Molecular detection of thyroid cancer: differentiating "signal" and "noise" in clinical assays.

Measurements of circulating serum thyroglobulin (Tg) and whole body radioiodine scanning, both of which rely on thyroid-specific gene transcription or function, are highly specific tests for detecting recurrent or residual thyroid cancer and are commonly used in clinical practice. With the development of more accurate Tg immunoassays, there is now a greater reliance on Tg monitoring in clinical paradigms (1). However, there are several clinically important limitations of serum Tg immunoassays: 1) circulating anti-Tg antibodies interfere with Tg measurement in approximately 20% of patients, and 2) TSH stimulation is required for adequate clinical sensitivity in patients at greatest risk of tumor recurrence (1). There has therefore been an interest in developing new assays for thyroid cancer detection that are even more sensitive and are not altered by anti-Tg antibodies. These seemingly straightforward goals have proven remarkably difficult to achieve using immunoassay detection methods. However, the development of molecular RT-PCR-based methods to diagnose viral diseases that identify specific mRNAs, rather than proteins, suggested that a highly sensitive method for detecting thyroid cancer that would be unencumbered by antibody interference could be developed. Now, 7 yr after the initial promising report, it is an appropriate time to make a critical appraisal of the status of these assays for thyroid cancer and to determine whether we are able to detect a reliable “signal” over a more “noisy” background than was initially recognized. The first Tg mRNA detection assay was reported by Ditkoff et al. (2) who isolated total RNA from 100 individuals, including 87 with thyroid cancer, six with benign thyroid disease following total thyroidectomy, and five normal subjects. Tg mRNA was detected in nine of nine patients with metastatic thyroid cancer, seven of 78 patients thought to be free of disease, and none of 11 patients having surgery for benign disease or normal control subjects. Although detailed clinical information and TSH levels were not reported, the investigators had clearly demonstrated that Tg mRNA could be amplified from peripheral blood and that its presence appeared to correlate with the stage of disease. Tallini et al. (3) subsequently reported data using RT-PCR assays for detecting Tg, thyroid peroxidase (TPO), and RET/PTC1 mRNA from peripheral blood isolated preoperatively, postoperatively, or at both time points, from 44 patients. Of those patients, 24 had thyroid cancer (16 with metastases and 8 free of disease) and 20 had benign nodules. Of the 24 thyroid cancer patients, 56% of the patients with either local or distant metastases had positive Tg mRNA assays, compared with 63% of those thought to be free of disease. Of those thought to be free of disease who had positive Tg mRNA assays, 80% had cervical adenopathy at diagnosis and were felt to be at high risk of tumor recurrence. Of the patients with benign disease, two of 20 patients had a positive Tg mRNA assay and both reverted to negative after surgery. We reported our initial work in this area in 1998 (4) using a different method for detecting Tg mRNA in peripheral blood. For these assays, total RNA was isolated from whole blood placed directly into an RNA-stabilization solution, a method that resulted in a more sensitive assay. Of the 87 individuals with thyroid cancer evaluated, Tg mRNA was detected in all 14 with cervical or distant metastases during l-T4 therapy, in 65% of patients with thyroid bed uptake, and in 20% of patients with no uptake on scan. These data suggested both a higher sensitivity and lower specificity of the assay than the prior studies. Of concern was that similar to Tallini et al. (3) and distinct from Ditkoff et al. (2), circulating Tg mRNA was detectable in normal subjects and in 20% of athyreotic patients. These results raised two possibilities: 1) a low assay specificity due to the presence of ectopically transcribed Tg or splice-variants of Tg in nonthyroid cells; or 2) a high assay sensitivity, i.e. detection of very early minimal residual or recurrent disease, a hypothesis supported by the lower percentage of positive results in athyreotic patients and the identification of rare cells expressing TSH receptor and Tg in peripheral blood of normal subjects (at that time believed to be evidence of thyroid cells). Tg mRNA and immunoassay results did not correlate, a factor common to all studies that could be related to lower specificity, higher sensitivity, or differences in the parameters being measured. Based on these data, we concluded that Tg mRNA was more sensitive than Tg immunoassay, particularly when TSH levels were low, and that the specificity may be acceptable for clinical practice, but long-term clinical follow-up was needed. Additional data have been published from many groups using similar approaches to amplify Tg mRNA and other “thyroid” mRNA transcripts from peripheral blood. The results have been remarkably variable, with some groups demonstrating excellent correlation between tumor stage and results (5–8), whereas others demonstrate no correlation with tumor stage (9–11). Some have demonstrated that the assay is more useful for papillary than follicular cancer (5), whereas others report optimal screening for tumor recurrence by combining Tg mRNA assays with Tg immunoassays (7). Taken together, nearly all groups have confirmed the finding that circulating Tg mRNA is detectable in peripheral blood of normal subjects and in a subset of athyreotic patients, suggesting that ectopic transcription of Tg or splice variants of Tg can be detected if the sensitivity is high Abbreviations: GAPDH, Glyceraldehyde-3-phosphate-dehydrogenase; Tg, thyroglobulin; TPO, thyroid peroxidase.