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Original Studies |
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
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: mxr9{at}mhg.edu
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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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.
We and others have previously reported qualitative Tg RT-PCR assays that detect circulating thyroid-specific transcripts (5, 6, 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.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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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 assay), Tg concentrations by either RIA (University of Southern California, Los Angeles, CA; n = 41) or Immunoradiometric (Kronus, San Clemente, CA; n = 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 µl 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 1x Taqman buffer, 5.5 mM MgCl, 500 µM each dNTP, 2.5 µM random hexamer primer, 0.4 U/µl ribonuclease inhibitor, and 1.23 U/µl 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 intron-spanning Tg-specific primers and the antisense fluorogenic probe labeled with a 5' 6-carboxy-fluoroscein (FAM) reporter dye and a 3' 6-carboxy-tetramethyl-rhodamine (TAMRA) quencher (10). To fluoresce, the probe must bind to its complementary sequence and be cleaved by the 5' 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, 5'-GTGCCAACGGCAGTGAAGT-3'; antisense, 5'-TCTGCTGTTTCTGTAGCTGACAAA-3'; probe, 5'-FAM-ACAGACAAGCCACAGGCCG-TCCTTAMRA-3'.
Each sample was assayed in triplicate with the following reaction
conditions: 50 
l total by volume; 1x Taqman buffer; 0.05% gelatin;
0.01% Tween 20; 8% glycerol; 5.5 mM MgCl; 200
µM dATP, dCTP, and dGTP; 400 µM dUTP; 200
µM of each Tg primer; 100 µM antisense
probe; 0.01 U/µl AmpErase UNG; and 0.025 U/µl 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
4.75% acrylamide, 8.3 M Urea, 1x 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
G
s 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/µg 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 
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 coefficient.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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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 = 33); thyroid bed uptake (n = 37); local/regional metastases (n = 21); and distant metastases (n = 16). Of these 107 patients, 84 were evaluated during T4 suppression alone, 14 were evaluated 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
= 0.009) or with detectable thyroid tissue at any location
(P = 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/µg 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 = 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 =
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/µg thyroid RNA and clinical stage, but
the number of individuals was too small (n = 23) for meaningful
conclusions. All normal subjects had detectable circulating Tg mRNA
(Fig. 1).
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). 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.
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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/µg 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 = 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 = 0.031), but this was less significant than the
relationship noted for Tg mRNA assay (Table 1). Five patients in the
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).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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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 immunoassays 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, 17, 18, 19, 20, 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/µg 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, 24, 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, 24, 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" 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 3' 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 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.
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Footnotes |
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Received April 29, 1999.
Revised August 12, 1999.
Accepted August 19, 1999.
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