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Original Studies |
Division of Endocrinology and Metabolism, The Johns Hopkins University School of Medicine (M.D.R., P.W.L., M.A.L.), Baltimore, Maryland 21287-4904; and Washington Hospital Center and Medlantic Research Institute (M.D.R.), Washington, D.C. 20010
Address all correspondence and requests for reprints to: Matthew D. Ringel, M.D., Washington Hospital Center and Medlantic Research Institute, Room 2A 46B, 110 Irving Street NW, Washington, D.C. 20010.
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Abstract |
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Thyroglobulin mRNA was amplified from peripheral blood of 77 patients who had undergone thyroidectomy for well differentiated thyroid cancer, 68 of whom while taking thyroid hormone for TSH suppression. Patient staging was based on the most recent radioiodine scan after thyroid hormone withdrawal. Ten normal control subjects were also studied.
Among patients taking T4, thyroglobulin mRNA was detected in 26 of 33 patients with either thyroid bed or metastatic iodine-avid tissue on most recent withdrawal scan (79%), whereas serum thyroglobulin was detected in 12 of these 33 patients (36%; P < 0.001). Thyroglobulin mRNA was detected in 7 of 35 patients (20%) with negative radioiodine scans, 12 of 19 patients (63%) with radioiodine uptake in the thyroid bed, and all 14 patients with metastases, including 2 patients with antithyroglobulin antibodies. Thyroglobulin mRNA was detected in all 10 normal subjects. Epithelioid cells that stained strongly with antithyroglobulin antibodies were identified in blood.
Detection of circulating thyroglobulin mRNA is a more sensitive marker of residual thyroid tissue or cancer than immunoassay for serum thyroglobulin, particularly in patients treated with thyroid hormone or who have circulating antithyroglobulin antibodies.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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To overcome these difficulties, we developed an assay based on the hypothesis that thyroid cells are present in the circulation of patients with residual or recurrent thyroid cancer. To detect circulating thyroid cells, we used RT-PCR to amplify messenger RNA (mRNA) encoding thyroglobulin, a protein synthesized exclusively by thyroid follicular cells and most differentiated thyroid cancers. mRNA assays employing RT-PCR have been used to identify micrometastases of thyroid (16, 17) and other tumors (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29) by amplification of cancer-specific mRNAs from peripheral blood. These assays can detect as few as 1 cancer cell in 105-106 white blood cells, and in thyroid cancer have been reported to detect cells in patients with metastatic disease.
We report a highly sensitive thyroglobulin mRNA detection assay that is able to detect circulating thyroid cells from patients with metastatic or residual neck thyroid tissue during thyroid hormone therapy and has comparable accuracy to thyroglobulin immunoassay performed after thyroid hormone withdrawal. We also report the identification of circulating thyroid cells in normal individuals.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We studied 77 patients with well differentiated thyroid cancer followed at The Johns Hopkins Hospital and 10 normal subjects with no history or clinical evidence of thyroid disease. Seventy-four of 77 patients had been treated with near-total thyroidectomy and radioactive iodine. Sixty-two patients had papillary carcinoma, and 15 had follicular carcinoma. Blood samples for thyroglobulin mRNA measurement by RT-PCR were obtained in conjunction with samples for measurements of serum TSH (Nichols TSH, 3rd Generation, Quest, Nichols Institute Diagnostics, San Juan Capistrano, CA) and thyroglobulin (Optiquant assay, Kronus, San Clemente, CA). This serum thyroglobulin assay has a sensitivity of 1 ng/mL. All serum samples were first screened to identify samples with interfering antithyroglobulin antibodies (Kronus). The protocol was approved by the Joint Committee on Clinical Investigation, and written informed consent was obtained from each study subject. Laboratory investigators were blinded to the clinical status of the patients.
RNA extraction
RNA was prepared from 3 mL fresh blood. Blood samples were immediately placed in sterile tubes containing 18 mL TRIzol LS (Life Technologies, Gaithersburg, MD) and 3 mL ribonuclease (RNase)-free water. Total RNA was isolated according to the manufacturer’s recommendations. RNA yields ranged from 30–60 µg/sample.
RT-PCR
One microgram of total RNA was heated for 10 min at 70 C and then incubated for 50 min at 42 C in a 19-µL reaction volume containing reverse transcriptase buffer, 200 U Moloney murine leukemia virus reverse transcriptase (Life Technologies), 75 pmol random hexamer primers [Pd (n)6, Pharmacia Biotech, Piscataway, NJ], 10 mmol/L dithiothreitol, 1 mmol/L of each deoxynucleotide triphosphate, and 10 U RNase inhibitor. RT-negative samples were prepared for each individual sample and served as controls for detection of assay contamination. The reaction was heat inactivated at 90 C for 5 min; after cooling to 37 C, 1 µL RNase H (Life Technologies) was added, and the sample was incubated for 20 min at 37 C.
PCR (30) was performed using 2 µL first strand complementary DNA
(cDNA) in a 25-µL volume containing Taq polymerase buffer
(1.5 mmol/L magnesium chloride), 1 mmol/L of each thyroglobulin primer
(Table 1
), 0.1 mmol/L of each
deoxynucleotide triphosphate, and 2.5 U Taq polymerase
(Perkin-Elmer, Foster City, CA). To prevent amplification
of residual genomic DNA, the thyroglobulin PCR primers corresponded to
nucleotide sequences in exons 2 and 4, which are separated by 2
introns that together contain more than 2 kbp (31). After initial
denaturation at 94 C for 4 min, 39 amplification cycles consisting of
denaturation for 1 min at 94 C, annealing for 1 min at 60 C, and
extension for 1 min at 72 C were performed. Final extension was for 4
min at 72 C.
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cDNA, transcripts
that are expressed in blood cells. The sequences of these
intron-spanning PCR primers are listed in Table 1
. RT-negative controls
for the RT-PCR were performed for each individual sample to evaluate
sample contamination. A distilled water template control was also
performed in each PCR reaction. Analysis of amplified thyroglobulin cDNA
After PCR, 8% polyacrylamide-TBE [Tris-borate-ethylenediamine tetraacetate (EDTA)] minigels were poured. Ten microliters of each reaction mixture were electrophoresed at 200 V for 25–30 min in 1 x TBE running buffer using a minigel system (Bio-Rad Laboratories, Hercules, CA) and were visualized by ethidium bromide staining. Restriction analysis using BglII (Life Technologies) and blot hybridization using a radiolabeled internal oligonucleotide probe (mdr 12; 1.5 x 106 cpm/mL) were performed according to previously described techniques (32, 33). Every sample was subjected to blot hybridization to confirm product identity. Several representative amplified DNAs were sequenced directly using a thermal cycle sequencing kit (Amersham, Arlington Heights, IL) and the exonic thyroglobulin PCR primers.
Isolation of circulating thyroglobulin-staining cells
One milliliter of venous blood from two normal subjects and one athyreotic patient with a negative 131I scan and no detectable thyroglobulin by immunoassay and mRNA assay was collected in EDTA tubes. Erythrocytes were lysed by the addition of 5 mL of a solution containing 155 mmol/L NH4Cl, 10 mmol/L KHCO3, and 0.1 mmol/L EDTA. Erythrocyte ghosts were removed by centrifugation at 300 x g at room temperature. The pellet was washed twice by resuspension in 10 mL buffer (5 mmol/L EDTA-0.5% BSA) and centrifugation. Approximately 107 cells were resuspended in 100 µL of a 1:100 dilution of monoclonal antibody to the human TSH receptor (NCL-TSH-R2, Novacastra, Burlingame, CA) for 10 min at 4 C. After incubation, the cells were washed in 10 mL buffer and resuspended in 100 µL of a 1:5 dilution of polyclonal goat antimouse IgG conjugated to paramagnetic microbeads (Miltenyi Biotec, Sunnyvale, CA). The bead/cell mixture was incubated at 4 C for 15 min and then applied to magnetic separating columns (mini-MACS, Miltenyi Biotec) as recommended by the manufacturer.
Isolated cells were collected onto glass microscope slides by centrifugation, air-dried, and washed with Tris-buffered saline. Slides were incubated with a polyclonal antiserum against human thyroglobulin (Immunotech, Westbrook, ME) at full strength, and antibody binding was detected by immunoperoxidase staining using the avidin-biotin-peroxidase technique (Vector Laboratories, Inc., Burlingame, CA). Control reactions were performed using normal human thyroid tissue as a positive control and substitution of saline for thyroglobulin antiserum as a negative control.
Statistical analysis
Statistical analysis was performed using SAS 6.12 (SAS Institute, Inc., Cary, NC). McNemar’s exact test was used to compare thyroglobulin RT-PCR and immunoassay results among three groups of patients: those with negative radioiodine scans, thyroid bed activity, and iodine-avid metastases. P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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To detect low levels of thyroglobulin mRNA, we optimized
conditions for both synthesis of cDNA and amplification of
thyroglobulin cDNA by PCR. One round (39 cycles) of amplification
yielded a product of 348 bp, the size predicted from the thyroglobulin
sequence (31) (Fig. 1). A smaller band of
approximately 300 bp was present in two samples and probably represents
a splice variant (34). Restriction digestion, hybridization with an
internal oligonucleotide probe (Fig. 1), and direct nucleotide sequence
analysis indicated that the 348-bp product was derived from
thyroglobulin mRNA (31).
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The identification of thyroglobulin mRNA in peripheral
blood suggested that thyroid cells might be present in the circulation.
In preliminary experiments, we used Ficoll-Hypaque gradients to
fractionate whole blood samples and were able to amplify thyroglobulin
mRNA from the erythrocyte cell pellet, but not from either the
mononuclear cell or plasma fractions (data not shown). Consequently, we
subjected whole blood from two normal subjects and one athyreotic
patient (as determined by radioiodine scan, thyroglobulin mRNA, and
thyroglobulin immunoassay) to cell sorting using an anti-TSH receptor
antibody and magnetic bead separation, a technique previously used to
identify circulating tumor cells in patients with other malignancies
(35, 36). The isolated cells were further characterized
immunocytochemically using antithyroglobulin antiserum and disclosed
approximately three thyroglobulin-staining epithelioid cells per mL
blood (Fig. 3) in the normal subjects. No
cells were identified in the athyreotic patient or with incubation of
blood from normal subjects with antibody diluted to less than
1:100.
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Seventy-seven adult patients (46 women and 31 men) with well differentiated thyroid cancer were evaluated. Seventy-four of 77 patients were treated with near-total thyroidectomy, and 75 of 77 received postoperative radioiodine ablation with 30–150 mCi before this study. Of the 3 patients treated with less than near-total thyroidectomy, 1 was subsequently treated with radioactive iodine. Diagnostic T4 withdrawal scans of these patients revealed thyroid bed uptake in 2 of 3 and no uptake in 1. Sixty-one of 77 patients had undergone radioiodine scans within 3 yr of evaluation. Four patients had antithyroglobulin antibodies.
Sixty-eight patients were studied while taking T4; 79% of these patients had a serum TSH concentration less than 0.5 mU/L (62% had TSH <0.1 mU/L). Forty-nine of these 68 patients were evaluated after their most recent diagnostic scan. None of these patients was treated with radioiodine in the interval between their most recent diagnostic scan and the time of the laboratory evaluation, and all were clinically stable. Nineteen of the 68 patients were evaluated before their most recent scan; 15 of these 19 patients were subsequently treated with radioiodine (100–200 mCi). Posttherapy scans confirmed the findings of the diagnostic scan in all 15 patients, including thyroid bed uptake in 5 patients and metastatic disease in 10 patients.
Synthesis of cDNA was achieved in all cases, as determined by
successful amplification of either Gs or
interleukin-2 cDNA in each sample. Thyroglobulin mRNA could be
amplified using cDNA prepared from all 10 normal subjects (Fig. 1).
Twenty-eight of 35 patients (80%) with either absent or minimal
residual thyroid bed radioiodine uptake (<0.1% when quantified) had
no detectable thyroglobulin mRNA, whereas 33 of these 35 patients
(94%) had undetectable levels of serum thyroglobulin
(P = 0.125, not significant; Fig. 4).
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Radioiodine scanning disclosed cervical metastases in 3 patients and extracervical metastases in 11 patients. All 14 of these patients had detectable thyroglobulin mRNA, whereas only 8 patients (53%) had detectable serum thyroglobulin concentrations. When 10 of these 14 patients were treated with radioiodine, posttherapy scans revealed metastases at the same locations.
Among patients who were studied while receiving thyroid hormone
therapy, identification of either eutopic or metastatic thyroid tissue
on radioiodine scanning correlated better with the thyroglobulin mRNA
assay than with serum thyroglobulin levels (79% vs. 36%;
P < 0.001; Fig. 4). In four patients with
antithyroglobulin antibodies, both patients with metastases and one of
the two patients with thyroid bed uptake had detectable thyroglobulin
mRNA.
Seven patients were studied both during T4 therapy and
after a 6-week period of withdrawal (Fig. 5). During T4 therapy, two of
these patients with metastases and undetectable serum thyroglobulin
levels had detectable thyroglobulin mRNA.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The assay for circulating thyroglobulin mRNA used in this study appears to have performance characteristics different from those of other assays recently reported. Ditkoff et al. detected circulating thyroglobulin mRNA in all 7 patients with metastatic thyroid cancer whom they studied, but in less than 10% of 78 patients without metastases (16). Moreover, they did not detect thyroglobulin mRNA in blood from normal subjects. Tallini et al. analyzed thyroglobulin mRNA in blood samples obtained from patients with thyroid cancer before surgery (17). Although these patients had intact thyroid glands, circulating thyroglobulin mRNA was detected in only 4 of 7 patients with thyroid cancer and in 4 of 17 patients with benign thyroid nodules before thyroidectomy. After thyroidectomy, thyroglobulin mRNA was detected in 13 of 22 patients with thyroid cancer and none of 6 patients with benign nodules. Among the patients with thyroid cancer, no correlation between clinical stage of disease and assay results was noted. The differences between these investigators’ findings and our own results probably relate to the greater sensitivity of our thyroglobulin mRNA assay. In the absence of TSH stimulation, Ditkoff et al. could not detect thyroglobulin mRNA in samples with fewer than 200 thyroid cells/mL blood. The detection limit for Tallini et al. was equivalent to approximately 50–100 thyroid cells/mL blood. By contrast, we calculated detection of thyroglobulin mRNA in as few as 10 normal thyroid cells/mL blood, a sensitivity similar to our experimental observation of approximately 3 thyroid cells/mL blood. The sensitivity of the assay in patients with thyroid cancer will depend on the level of thyroglobulin expression in the circulating cancer cells and may differ from the calculated sensitivities. The difference in analytical sensitivity between these assays may reflect differences in the design of the oligonucleotide primers, conditions for synthesis of first strand cDNA and PCR, and preparation of RNA. For example, Tallini et al. isolated RNA from the buffy coat layer rather than whole blood. In preliminary experiments, we could detect thyroglobulin mRNA from unfractionated blood samples from normal subjects, but not from the buffy coat fraction (data not shown).
Several lines of evidence argue that the thyroglobulin mRNA we detect in peripheral blood arises from circulating thyroid cells rather than from lymphocytes, in which thyroglobulin gene expression might represent ectopic transcription (37, 38). First, thyroglobulin mRNA was undetectable in RNA samples prepared from Epstein-Barr virus-transformed lymphoblasts and from the blood of most patients who lacked thyroid tissue. Second, thyroglobulin mRNA can be detected in peripheral blood RNA after just one round of amplification. By contrast, detection of the extremely low levels of ectopically expressed mRNAs typically requires two consecutive rounds of nested amplification. Third, we have identified circulating epithelioid cells that express thyroglobulin protein in blood samples from normal subjects. These observations indicate that small numbers of thyroid cells are normally present in the circulation and are probably the source of the thyroglobulin mRNA that we detect by RT-PCR. The thyroglobulin mRNA assay correctly identified all patients with iodine-avid metastatic thyroid cancer on their most recent diagnostic scan while they were receiving thyroid hormone therapy. Moreover, thyroglobulin mRNA was detected in blood samples from 63% of patients who had radioiodine uptake in the thyroid bed. Finally, thyroglobulin mRNA was undetectable in 80% of patients who lacked significant radioiodine uptake. Overall, the correlation between the thyroglobulin mRNA assay performed during thyroid hormone therapy and radioiodine scanning after withdrawal of thyroid hormone was 79%. Similar degrees of correlation have been reported for thyroglobulin immunoassay, but only after withdrawal of thyroid hormone (5, 7, 8, 9, 39, 40). In the seven subjects studied prospectively, thyroglobulin mRNA detection also appeared more sensitive than thyroglobulin immunoassay.
In our series, several patients showed discordance between the thyroglobulin mRNA assay and radioiodine scans. Similar discrepancies between serum thyroglobulin immunoassay and radioiodine imaging findings have been previously described (41, 42) and have led clinicians to rely upon both techniques to monitor thyroid cancer patients. The majority of patients with detectable serum thyroglobulin and negative diagnostic scans will display iodine avid tissue on whole body iodine scans performed after subsequent therapy (41, 42). Therefore, the lower specificity of the thyroglobulin mRNA assay when using diagnostic scans as a "gold standard" for the presence of disease may be due in part to the low sensitivity of the diagnostic scans. Discordance between these two markers, typically detectable serum thyroglobulin in a patient with a negative radioiodine scan, may reflect changes in the pattern of expression of genes encoding thyroglobulin and the sodium iodine symporter (43, 44). Although we evaluated only patients with differentiated thyroid carcinoma, tumors that are most likely to express thyroglobulin and to transport iodine, thyroid cancers can dedifferentiate over time and lose the ability to express thyroid-specific genes. Discordant expression of the sodium iodide symporter and thyroglobulin genes may also account for some of the apparently conflicting results we observed between the thyroglobulin mRNA assay and radioiodine imaging. Consequently, monitoring approaches that rely upon expression of only one thyroid-specific gene may underestimate the tumor burden of some patients.
We also observed discrepancies in some patients between results of the thyroglobulin mRNA assay and the serum thyroglobulin immunoassay. Two patients had no detectable thyroglobulin mRNA, but had measurable thyroglobulin concentrations while taking thyroid hormone, and one patient had a similar pattern after T4 withdrawal. In two of these three patients, the serum thyroglobulin concentration was between 3–7 ng/mL, a range over which there is variable reliability in thyroglobulin immunoassays (14). Alternatively, these patients may have had too few circulating thyroid cells for us to detect thyroglobulin mRNA.
Thyroglobulin mRNA detection has been reported to identify lymph node metastases in patients with thyroid cancer and cervical adenopathy (45). Although we concentrated on detection of circulating cells in peripheral blood, the current assay could be used to identify metastases in lymph nodes, bone, or other tissues from which RNA can be extracted in a similar manner. Thus, detection of thyroglobulin mRNA may enhance the sensitivity of cytology alone in identifying metastatic thyroid tissue.
RT-PCR assays are currently used clinically to measure circulating levels of hepatitis C virus (46) and immunodeficiency virus (47, 48), and provide information that is helpful in the management of patients. Although the clinical utility of RT-PCR assays for cancer is less certain, these assays may complement other conventional tests used to evaluate and monitor patients. The thyroglobulin mRNA assay may be particularly valuable in the approximately 10–25% of thyroid cancer patients who have antithyroglobulin antibodies that interfere with thyroglobulin immunoassays (49, 50, 51). Among our four study patients with detectable serum antithyroglobulin antibodies, the two with metastatic disease were correctly identified even while taking thyroid hormone. Larger studies of patients with antithyroglobulin antibodies are needed to confirm this apparent advantage of mRNA detection. Another advantage of the thyroglobulin mRNA assay is its high predictive value in patients who are still taking doses of thyroid hormone that suppress TSH secretion. Because the discovery of any thyroid tissue in a previously athyreotic thyroid cancer patient has clinical importance, further clinical assessment and radioiodine scanning may be indicated in patients who have detectable thyroglobulin mRNA. By contrast, we believe that low risk patients with undetectable thyroglobulin mRNA may require less frequent radioiodine scanning and may benefit from fewer periods of induced hypothyroidism. Quantitation of thyroglobulin mRNA detection will be needed for serial measurements in patients to identify disease progression, which is required for optimal utility of the assay (52). Confirmation of this approach will require large prospective trials.
In conclusion, thyroglobulin mRNA is detectable in the blood of normal subjects as well as most patients with residual thyroid cancer who are taking thyroid hormone. Our thyroglobulin RT-PCR assay is highly sensitive and appears to circumvent two limitations of contemporary serum thyroglobulin immunoassaysL the need for TSH stimulation to enhance assay sensitivity and analytical interference by thyroglobulin autoantibodies. With further assay refinement, particularly the development of a quantitative assay, measurement of thyroglobulin mRNA in peripheral blood could become widely available as a sensitive technique for the detection of well differentiated thyroid cancer tissue in previously treated patients.
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Acknowledgments |
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Footnotes |
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Received July 21, 1998.
Revised August 28, 1998.
Accepted September 3, 1998.
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References |
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subunit of the stimulatory G
protein of adenylyl cyclase in Albright hereditary osteodystrophy. J Clin Endocrinol Metab. 76:1560–1568.[Abstract]
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1 mL blood; A) or per 1 mL blood (B)]. By ethidium bromide
staining, thyroglobulin mRNA equivalent to 10 cells/mL blood was
visualized (B). In the absence of added thyroid tissue RNA (indicated
as 0 addition), thyroglobulin mRNA was not detected in RNA from the
lymphoblastoid cells (A) or from whole blood from the athyreotic
patient (B). A positive control (thyroid RNA) and negative control
reactions for both thyroid and lymphoblastoid RNA in which reverse
transcriptase (RT negative) was omitted or water was substituted for
RNA (H2O) are shown.
