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Abstracts of Oak Ridge Posters |
Departments of
1
Clinical Pathology,
2 Endocrinology, and
3 General Surgery, The Cleveland Clinic Foundation, Cleveland, OH 44195
aaddress correspondence to this author at: Department of Clinical Pathology, L-30, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195; fax 216-445-9444, e-mail guptam{at}ccf.org
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 pairs with specificity for thyroid tissue and no reactivity with normal peripheral blood mononuclear cells (PBMCs) were further tested to evaluate the potential for clinical utility on detection of circulating thyroid cells.
Four TSHR primer pairs were tested against a panel of normal PBMC RNA and thyroid cancer tissue RNA. One of these was designed to amplify a segment in exons 9 and 10, starting at nucleotide 873 and ending at nucleotide 1371 (TSHR-1; 498 bp). Two were designed to amplify segments in exon 10, spanning nucleotides 1365–1851 (TSHR-2; 486 bp) and 1813–2316 (TSHR-3; 503 bp). The fourth primer pair was targeted to amplify a segment spanning exons 6–9 (14), starting at nucleotide 555 and ending at nucleotide 767 (TSHR-4A; 212 bp). The primer sequences for TSHR were as follows:
For comparison we also tested three previously described (6)(7)(14) Tg primer pairs for specificity, all targeted to exons 1–5 and spanning nucleotides 99–447 (Tg-1; 348 bp), 112–519 (Tg-2; 407 bp), and 33–562 (Tg-3; 529 bp). The primer sequences were as follows:
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a ubiquitously expressed control gene, was also analyzed to confirm the success of RNA extraction and the reverse transcription and PCR reactions (4).
This study was approved by the Institutional Review Board.
Venous blood (5–7 mL into an EDTA tube) was collected from 27 healthy individuals (13 males and 14 females), and mononuclear cells were separated by Ficoll– Hypaque gradient. mRNA was extracted with Trizol reagent (Life technologies). One microgram of RNA was reverse-transcribed with the superscript II preamplification system (Invitrogen). PCR was carried out using primers for 30 cycles [94 °C for 1 min (first cycle for 2 min), 62 °C for 1 min, and 72 °C for 1 min (10 min for the last cycle)]. RT-PCR products were resolved by 2% gel electrophoresis and visualized by ethidium bromide staining.
During the initial testing we observed that all four TSHR primer pairs amplified specific signals from the thyroid cancer tissue. However, the three primer pairs targeted to exons 9 and 10 also amplified signals from normal PBMCs (Fig. 1A
). In contrast, primer pair TSHR-4 (exons 6–9; product size, 212 bp) failed to amplify any signal in the PBMCs from any healthy individuals tested (Fig. 1A
). Similar results were obtained with Tg primer pairs; whereas all Tg primer pairs amplified specific signals from the thyroid tissues, only two of these (Tg-1 and Tg-3) amplified signals from PBMCs of healthy individuals (data not shown). One primer pair (Tg-2) was specific for thyroid tissue only and showed no reactivity in PBMCs from healthy individuals. On the basis of these findings, the TSHR-4 and Tg-2 primer pairs were selected for further testing in clinical samples and to further refine the sensitivity of these assays.
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Assay sensitivity was tested by serial dilution of the thyroid cancer tissue RNA with the RNA from normal PBMCs. Assuming 10–20 pg total RNA per thyrocyte, mRNA from 
50–100 cells/mL of blood was detected by ethidium bromide staining. The lower limit of detection was further improved by increasing the template amount from 1 µg to 5 µg and by increasing the number of PCR cycles from 30 to 38. With these modifications, the estimated detection limit was 
10 cancer cells/mL of blood (Fig. 1
). Even with this enhancement in sensitivity, the selected primer pairs for both TSHR-4 and Tg-2 showed no reactivity with PBMCs from healthy individuals when retested.
Clinical validation of these RT-PCR assays was then performed by analyzing blood from 10 healthy euthyroid individuals, 8 patients with benign thyroid diseases, and 27 patients with treated thyroid cancer. All thyroid cancer patients were on thyroid suppressive therapy at the time of testing and had diagnostic WBS within the previous 2 years. Twenty-one of these patients were negative for residual disease, 3 had distant metastases, and 3 showed local recurrences.
No TSHR or Tg mRNA signals were detected in 10 euthyroid individuals and in 7 of 8 patients with benign thyroid disease (5 with hyperthyroidism and 2 with multinodular goiter). One patient with a large goiter, and tracheal compression was positive for both TSHR and Tg mRNA. TSHR and Tg mRNA were detected in the three patients with distant metastases and in two of the three with thyroid bed/neck uptake [total, five of six (83%)]. Of 21 patients with negative WBS, only one (5%) was positive for both TSHR and Tg mRNA (Fig. 1B
and Table 1
). Tg mRNA was detected in two more patients with negative WBS whose serum Tg concentrations were undetectable. It remains to be seen whether these three WBS- and serum Tg-negative patients eventually will become positive on follow-up testing, which in turn will confirm the early detection capability of the TSHR and Tg mRNA assay.
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Our results suggest that the specificity of RT-PCR depends to a greater extent on the design of the oligonucleotide primers. Like Francis et al. (15), we also detected TSHR transcripts in normal lymphocytes with primers amplifying the exon 10 segment. However, in PBMCs from healthy individuals and patients with benign thyroid diseases, the segment spanning exons 6–9 failed to amplify. Similarly, many investigators have detected Tg mRNA transcripts in blood from healthy euthyroid individuals (7)(8), whereas others failed to detect any signals in blood from healthy individuals (6). Ringel et al. (7) related these differences to the greater sensitivity of their RT-PCR assay. Our results using different primer sets suggest that these differences relate to primer pair selection. Using the same primer pair used by Ringel et al., we also detected positive signals in some healthy individuals, but failed to do so with another primer pair (even after significant enhancement of sensitivity) that showed specific signals selectively in those thyroid cancer patients with positive scans. As discussed previously by others, the discordance between different primers can be explained by the limitation of PCR-based techniques to detect only the alternatively spliced variants amplified by the selected primers (9)(11).
In summary, our results confirm previous findings that TSHR mRNA is detected by RT-PCR in PBMCs from healthy individuals. However, we found that this positivity was dependent on the primer pairs used and that, with careful selection of the primer pair, the presence of mRNA was specific for thyroid cancer and corresponded to the presence of extrathyroidal disease and the presence of Tg mRNA transcripts. With further refinement of this assay and with the development of a quantitative real-time PCR procedure, the measurement of TSHR mRNA by RT-PCR may serve as a specific and sensitive tool for monitoring of thyroid cancer.
Acknowledgments
This study was supported by a grant from The Cleveland Clinic, Research Projects Fund. This work was presented at the 34th Annual Oak Ridge Conference, April 25–26, 2002, La Jolla, CA.
References
The following articles in journals at HighWire Press have cited this article:
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  S.-Y. Chia, M. Milas, S. K. Reddy, A. Siperstein, M. Skugor, J. Brainard, and M. K. Gupta Thyroid-Stimulating Hormone Receptor Messenger Ribonucleic Acid Measurement in Blood as a Marker for Circulating Thyroid Cancer Cells and Its Role in the Preoperative Diagnosis of Thyroid Cancer J. Clin. Endocrinol. Metab., February 1, 2007; 92(2): 468 - 475. [Abstract] [Full Text] [PDF]
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K. Wagner, R. Arciaga, A. Siperstein, M. Milas, I. Warshawsky, S. Sethu, K. Reddy, and M. K. Gupta Thyrotropin Receptor/Thyroglobulin Messenger Ribonucleic Acid in Peripheral Blood and Fine-Needle Aspiration Cytology: Diagnostic Synergy for Detecting Thyroid Cancer J. Clin. Endocrinol. Metab., April 1, 2005; 90(4): 1921 - 1924. [Abstract] [Full Text] [PDF]
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P. Chinnappa, L. Taguba, R. Arciaga, C. Faiman, A. Siperstein, A. E. Mehta, S. K. Reddy, C. Nasr, and M. K. Gupta Detection of Thyrotropin-Receptor Messenger Ribonucleic Acid (mRNA) and Thyroglobulin mRNA Transcripts in Peripheral Blood of Patients with Thyroid Disease: Sensitive and Specific Markers for Thyroid Cancer J. Clin. Endocrinol. Metab., August 1, 2004; 89(8): 3705 - 3709. [Abstract] [Full Text] [PDF]
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M. D. Ringel Molecular Detection of Thyroid Cancer: Differentiating "Signal" and "Noise" in Clinical Assays J. Clin. Endocrinol. Metab., January 1, 2004; 89(1): 29 - 32. [Full Text] [PDF]
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