroid-specific transcripts from tumor cells. To evaluate the efficacy of these tests in the assessment of disease status of DTC patients, each assay, carried out either Abbreviations: DTC, differentiated thyroid carcinoma; NIS, sodium/ iodide symporter; PDS, Pendred syndrome; TG, thyroglobulin; THST, thyroid hormone suppressive therapy; TPO, thyroperoxidase; TSH, thyroid stimulating hormone; TSHR, TSH receptor; rhTSH, recombinant human TSH; WBS, whole body scan. Grant sponsor: MIUR; Grant number: 2001061979, 2002062741. *Correspondence to: Department of Histology, Microbiology and Med- ical Biotechnologies, University of Padova, via Gabelli 63, I-35121 Padova, Italy. Fax: +39-049-8272355. E-mail: giorgio.palu@unipd.it Received 31 October 2003; Revised 24 December 2003; Accepted 20 January 2004 DOI 10.1002/ijc.20182 Published online 24 March 2004 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 110, 914 –920 (2004) © 2004 Wiley-Liss, Inc. Publication of the International Union Against Cancer