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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 10 3623-3627
Copyright © 2000 by The Endocrine Society

Original Studies

Detection of Recurrent Thyroid Cancer by Sensitive Nested Reverse Transcription-Polymerase Chain Reaction of Thyroglobulin and Sodium/Iodide Symporter Messenger Ribonucleic Acid Transcripts in Peripheral Blood1

Rosa Paula M. Biscolla2, Janete M. Cerutti and Rui M. B. Maciel

Laboratory of Molecular Endocrinology, Division of Endocrinology, Department of Medicine, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, 04039-032 Sao Paulo, Brazil

Address all correspondence and requests for reprints to: Rui M. B. Maciel, M.D., Ph.D., Laboratory of Molecular Endocrinology, Division of Endocrinology, Department of Medicine, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, Rua Pedro de Toledo 781, 12th Floor, 04039-032 Sao Paulo, Brazil. E-mail: rmbmaciel-endo{at}pesquisa.epm.br


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
To investigate whether circulating thyroglobulin (Tg) messenger ribonucleic acid (mRNA) and sodium/iodide symporter (NIS) mRNA transcripts in peripheral blood are valuable in the follow-up of patients with thyroid cancer, we developed highly sensitive nested Tg and NIS mRNA detection assays and compared their accuracy with serum thyroglobulin (sTg) and whole body scan with 131I during the monitoring of 34 patients with well differentiated thyroid carcinoma who had undergone total thyroidectomy (17 of 34 also submitted to thyroid ablation with radioiodine) and were taking T4.

Circulating Tg mRNA was found in 13 of 34 patients, 5 of 13 with detectable and 8 of 13 with undetectable sTg. From these 8 patients with undetectable Tg, 6 showed no cervical radioiodine uptake, and 3 presented proven metastatic disease (2 of them positive for antithyroglobulin antibodies). NIS mRNA was detected in 11 of 34 patients, but its measurement did not improve the ability to detect patients with metastases. Overall, identification of metastatic thyroid cancer was better associated with Tg mRNA than with NIS mRNA, sTg, or whole body scan (83% vs. 16.6% vs. 50% vs. 50%; P < 0.001).

These data showed that circulating Tg mRNA is not only a more sensitive marker of residual thyroid tissue or thyroid cancer than sTg, particularly in patients during T4 therapy and with positive antithyroglobulin antibodies, but also was more sensitive than NIS mRNA in all patients.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
SERUM THYROGLOBULIN (sTg) is a very useful tumor marker to discover residual, recurring, or metastatic disease in patients with differentiated thyroid carcinoma after total thyroidectomy and ablative radioiodine (1, 2, 3). sTg assays, however, present some technical dilemmas that decrease its clinical utility: insufficient sensitivity of many commercially available methods, "hook" effect, high interassay variation, absence of international standardization, and, mainly, interference by the presence of antithyroglobulin antibodies (TgAb), present in 15% of the patients (3, 4, 5). Furthermore, to obtain the best possible sensitivity, many physicians recommend collection of blood after thyroid hormone removal, which causes symptomatic hypothyroidism and the possibility of tumor growth (5, 6, 7). To solve these problems, alternative methods have been devised, and some researchers have developed sensitive RT-PCR assays to amplify Tg messenger ribonucleic acid (mRNA) that were able to detect circulating thyroid cells from patients with metastatic or residual neck thyroid tissue (8, 9, 10, 11).

The plasma membrane protein sodium/iodide symporter (NIS), which mediates the ability of the thyroid gland to concentrate iodine (12), may also be used as a thyroid-specific marker. The decreased radioiodine uptake observed in some thyroid carcinomas may be due, as recently shown by several researchers, to the reduced expression and/or stability of NIS mRNA found in human thyroid tumors (13, 14, 15). Therefore, the discordance between sTg and whole body scan (WBS) frequently observed in clinical practice may well indicate variations on the expression of genes connected to Tg and NIS.

To investigate whether Tg mRNA and NIS mRNA are valuable in monitoring patients with thyroid cancer during T4 therapy, we developed highly sensitive Tg and NIS mRNA detection assays and compared their accuracy with sTg and WBS.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

We studied 34 patients with well differentiated thyroid carcinoma at follow-up at Hospital Sao Paulo, a teaching hospital from Universidade Federal de Sao Paulo, Brazil, and 6 normal subjects with no history or clinical evidence of thyroid disease. All 34 patients (31 women and 3 men) had been treated with near-total thyroidectomy, 17 of whom also received radioiodine ablation, according to the current clinical protocols in use at our institution. Twenty-four patients had papillary carcinoma, and 10 had follicular carcinoma, classified according to the recommendations of the WHO (16). All patients were studied during T4 therapy. The most recent WBS after T4 withdrawal of each patient was used for comparison. WBS radioiodine scanning was performed 48–72 h after a 2- to 5-mCi dose of 131I; negative scans were those with no detectable or less than 1% of measured radioiodine uptake. Blood samples for Tg and NIS mRNA measurements by RT-PCR were obtained in combination with samples for determinations of serum TSH (Delphia immunofluorometric assay, third generation; Delphia, Turku, Finland) (17), sTg (Brahms immunoradiometric assay, Berlin, Germany; assay with a sensitivity of 1 ng/mL) (18), and TgAbs (19); 21 of 34 patients (61.7%) had TSH concentrations below 0.1 mU/L, 26 of 34 (76.4%) had TSH levels below 5 mU/L, and the remaining 8 patients, although clinically euthyroid, presented TSH concentrations between 6–17 mU/L. The study was approved by the ethical committee of Hospital Sao Paulo, and written informed consent was obtained from each subject.

RNA extraction

Total RNA was isolated from 1 mL venous blood obtained from standard venipuncture and transferred immediately into sterile tubes containing 3 mL TRIzol LS reagent (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s recommendations (20). RNA isolated from 50–100 mg thyroid tissue obtained from patients undergoing thyroidectomy was used as positive control.

RT-PCR

Total RNA (10 µg) was reverse transcribed to complementary DNA (cDNA) in a final volume of 40 µL using 250 ng random hexamer primers (Life Technologies, Inc.), 10 U ribonuclease inhibitor (Life Technologies, Inc.), 200 U Superscript II (Life Technologies, Inc.), 50 mmol/L Tris-HCl (pH 8.3), 75 mmol/L KCl, 3 mmol/L MgCl2, 500 µmol/L of each deoxy-NTP, and 10 mmol/L dithiothreitol. Reverse transcriptase-negative samples were prepared for each individual reaction and served as controls for detection of assay contamination.

PCR was performed using 5 µL first strand cDNA in a 25-µL reaction volume containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2, 200 µmol/L of each deoxy-NTP, 1 U Taq polymerase (Life Technologies, Inc.), and 40 pmol of each specific primer (ß-actin, Tg, and NIS; Table 1Go). Primers for ß-actin (21), Tg (8), and NIS (22) were described previously.


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  		Table 1. RT-PCR details for Tg, NIS, and ß-actin

 
The cycling conditions used to generate Tg products included an initial phase of 5 min at 94 C, followed by 25 cycles of 45 s at 94 C, 90 s at 60 C, and 2 min at 72 C. After that, 1/10th of the PCR product of the first amplification was used as a template for nested PCR (23). The reamplification was carried out in a volume of 25 µL, using 66 C as the annealing temperature during 2 min for 20 more cycles.

To generate NIS products, an initial phase of 1 min at 95 C was followed by 45 cycles of 15 s at 95C, 15 s at 61 C, and 1 min at 72 C. As above, 1/10th of the PCR product was used as a template for a nested PCR (22). The reaction was submitted to 35 PCR cycles employing the previous conditions, except for the annealing temperature of 62 C for 15 s.

RNA integrity was verified using specific primers to ß-actin. Amplification of normal thyroid tissue was used as a positive control. Each sample was analyzed at least twice.

Analysis of amplified Tg and NIS cDNAs

After the amplification, 5 µL of each PCR reaction were electrophoresed through 2% agarose gels and visualized with ethidium bromide. To confirm the identity of nested RT-PCR-amplified Tg products, samples were digested with the restriction enzyme BglII (Life Technologies, Inc.) at 37 C overnight, analyzed on 2% agarose gels, and visualized with ethidium bromide. In addition, the specificity of each Tg and NIS amplification was verified by manual sequencing (Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit, Amersham Pharmacia Biotech, Cleveland, OH) of the representative amplified product, using a 8% acrylamide denaturing gel in a glycerol tolerant gel buffer (Amersham Pharmacia Biotech, Arlington Heights, IL).

Statistical analysis

The association between the presence of metastases (confirmed by either fine needle aspiration cytology or surgery) and RT-PCR positivity for Tg and NIS mRNAs, sTg levels, and positive scans (thyroid bed uptake and metastases) was determined using the measurement of {kappa} agreement by the STATA program (24). P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Detection of Tg and NIS mRNAs from peripheral blood by nested RT-PCRs

To detect low levels of circulating Tg and NIS mRNA transcripts, we used sensitive RT-PCR assays, optimized for both synthesis of cDNA and amplification of Tg and NIS cDNAs by nested RT-PCRs. For Tg and NIS mRNA, two rounds of amplification yielded products of 448 and 203 bp, respectively (Fig. 1Go). Restriction digestion and direct nucleotide sequencing analysis indicated that 448- and 203-bp products were derived from Tg and NIS mRNAs. Synthesis of cDNA was achieved in all cases, as determined by successful amplification of ß-actin in each sample (Fig. 1Go). Furthermore, Tg and NIS mRNAs were also amplified using cDNAs prepared from all six normal subjects.



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  		Figure 1. Representative nested RT-PCR products for Tg, NIS, and ß-actin in agarose gel stained with ethidium bromide. The total RNA from the same samples was used in the RT-PCR for Tg, NIS, and ß-actin. Patients 1 and 2 had no uptake on the most recent radioiodine scan, patients 3 and 4 had thyroid bed uptake, and patient 5 had pulmonary metastases.

 
Clinical evaluation of the results of RT-PCRs for Tg and NIS mRNAs

To compare the expression of the 2 thyroid genetic markers analyzed in this study, the 34 patients were ordered according to clinical stage into 1 of 3 classes, defined by the most recent WBS or the occurrence of metastases: no uptake (n = 23), thyroid bed uptake (n = 8), and distant metastases (n = 3). Table 2Go and Fig. 2Go depict the relationship between the several markers of thyroid tumors studied and the present disease status.


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  		Table 2. Status of disease, histology, WBS, sTSH and sTg levels, detection of Tg and NIS mRNAs, and anti-Tg in 34 patients taking L-T4

 


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  		Figure 2. Comparison among sTg, WBS, and Tg and NIS mRNA assays in 34 patients during T4 therapy. Patients are classified into 3 groups, according to the most recent whole body scan, on the x-axis. sTg values are plotted on a logarithmic scale on the y-axis. sTg concentrations below 1 ng/mL were considered undetectable.

 
The upper part of Table 2Go and the left portion of Fig. 2Go demonstrate the results of the 23 patients who presented no residual thyroid radioiodine uptake; 22 of 23 also showed undetectable sTg measurements. However, 7 of 23 patients presented with a positive Tg mRNA assay. Of these 7 patients, 1 had detectable sTg (2.5 ng/mL); in the remaining 6 patients, we were able to observe, under careful ultrasound evaluations, the presence of suspicious metastatic cervical lymph nodes in 3 of them, confirmed by either aspirative cytology or surgery (patients 11, 12, and 22). These 3 patients were, however, negative for NIS mRNA, which may explain the lack of radioiodine uptake. The other 3 patients were positive for both Tg and NIS mRNAs, but they had undetectable sTg and suppressed serum TSH (patients 13, 14, and 15), and will need long term follow-up to demonstrate the meaning of these results. To date, they have shown no positive marker of residual or recurrent disease.

The central portion of Fig. 2Go (patients 24–31 in Table 2Go) illustrates the results of eight patients with thyroid remnants; four of them had positive Tg mRNA (three of whom also had positive NIS mRNA); two of four showed detectable and two of four showed undetectable sTg measurements. Ultrasound evaluation was negative in all of them. Despite the fact that they were receiving T4 therapy, the serum TSH values were not completely suppressed in two patients (6.9 and 8.4 mU/L), which could stimulate Tg and NIS gene expression. These cases will also need long term follow-up to demonstrate the meaning of these results.

On the right of Fig. 2Go and at the bottom of Table 2Go (patients 32–34) we presented the results of patients who disclosed distant metastases (two of them with lung and one with bone metastases). Two of these patients had an aggressive form of the disease, with very elevated sTg levels and positive Tg mRNA. The remaining patient is an interesting case of a negative result for Tg and NIS mRNA. This patient, after total thyroidectomy and radioiodine ablation, started to show an elevation of sTg despite negative scans. It was then decided to treat him with an elevated dose of radioiodine, and after this dose, a new scan revealed uptake in the lung.

Therefore, among patients studied while receiving thyroid hormone therapy, the sensitivity and specificity of markers to identify metastatic thyroid disease were, respectively: Tg mRNA, 83% and 71%; sTg, 50% and 89%; NIS mRNA, 16.6% and 54%; and WBS, 50% and 71%.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Our study demonstrates that circulating Tg mRNA is a more sensitive marker of the presence of residual thyroid tissue or thyroid cancer than NIS mRNA, sTg, and WBS, particularly in patients receiving T4 therapy and with positive anti-Tg antibodies. This observation is valuable in clinical practice, because it offers a new method capable of increasing our ability to detect residual disease without the removal of T4 therapy and/or the use of recombinant human TSH, which is advantageous to patients. In contrast, the detection of circulating NIS mRNA in this particular setting of patients taking T4 did not improve the ability to detect those with active disease and was not always concordant with Tg mRNA.

We have developed highly sensitive nested RT-PCRs to identify mRNA encoding Tg and NIS in blood of individuals with normal and malignant thyroid tissue. The characteristics of these assays are distinct from those of others recently reported in several aspects: the design of nucleotide primers, the conditions for synthesis of cDNA and for PCR, and the preparation of total RNA (8, 9, 10, 11). Thus, our assays use nested PCR as a method to improve sensitivity, with a technique similar to that employed for other tumors, such as circulating colo-rectal, prostatic, and biliary-pancreatic tumor cells (25, 26, 27). In fact, in the majority of our patients with positive results, we were able to detect the Tg or NIS mRNAs only after the use of a second round of PCR. We do not presume that these amplifications could be due to ectopic or illegitimate transcriptions, especially in the case of Tg mRNA, because Tg promoters show striking cell type-specific transcriptional activity and are only active in differentiated thyroid follicular cells. In addition, the molecular events involved in the differentiation of thyroid follicular cells are very specific and are dependent on the activation of a combinatorial sequence of at least three transcription factors, TTF-1, TTF-2, and PAX-8, which control their migration, growth, and differentiation (28). Moreover, we have already had confirmation of the presence or recurrence of the tumor in three of six patients consistently negative for sTg and WBS.

In comparison with simultaneously performed NIS mRNA detection assay, sTg measurement, and WBS, the detection of Tg mRNA was more sensitive, identifying additional patients with recurrence of disease. Thus, as indicated by the results for those patients showing no radioiodine uptake, we were able to detect metastases in three patients receiving T4 therapy, who were consistently negative for WBS and sTg measurements during follow-up. This demonstrates a superior sensitivity of Tg mRNA compared to other markers. It is important to stress that after the suspicion elicited by the positive results of Tg mRNA in these cases, the use of neck ultrasound was instrumental in discovering residual disease.

The false negative case shown on the left of Fig. 2Go is in agreement with cases previously described by Tallini et al. (9) and Ringel et al. (10) of patients positive for sTg and negative for Tg mRNA, suggesting that these two tests should be used in conjunction to evaluate diverse properties of the tumors, such as the amount of circulating cancer cells released by the tumor and its potential to synthesize dissimilar proteins. Another possible explanation for this finding is the presence of polymorphisms or splice variants in the Tg molecule produced by the tumor. This would create primer mismatches impairing the appropriate annealing of primers employed in the RT-PCR. Conversely, sTg would probably be able to measure the modified protein, because the antibody used in the sTg assay would react against several Tg molecular forms.

The interpretation of negative NIS results in patients taking T4 should be cautiously analyzed, because NIS gene expression by thyroid cells is subjected to numerous influences, mainly the induction by TSH (29), which was suppressed in the majority of our patients. Therefore, it is difficult to differentiate whether the negative results are related to the absence of NIS expression due to genetic changes in the tumor cell, the suppressed TSH, or both. On the other hand, NIS production is not restricted to the thyroid; it is also expressed in salivary gland, mammary gland, and gastric mucosa. Therefore, positive NIS mRNA results can be derived from extrathyroid tissues (30). Based on the false negative results shown in our studies and the possibility of false positive results due to NIS mRNA expression in nonthyroid cells, we do not suggest the use of the NIS mRNA assay for detection of circulating thyroid tumor cells.

In conclusion, the most prominent benefit of Tg mRNA detection compared to sTg measurement is the lack of interference by anti-Tg antibodies in circulation and the high sensitivity of the assay during T4 therapy.


   Acknowledgments
 
We are grateful to Magnus R. D. Silva, M.D., for statistical advice, and to Rogério Rabelo, M.D., and José Gilberto H. Vieira, M.D., for helpful discussions and careful review of the manuscript.


   Footnotes
 
1 This work was supported by Grants 97/00145–4 and 99/03688–4 from the Sao Paulo State Research Foundation (to R.M.B.M.). Back

2 Research fellow supported by the Brazilian Research Council (CNPq Grant 133504/1997-2). Back

Received February 11, 2000.

Revised June 27, 2000.

Accepted July 3, 2000.


   References
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 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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