0021-972X/99/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 1999 by The Endocrine Society
Vol. 84, No. 11 Printed in U.S.A.
Quantitative Reverse Transcription-Polymerase Chain Reaction of Circulating Thyroglobulin Messenger Ribonucleic Acid for Monitoring Patients with Thyroid Carcinoma* MATTHEW D. RINGEL, PINA L. BALDUCCI-SILANO, JEFFERY S. ANDERSON, CAROLE A. SPENCER, JILL SILVERMAN, YVONNE H. SPARLING, GARY L. FRANCIS, KENNETH D. BURMAN, LEONARD WARTOFSKY, PAUL W. LADENSON, MICHAEL A. LEVINE, AND R. MICHAEL TUTTLE Department of Medicine (M.D.R., P.L.B-S., Y.H.S., K.D.B., L.W.), Washington Hospital Center and Medstar Research Institute, Washington, DC 20010; Department of Clinical Investigation (J.S.A., G.L.F., R.M.T.), Walter Reed Army Medical Center, Washington, DC 20307; Department of Medicine (C.A.S., J.S.), University of Southern California, Los Angeles, California 90033; Departments of Medicine (P.W.L.) and Pediatrics (M.A.L.), Johns Hopkins University, Baltimore, Maryland 21205 ABSTRACT Patients with thyroid cancer are monitored for disease recurrence by measurement of serum thyroglobulin (Tg) and iodine-131 (131I) scanning. To enhance sensitivity and to circumvent antibodies that interfere with Tg immunoassays, we have developed RT-PCR assays that detect circulating thyroid messenger RNA (mRNA) transcripts. We now report results using a sensitive quantitative Tg mRNA assay (Taqman; ABI, Foster City, CA) in comparison with immunoassay in patients previously treated for thyroid cancer. We evaluated 107 patients: 84 during T4 therapy, 14 after T4 withdrawal, and 9 at both time points. All patients had near-total thyroidectomy, and 92% received postoperative 131I. Serum TSH, Tg protein, and Tg mRNA were measured. Patients were grouped based on most recent 131I scan or pathologically confirmed disease as having no detectable thyroid tissue (n 5 33), thyroid bed uptake (n 5 37), cervical/regional adenopathy (n 5 21), or distant metastases (n 5 16). During T4 therapy, median (range) Tg mRNA values (pg Tg Eq/mg thyroid RNA) for the groups were 1.5 (0 –26.8), 9.4 (0.5–90.0), 15.4 (0.2–92), and 12.4 (1.9 – 16.6), respectively. Using a value of 3 pg Tg Eq/mg thyroid RNA as cut-point, Tg mRNA was positive in 38% of patients with no uptake, 75% with thyroid bed uptake, 84% with cervical/regional disease, and 94% with distant metastases. The median Tg mRNA value for pa-
T
HYROID cancer treatment frequently includes neartotal thyroidectomy and radioiodine therapy, followed by long-term suppression of TSH, although there is debate regarding use of this paradigm for patients with small, welldifferentiated tumors (1). With appropriate therapy, patients with thyroid cancer enjoy an excellent prognosis; however, recurrence develops in 20 – 40% of patients over decades of Received April 29, 1999. Revision received August 12, 1999. Accepted August 19, 1999. Address correspondence and requests for reprints to: Matthew D. Ringel, M.D., Co-Director Laboratory of Molecular Endocrinology, Section of Endocrinology, Washington Hospital Center, 110 Irving Street, NW, Room 2A-46B, Washington, DC 20010. E-mail:
[email protected]. * Funded by grants from Medlantic Research Institute, Knoll Pharmaceutical Co. (TRAC), and the Department of Defense. These data were presented in part at the 71st annual meeting of the American Thyroid Association (1998) and the 81st Annual Meeting of the Endocrine Society (1999).
tients with no uptake was lower than the median values for patients with thyroid bed uptake (P 5 0.009) or with detectable thyroid tissue at any site (P 5 0.010). Patients with negative 131I whole body scans were also less likely to have detectable Tg mRNA levels than were patients with thyroid bed uptake (P , 0.001) or any detectable thyroid tissue at any location (P , 0.001). Similar differences between these groups were seen after T4 withdrawal and for the 23 patients with circulating anti-Tg antibodies, when analyzed separately. Eight of the nine patients studied with low and high TSH concentrations displayed greater amounts of circulating Tg mRNA after T4 withdrawal. In three patients followed prospectively, the amount Tg mRNA correlated with the presence and absence of cervical metastases. In conclusion, we have demonstrated that a quantitative Tg mRNA assay can identify thyroid cancer patients with recurrent or residual thyroid tissue with greater sensitivity and similar specificity to Tg immunoassay during T4 therapy. The assay was unaffected by anti-Tg antibodies, responded to TSH-stimulation, and was reduced after surgical removal of metastases. These data suggest that this quantitative Tg mRNA assay may be a sensitive marker of tumor recurrence or response to therapy, particularly in patients with anti-Tg antibodies. (J Clin Endocrinol Metab 84: 4037– 4042, 1999)
follow-up (2, 3). It is, therefore, necessary to monitor patients with thyroid cancer to detect recurrent disease. Patients are monitored for thyroid cancer recurrence or progression primarily by periodic radioiodine scanning and serum thyroglobulin (Tg) measurement, techniques that rely on expression of thyroid-specific genes that are stimulated by TSH. Serum Tg measurement is limited by a low sensitivity during TSH suppression therapy and by the presence of circulating anti-Tg antibodies that interfere with Tg immunoassays in as many as 15–25% of patients with thyroid cancer (4). In an effort to both avoid antibody interference and enhance the sensitivity of Tg testing, there has been an effort to develop alternative methods to identify thyroid tumor recurrence using nonimmunoassay techniques. Identification of circulating thyroid cells using RT-PCR amplification of thyroid-specific messenger RNA (mRNA) transcripts represents a promising alternative assay system.
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We and others have previously reported qualitative Tg RT-PCR assays that detect circulating thyroid-specific transcripts (5–7). We have previously reported that during T4 therapy, our qualitative Tg mRNA assay was more sensitive and had similar specificity compared with immunoassay (7). We were surprised to determine that although most athyreotic individuals had negative qualitative Tg mRNA assays, some patients with thyroid bed uptake and all normal subjects had detectable circulating Tg mRNA. To confirm this finding, we detected circulating thyroid cells by magnetic cell sorting in peripheral blood of normal individuals (7). The identification of presumably normal circulating epithelial cells has been recently reported by several other groups using similar cell-sorting techniques (8, 9). These qualitative assays, although potentially useful, are not automated and are limited by the inability to monitor progression of disease or response to therapy for individual patients. To expand the potential usefulness of this new detection assay, we developed and reported a quantitative Tg RT-PCR assay using an in-cycle fluorescent detection system (Taqman; ABI) in normal subjects (10). This RT-PCR assay system measures Tg mRNA using a gene-specific probe that fluoresces only when Tg template is being replicated. Thus, by continuously measuring the amount of fluorescence in a reaction sample, the cycle at which the PCR curve is most linearly dependent on starting mRNA amount (threshold cycle) can be determined. Use of several samples of known starting Tg mRNA concentrations allows for creation of standard curves over a broad range. Threshold cycles for simultaneously amplified patient samples are then plotted on the standard curve to determine the amount of circulating Tg mRNA. By contrast, typical PCR-enzyme-linked immunosorbent assays are completed and measured at the end of a fixed cycle; therefore, the assays may lose linearity of input/product relationship at low or high template concentrations (11). We believe the reliability of this system along with its automated format may make it more applicable for potential clinical use. In the present study, we report the first data using this in-cycle quantitative Tg mRNA RT-PCR detection system in 107 patients with thyroid cancer, including 23 patients with anti-Tg antibodies. Subjects and Methods Subjects We evaluated 107 patients with thyroid cancer from The Washington Hospital Center, The Johns Hopkins Hospital, and the University of Southern California. All patients had prior near-total thyroidectomy, and 92% had postoperative radioiodine ablation. Of the 107 patients, 87 had papillary carcinoma, 19 had follicular carcinoma, and 1 had anaplastic thyroid cancer. There were 39 men and 68 were women. A total of 84 patients were evaluated only during T4 therapy, 14 were evaluated only after T4 withdrawal, and 9 patients were evaluated before and after T4 withdrawal. Sixty-eight of the 84 patients (81%) studied during T4 therapy only had diagnostic scans within 3 years of the sample (64% within 12 months of the sample). The remaining patients either had distant metastases and were followed by nonradioiodine imaging after therapy with over 800 mCi of iodine-131 (131I) or were disease-free for greater than 10 years and were followed with less frequent radioiodine imaging with at least yearly measurements of serum Tg concentrations and physical examination. Samples were obtained for simultaneous measurement of serum TSH concentrations (Nichols third generation
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assay), Tg concentrations by either RIA (University of Southern California, Los Angeles, CA; n 5 41) or Immunoradiometric (Kronus, San Clemente, CA; n 5 66), and whole blood Tg mRNA. All serum samples were screened for presence of anti-Tg antibodies (Kronus). Seventy of 84 patients (83%) had TSH concentrations below 0.1 mU/L, and 96% had values below 1.5 mU/L. All patients who were studied after T4 withdrawal had TSH concentrations more than 30 mU/L. Tg mRNA assay was also performed on whole blood obtained from 16 normal subjects with no known history of thyroid disease. Serum TSH concentrations were measured on 7 of 16 patients and were normal in all cases. Whole body radioiodine scanning was performed 48 –72 h after a 2–5-mCi dose of 131I. Negative scans were those with no detectable uptake on visual inspection by the nuclear medicine physician or with less than 0.1% uptake when available. No patients in this study were evaluated after administration of recombinant human TSH. Laboratory investigators were blinded to the clinical status of each subject. The protocol was approved by the institutional review boards at the participating institutions, and informed consent was obtained.
Tg RT-PCR Total RNA was isolated from 3 mL whole blood by immediately placing the sample into 18 mL TRIzol LS (Life Technologies, Inc., Gaithersburg, MD) and 3 mL ribonuclease-free water, shaking vigorously for 30 sec, and extracting the RNA after the manufacturer’s suggested procedure. Whole blood total RNA (250 ng) was reverse transcribed to complementary DNA (cDNA) in 20 ml using random hexamer primers as per the manufacturer’s recommendations for the Taqman system (PE Applied Biosystems, Foster City, CA) (11). Final reaction conditions were 13 Taqman buffer, 5.5 mm MgCl, 500 mm each dNTP, 2.5 mm random hexamer primer, 0.4 U/ml ribonuclease inhibitor, and 1.23 U/ml Multiscribe reverse transcriptase (all purchased from PE Applied Biosystems). Reaction mixture was incubated at 25 C for 10 min, 48 C for 30 min, and heat-inactivated at 95 C for 5 min. Twenty-five percent of the cDNA was used in the subsequent quantitative PCR reactions. To develop a quantitative RT-PCR assay for Tg, we used the PRISM 7700 detection system (PE Applied Biosystems), in which detection of PCR products is accomplished using a sequence-specific oligonucleotide probe. Quantitative PCR is performed in 96-well plates using intronspanning Tg-specific primers and the antisense fluorogenic probe labeled with a 59 6-carboxy-fluoroscein (FAM) reporter dye and a 39 6-carboxy-tetramethyl-rhodamine (TAMRA) quencher (10). To fluoresce, the probe must bind to its complementary sequence and be cleaved by the 59 exonuclease activity of Taq polymerase. A signal is detected only if the probe binds and the template is active. The amount of specific product is measured throughout each cycle and recorded, allowing for determination of the cycle at which the slope of the PCR curve is most dependent on initial concentration of cDNA template (threshold cycle). This threshold cycle occurs at the most linear portion of the logarithmic phase of the PCR curve and allows for creation of standard curves based on threshold cycle and starting concentration. The primers used amplify an 87 bp product from bp 262 to 348 of the Tg cDNA sequence. The primer and probe sequences were: sense, 59GTGCCAACGGCAGTGAAGT-39; antisense, 59-TCTGCTGTTTCTGTAGCTGACAAA-39; probe, 59-FAM-ACAGACAAGCCACAGGCCGTCCTTAMRA-39. Each sample was assayed in triplicate with the following reaction conditions: 50 }l total by volume; 13 Taqman buffer; 0.05% gelatin; 0.01% Tween 20; 8% glycerol; 5.5 mm MgCl; 200 mm dATP, dCTP, and dGTP; 400 mm dUTP; 200 mm of each Tg primer; 100 mm antisense probe; 0.01 U/ml AmpErase UNG; and 0.025 U/ml Amplitaq Gold (all purchased from PE Applied Biosystems). Reaction mixtures were incubated for 2 min at 50 C, denatured for 10 min at 95 C, and subjected to 40 cycles of a two-step PCR consisting of a 15-sec denaturation step at 95 C and a 1-min annealing/extension step at 60 C. To ensure amplification of the appropriately sized product, samples were electrophoresed through 3% agarose gels and visualized with ethidium bromide. Samples omitting reverse transcriptase or template were included as negative controls in each set of reactions. To further confirm product identity, forward and reverse strands of several Tg RT-PCR products were sequenced with a cycle sequencing kit (PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit; PE Applied Biosystems). Sequencing was performed using a denaturing gel of
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4.75% acrylamide, 8.3 m Urea, 13 TBE buffer run at 2500V at 30 C for 10 h on an automated sequencer (373 DNA Sequencer; PE Applied Biosystems). To evaluate for cDNA quality, 25% of the cDNA in each sample was used to amplify Gas as a control template, as described previously (7).
Assay Quantitation To quantify the amount of circulating Tg mRNA, we created a standard curve to calibrate the assay. Total RNA was isolated from normal thyroid gland removed at surgery for a benign thyroid nodule using TRIzol (Life Technologies, Inc.), as per the manufacturer’s recommendations. Serial dilutions were made to produce a standard curve that ranged from 1.0 –10,000 pg of thyroid RNA using six concentrations. Standard (calibration) curves using the six concentrations were performed in triplicate for six separate reactions, as were quality control standards for each concentration point. The values were calculated as described in detail previously (10). Briefly, the mean interassay coefficient of variation between the six PCR reactions was 1.6%. The intraassay variability was less than 1% at amounts 3.2 pg and greater. This value rose to 3– 6% for concentrations less than 1 pg. The standard curve displayed a strong linear relationship when plotted as threshold cycle vs. concentrations, with r values of 0.996 – 0.998. Intra-assay variability was 17–22% when calibrated to the actual blood draw, thus, including the phlebotomy, RNA isolation, and RT (10). Triplicate six-point calibration standard curves were run in each 96-well plate used in this experiment, and average values were determined. Samples were also run in triplicate, averaged, and quantified using the threshold cycle curve created with the total thyroid RNA, as described previously (10). Results are reported as pg Tg mRNA Eq/mg thyroid mRNA.
Statistical Analysis Statistical analysis was performed using SAS 7.0 (SAS Institute, Inc., Cary, NC). Differences in the distribution of Tg mRNA values between stages were examined with the median test, and the proportion of patients with detectable values were compared between stages by Z scores. Agreement between Tg mRNA and immunoassay was assessed by the simple k coefficient (12). Patients had one of two immunoassays performed, as noted above. Quantitation using these two assays correlate well in the absence of anti-Tg antibodies (C. A. Spencer, unpublished data) with r2 values of ;0.99 in the range of detectable to 1000 ng/mL. Similar levels of agreement for each immunoassay with Tg mRNA detection were seen. To minimize an effect of using two immunoassays, mRNA assay and immunoassay results were compared only by the number of individuals with detectable levels, rather than actual values. Agreement of Tg mRNA assay results between patients with and without anti-Tg antibodies was evaluated by the simple k coefficient.
Results Clinical Validation of Quantitative Tg mRNA Assay
To clinically validate the Tg mRNA assay, the 107 subjects were grouped by clinical stage based on most recent radioiodine scan or presence of pathologically defined metastases into one of four categories: no uptake (n 5 33); thyroid bed uptake (n 5 37); local/regional metastases (n 5 21); and distant metastases (n 5 16). Of these 107 patients, 84 were evaluated during T4 suppression alone, 14 were evaluated
FIG. 1. Tg mRNA by diagnostic radioiodine scan stage during T4 therapy. A total of 93 samples were obtained during T4 therapy. Individual data points for patients in each stage are depicted as clear circles, and median values are depicted as a solid line in each group. Some data points overlap; median values and ranges are shown in Table 1. The reference (cut-point) line for assay positivity is at 3 pg Tg mRNA Eq/mg thyroid RNA.
after T4 withdrawal, and 9 were evaluated at both time points. Median Tg mRNA values for the different groups of patients evaluated during T4 suppression were analyzed. Data from the 93 patients studied during T4 therapy are included (see above). Patients with no uptake had lower circulating levels of Tg mRNA than those with thyroid bed uptake (P 5 0.009) or with detectable thyroid tissue at any location (P 5 0.001). Fig. 1 displays the actual data points for these patients separated into groups by location of iodine uptake on diagnostic scan. Using a value of 3 pg Tg mRNA Eq/mg thyroid RNA as a cut point, only 38% of patients with no uptake had positive Tg mRNA levels compared with 75% of patients with thyroid bed uptake (P 5 0.001) and 84% of patients with thyroid tissue (P , 0.001) at any location (Table 1). Median Tg levels were not statistically different for patients with thyroid bed uptake and patients with local or distant metastases (P 5 0.467; Table 1). Patients evaluated after T4 withdrawal showed a similar correlation between Tg mRNA assay results greater than 3 pg Tg mRNA Eq/mg thyroid RNA and clinical stage, but the number of individuals was too small (n 5 23) for meaningful conclusions. All normal subjects had detectable circulating Tg mRNA (Fig. 1). Twelve patients were studied prospectively: nine during T4 suppression and after T4 withdrawal, 2 before and after surgical removal of cervical metastases, and one before and after presentation with new cervical metastases. Samples from patients with multiple tests were run on the same assay
TABLE 1. Tg mRNA by diagnostic radiodine scanning during L-T4 therapy in the presence or absence of anti-Tg antibodies Group
No uptake Thyroid bed Local/regional Distant metastases
Number
Tg mRNA median (range)
Tg Ab1
Tg Ab2
Tg Ab1
Tg Ab2
2 4 8 1
24 28 10 15
0.9 (0.5–1.3) 31.9 (9.4 –90.0) 23.5 (2.8 –92.0) 13.4 (—)
2.2 (0.0 –26.8) 7.3 (0.5–32.4) 11.2 (0.2–54.0) 11.6 (1.9 –54.0)
Tg mRNA .3.0
Serum Tg protein (% detectable, Tg Ab2)
38% 75% 89% 94%
23% 15% 60% 86%
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to eliminate interassay variations. Eight of 9 patients studied who had low and high TSH values had an increase in circulating Tg mRNA when TSH was elevated (Fig. 2). Both patients who had surgical removal of cervical metastases displayed a reduction in the subsequent Tg mRNA determination, whereas the patient with new neck metastases was found to have a rise in Tg mRNA (Fig. 3). These prospective data suggest that circulating Tg mRNA levels correlate with the presence and absence of thyroid tissue and that TSH responsiveness is maintained. Comparison between Tg mRNA Assay and Immunoassay
To further clinically validate Tg mRNA measurement, results were compared with simultaneously measured Tg protein by immunoassay (Table 1). The percentage of individuals with positive Tg mRNA assays (.3 pg Eq Tg mRNA/mg thyroid RNA) were compared with those with detectable Tg immunoassay results. Patients with circulating anti-Tg antibodies were excluded from the immunoassay group. The percentage of patients with no detectable uptake who had positive Tg determinations during T4 therapy were similar for the mRNA assay and the immunoassay (38% and 23%, respectively); however, there was poor agreement between the two methods. The discordant results were primarily individuals with a positive Tg mRNA assay and a negative immunoassay. This discrepancy was most evident in the number of patients with positive Tg mRNA assays and immunoassays in the group with thyroid bed uptake (75% vs. 15%, respectively) and local/regional metastases (89% and 60%, respectively), underscoring the greater sensitivity of the mRNA assay during T4 suppression (Table 1). In contrast to the Tg mRNA assay, no statistical difference in the number of patients with detectable Tg immunoassays was identified between patients with no uptake vs. thyroid bed uptake (P 5 0.221) during T4 therapy. The percentage of patients with detectable Tg protein was greater for patients with thyroid tissue detected at any location vs. those with no uptake (P 5 0.031), but this was less significant than the relationship noted for Tg mRNA assay (Table 1). Five patients in the
FIG. 3. Quantitative Tg mRNA assay with and without cervical metastases. Two patients underwent surgical removal of cervical metastases, and one patient developed new cervical metastases during the course of the study. Quantitative Tg mRNA levels correlated with the presence and absence of metastases, including in the two patients with anti-Tg antibodies.
group with no uptake had positive Tg immunoassay results and negative mRNA assay results. Four of these five individuals had serum Tg protein concentrations below 5 ng/ mL, values of uncertain clinical or biochemical significance. The relationship between the two assays and clinical stage was similar for the 23 sets of values obtained after T4 withdrawal. Two patients with metastases had marked elevations in Tg immunoassay, but negative or only modest elevations in Tg mRNA levels. This observation supports the notion that these two tests may be complementary and might reflect different tumor characteristics, such as the number of circulating cancer cells associated with the tumor or the ability of a tumor to manufacture and secrete proteins. Alternatively, these tumors may have splice variants of Tg or have polymorphisms in the Tg gene that do not allow proper annealing of these particular PCR primers. Tg mRNA in patients with anti-Tg antibodies
Because the Tg mRNA assay may be particularly useful for patients with anti-Tg antibodies, we evaluated this group of 23 individuals. Of the 23 patients, 5 had no evidence of disease, 4 had thyroid bed uptake, 9 had local or regional disease, and 5 had distant metastases; fifteen patients were studied during T4 therapy, and eight patients were studied only after T4 withdrawal. The percentage of patients with anti-Tg antibodies who had positive Tg mRNA levels was similar to patients without anti-Tg antibodies by stage of disease. Prospectively, the Tg mRNA assay retained TSH responsiveness in two patients with antibodies (Fig. 2) and correlated with the presence and absence of cervical metastases in two patients with antibodies (Fig. 3). FIG. 2. Quantitative Tg mRNA assay on and off L-T4. Nine patients were evaluated during TSH suppression and then after subsequent T4 withdrawal in preparation for radioiodine scanning. All patients had TSH concentrations more than or equal to 30 IU/L, and mRNA samples were run in triplicate in the same assay. Eight of nine patients displayed a rise in circulating Tg mRNA in response to TSH stimulation, including both with anti-Tg antibodies, depicted as clear diamonds.
Discussion
Thyroid cancer has accounted for ;1% of all malignancies diagnosed in the United States since 1985 (13). However, the number of individuals with a history of thyroid cancer at risk for recurrence is much greater due to the young age of presentation and the low tumor-specific mortality. The Tg im-
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munoassays currently used to detect recurrences are limited by poor sensitivity while patients are on T4 therapy and by interfering anti-Tg antibodies. Therefore, more sensitive techniques that are unaffected by anti-Tg antibodies are needed. Molecular diagnostic assays that measure tissue-specific gene products have created a new level of sensitivity for monitoring patients with a variety of diseases including human immunodeficiency virus, hepatitis, and cancer. In viral diseases, the amount of circulating virus, or viral load, corresponds with response to therapy and prognosis (14, 15). In cancer, RT-PCR assays have detected metastatic circulating cells and also lymph node and bone marrow metastases (16 –21). To maximize the specificity of these highly sensitive assays, it is preferable to amplify tissue-specific genes that are highly expressed only in the cell type of interest. Moreover, the specificity for cancer is enhanced by the complete eradication of all normal tissue expressing the gene or by amplification of a tissue and tumor-specific transcript. For these reasons, thyroid cancer serves as an excellent model to test this type of assay. Initial thyroid mRNA assays amplified several different thyroid tissue transcripts qualitatively, the most sensitive and highly expressed of which was Tg (6, 7, 22). Our particular qualitative Tg mRNA assay was more sensitive at detecting recurrent or residual thyroid cancer than Tg immunoassay, and it seemed to be unaffected by anti-Tg antibodies (7). In the present study, we report the initial results of a clinical validation study of a new quantitative Tg mRNA assay using Taqman (ABI) technology in thyroid cancer. We initially developed standard calibration curves and tested the characteristics of the assay over broad concentration ranges (10). These studies revealed that the assay was highly accurate and was extremely sensitive over a range from 1–1500 pg Tg mRNA Eq/mg thyroid RNA. This variability increases when one performs calibration curves starting at the level of phlebotomy and RNA isolation (10). Based on the encouraging characteristics of this assay, in the present study, we evaluated 84 patients with thyroid cancer during T4 therapy and compared the results with the most recent scan or pathology data in similar manner to or prior study (7). In addition, 12 patients were evaluated prospectively. In comparison with simultaneously performed Tg immunoassays, the mRNA detection method was more sensitive, detecting more patients with thyroid bed uptake and local/ regional disease than the immunoassay. Using either technique, there were several patients with positive Tg assays and negative diagnostic scans. The discrepancy of positive Tg immunoassay and negative diagnostic scan is well-described (23–25), and the clinical significance is debated (26, 27). Several studies have demonstrated eradication of radiographic evidence of recurrent thyroid cancer and a reduction in subsequent serum Tg concentrations after 131I therapy for patients with these results (23–25). Based on these observations, it is not yet clear whether the positive Tg mRNA in these patients is clinically relevant or represents a “false positive” result. It is possible that some of these positive assays reflect the detection of Tg transcribed in nonthyroidal cells (ectopic or illegitimate transcription). The use of a single PCR reaction of 40 cycles rather than a two-step “nested”
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protocol makes this less likely. In addition, the significant relationship between Tg mRNA levels and extent of disease on diagnostic 131I scan argues against this possibility. To further evaluate the clinical relevance of the quantitative assay in individual patients, we evaluated a small number of subjects prospectively. Eight of nine individuals studied during T4 suppression and subsequently after T4 withdrawal displayed a rise in circulating Tg mRNA levels, including both patients with anti-Tg antibodies. Six of the seven patients without anti-Tg antibodies, but neither of the two patients with anti-Tg antibodies had a rise in Tg immunoassay. Tg mRNA samples were run on the same assays to minimize any potential interassay differences. The reason for the apparent TSH responsiveness is not yet certain; it could reflect either an increase in the number of circulating cells or TSH receptor signaling in the circulating cancer cells. If it is related to an enhancement of Tg gene transcription in response to TSH, an acute rise in TSH, such as that seen with administration of rhTSH, may be expected to cause a greater elevation of Tg mRNA than T4 withdrawal. Two patients had surgery for radiographic and clinically detected cervical metastases and one individual developed new cervical metastases during the study. Tg mRNA correlated with the presence or absence of disease in all three cases, even in the presence of anti-Tg antibodies in two of the three cases. Some patients evaluated during T4 therapy had detectable Tg mRNA levels and undetectable Tg immunoassays. The majority of these individuals had thyroid bed uptake on diagnostic scan. We believe this likely reflects the greater sensitivity of the mRNA assay. In a few cases, patients had detectable Tg immunoassay measurements but undetectable mRNA levels. Most of these patients had no uptake on diagnostic scans and had immunoassay values in the 0.5–2.0 range, perhaps reflecting false positive immunoassays. However, one individual had distant metastases and a highly positive Tg immunoassay. This individual had negative qualitative and quantitative mRNA assays on several different RNA samples with positive control amplification. The qualitative and quantitative assays share an antisense primer, but have different sense primers. Possible reasons for this discrepant result include the presence of a splice variant of Tg mRNA or the presence of a polymorphism at the 39 end of the antisense primer. Alternatively, the result may be accurate and the patient’s cancer cells may efficiently secrete Tg protein, but are no longer circulating. The most obvious advantage to the Tg mRNA assay compared with immunoassay is the absence of interference by circulating anti-Tg antibodies. Although some laboratories have reported accurate measurement of Tg protein in the presence of antibodies using Tg recovery techniques (28, 29), these techniques are not widely used and their accuracy is debated (4, 30). Using the Tg mRNA assay, we identified positive correlations between the extent of iodine-avid tissue on scan and the mRNA level in the presence or absence of antibodies. Similarly, our prospective data suggest a quantitative correlation with the presence or absence of thyroid cancer in the presence of antibodies. These data, and the method itself, which includes the separation of the RNA from
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protein, the transcription of RNA, and the specific amplification of Tg DNA, make antibody interference improbable. Several refinements in the assay are required. Multiplex quantitative PCR amplification of Tg using at a variety of different primer pairs, as well as a control sequence, may provide the most clinical information. We have quantitatively amplified the Na, I symporter (22) and the TSH receptor (unpublished data) from peripheral blood samples from patients and normal subjects. Although these transcripts are not completely thyroid-specific (31, 32) and seem to be less sensitive than Tg, they may provide confirmatory evidence of thyroid tissue and treatment-related information. Conversion of the thyroid RNA standard curve to Tg mRNA detected using in vitro transcribed Tg mRNA is ongoing. This will allow for determination of an actual amount of Tg transcript and alleviate reliance on surgical thyroid tissue. The goal of amplifying a thyroid cancer-specific transcript is more problematic. There seems to be no one thyroid cancer-specific gene, although the presence of rearrangements involving the ret-proto-oncogene (PTC genes) may be relatively specific (6). Single-cell RT-PCR of isolated circulating thyroid cells for more general cancer specific transcripts may be another possibility to allow for specific detection of cancer cells in peripheral blood. In conclusion, we describe a new quantitative Tg RT-PCR assay for the detection of recurrent or residual thyroid cancer that is highly reproducible and has excellent performance characteristics. Quantitative detection of circulating Tg mRNA using this method corresponds to the presence of iodine uptake on diagnostic scan. Moreover, Tg mRNA assay seems to be more sensitive than concomitantly performed Tg immunoassay in detecting iodine avid tissue, and its accuracy is unaffected by circulating anti-Tg antibodies. For individual patients followed prospectively, quantitative levels increase in response to TSH stimulation in the majority of individuals tested and correlates with the removal or development of new recurrence. These data suggest a potentially important role for quantitative Tg mRNA assay in the management of patients with thyroid cancer, particularly in the presence of anti-Tg antibodies. Additional prospective clinical studies of this new assay system are warranted. References 1. Singer PA, Cooper DS, Daniels GH, et al. 1996 Treatment guidelines for patients with thyroid nodules and well-differentiated thyroid cancer. Arch Intern Med. 156:2165–2172. 2. Mazzaferri EL, Jhiang SM. 1994 Long-term impact of initial surgical and medical therapy on papillary and follicular thyroid cancer. Am J Med. 97:418 – 428. 3. Hay I. 1990 Papillary thyroid carcinoma. Endocrinol Metab Clin North Am. 19:545–576. 4. Spencer CA, Wang CC. 1996 Thyroglobulin measurement: techniques, clinical benefits and pitfalls. Endocrinol Metab Clin North Am. 24:841– 864. 5. Ditkoff BA, Marvin MR, Yemul S, et al. 1996 Detection of circulating thyroid cells in peripheral blood. Surgery. 120:959 –965.
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