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Endocrine Care |
Institute of Endocrine Sciences (L.F., N.C., M.B., D.M., G.V., P.B.-P.), University of Milan, Ospedale Maggiore Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS); Istituto Auxologico Italiano IRCCS (L.P., E.G.); and Laboratory of Molecular Biology (A.M., M.R., E.P., A.M.D.B.), Istituto Auxologico Italiano IRCCS, 20122 Milan, Italy
Address all correspondence and requests for reprints to: Paolo Beck-Peccoz, M.D., Institute of Endocrine Sciences, Ospedale Maggiore IRCCS (Padiglione Granelli), Via Franceses Sforza 35, Milan 20122, Italy. E-mail: . paolo.beckpeccoz{at}unimi.it
Abstract
We studied the potential role of innovative diagnostic tools for the management of patients with differentiated thyroid cancer (DTC). Several methods for the detection of the tumor marker thyroglobulin (Tg) have been employed in 36 patients in apparent remission at the moment of the study. All patients had negative anti-Tg antibodies and were evaluated during L-T4 suppressive therapy before and after stimulation with recombinant human TSH (rhTSH). Serum Tg was measured by means of conventional [nonhighly sensitive (nhs)] or highly sensitive (hs) immunoassays with positive cut-off values set at 1.0 and 0.18 µg/liter, respectively. The RT-PCR conditions for the qualitative determination of Tg mRNA from peripheral blood were optimized to prevent interference by illegitimate transcription. The patients have been classified on the basis of a hs-basal Tg testing by taking into account the results of their baseline samples in hs immunoassay and RT-PCR method; hs-basal Tg testing was considered positive when the marker was detectable in at least one of the two tests. The predictive value of hs-basal Tg testing was estimated on the basis of a global clinical evaluation, including serum Tg response after rhTSH stimulation and reports of contemporary 131I scan and neck ultrasound. The clinical evaluation was considered positive when at least one of these criteria yielded positive results. Although nhs-Tg measurement was poorly predictive of the clinical status, basal hs-Tg evaluation was found to be concordant with the clinical evaluation in 71% of cases. Results of basal Tg mRNA detection did not vary after rhTSH stimulation and were concordant with the clinical evaluation in 66% of cases. Tg mRNA evaluation alone showed 10 apparently false-positive results, and serum basal hs-Tg was falsely negative in 11 additional cases, suggesting that a suitable predictability could be obtained by the association of these 2 parameters. Indeed, the combination of hs-Tg assay and mRNA detection in the hs-basal Tg testing allowed the identification of 22 patients with a positive persistent/recurrent disease or normal thyroid residue, as well as identification of all 6 patients with a negative clinical evaluation. In conclusion, the combined evaluation of circulating Tg mRNA and serum Tg by means of hs noncompetitive immunoassay (hs-basal Tg testing) can give useful information on the clinical status of patients with DTC who are apparently disease-free, even on L-T4 TSH-suppressive therapy. Therefore, these combined evaluations retain a potential role in the clinical monitoring of DTC patients. In particular, a negative hs-basal Tg testing would indicate disease remission and the opportunity to lengthen the intervals between rhTSH stimulations and/or to shift patients to a less profound TSH suppression with L-T4.
PATIENTS WITH THYROID carcinoma, previously treated with near-total thyroidectomy and residue 131I ablation, require lifelong monitoring for recurrent disease. Two diagnostic tests play a central role in the follow-up of these patients, radioiodine total body scan (TBS) and serum thyroglobulin (Tg) measurement. For various reasons, both of them have a higher diagnostic value during TSH stimulation. This condition is usually obtained by L-T4 withdrawal or, more recently, by administration of recombinant human TSH (rhTSH). This latter possibility, avoiding the appearance of poorly tolerated hypothyroidism, has been demonstrated to be effective in the stimulation of 131I uptake and Tg production by normal residual tissue, local relapse, or metastases (1, 2, 3). Recent clinical trials have shown that the sensitivity of combined rhTSH-stimulated radioiodine scanning and serum Tg measurement is similar to that achieved after thyroid hormone withdrawal (4, 5).
Tg measurement shows some advantages compared with TBS in the follow-up of thyroid cancer. Indeed, in the absence of circulating thyroid antibodies, serum Tg measurement by immunometric noncompetitive methods is usually very well correlated to the clinical status. When measured after L-T4 withdrawal or rhTSH administration, serum Tg shows a sensitivity higher than that of TBS and lacks false-positive readings. Moreover, it is simple, speedy, inexpensive, precise, and widely available. Tg is measurable in the serum of almost all patients with normal residues or persistent/recurrent disease and local/distant metastases, with a lower percentage of false-negative results when compared with TBS. Due to the lower limit of detection of the currently most widely diffuse methods for serum Tg measurements, negative cut-off values indicating disease remission have been set at 1.0 (6) or 2.0 µg/liter (4) after TSH stimulation.
In the search for a more sensitive test for detecting minimal disease, a RT-PCR assay for Tg mRNA in peripheral blood has been developed in recent years (7, 8, 9, 10, 11, 12, 13, 14). This test has been claimed to detect circulating thyroid cells from patients with metastatic or residual neck thyroid tissue and has been proposed as a tumor marker with comparable or even higher sensitivity than conventional serum Tg assays, particularly useful in patients with positive anti-Tg autoantibodies (9, 10, 11, 15, 16). The elevated sensitivity of circulating Tg mRNA detection has been demonstrated by both qualitative and quantitative methods. Various protocols, including nested PCR or buffy coat methods, have been used by different authors, and presently there is a lively discussion about the actual significance of positive Tg mRNA detection. Extensive amplification protocols must be avoided because of the possible interference by illegitimate Tg gene transcription in blood cells (14), and quantitative methods may be necessary to give a precise interpretation to positive results because circulating Tg mRNA is usually found also in normal or non-differentiated thyroid carcinoma (DTC) subjects. For this reason, some authors have recently suggested that circulating Tg mRNA detection could be helpful only in selected cases (12, 13). So far, no relevance has been given to undetectable circulating Tg mRNA. Furthermore, in the large majority of these studies (7, 8, 9, 10, 12, 13), Tg mRNA results have been correlated with scans performed several months before and with serum Tg results obtained by means of conventional immunoassays (with lower limit of detection at 1.0 µg/liter) in samples collected during TSH suppression.
The aim of the present study was to test the value of the highly sensitive (hs) methods recently developed for Tg detection in the identification of the patients at risk of tumor recurrence or considered cured. To this purpose, we studied 36 patients during suppression of endogenous TSH secretion with L-T4 both before and during rhTSH test, and we compared Tg results with other clinical parameters concomitantly collected. Because the need for a more sensitive Tg determination arises mostly in patients without apparent metastatic disease, the patients enrolled in the present study have been selected, on the basis of the clinical history and previous TBS and serum Tg results, as those apparently free of disease.
Patients and Methods
We studied 36 patients (28 females and 8 males) with DTC at the moment of follow-up evaluation (Table 1
). Two patients (15 and 28) have been evaluated twice with 1-yr interval. The age range at the diagnosis of thyroid cancer was 11–66 yr. All 36 patients were previously treated with total thyroidectomy. The histological diagnosis was papillary carcinoma in 33 and follicular carcinoma in 3 patients, according to World Health Organization recommendations (17). In the majority of patients, a tumor node metastasis (TNM) classification was available. In particular, tumors were T1 in 4 patients, T2 in 14 patients, T3 in 1 patient, and T4 in 12 patients. Lymph-node metastases at the histological examination were present in 12 patients, whereas in none were distant metastases detected at the time of the diagnosis. The TNM system, combined with the age at diagnosis, defines four stages with increasing risk of cancer-related death. The majority of patients (n = 26) were classified as stage I, four patients were at stage II, six patients were at stage III, and no patients were at stage IV.
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Clinical and biochemical evaluation
In all patients, the scanning dose of 131I was of 4 mCi (148 MBq). Patients remained on suppressive doses of L-T4 and received im injections of 0.9 mg rhTSH (Thyrogen, TSH 
, Genzyme Corp., Cambridge, MA) 24 and 48 h before the administration of the tracer dose. Patients were asked to avoid iodine-containing drugs. Whole-body counts were obtained at 48 h after 131I administration. Negative TBSs were those with no detectable radioiodine uptake. In all patients, an accurate ultrasound examination of the neck was carried out at d 5, and the presence of thyroid residue and/or suspicious lymph nodes (SLNs) was recorded.
Serum TSH, free T4 (fT4), and free T3 (fT3) levels were measured with Axsym System (Abbott Laboratories Diagnostics Division, Abbott Park, IL); the normal values were 0.25–4.2 mU/liter for TSH, 9–20 pmol/liter for fT4, and 2.5–5.3 pmol/liter for fT3. Anti-Tg and anti-thyroperoxidase (TPO) antibodies were measured with Liaison Kit (Byk-Sangtec Diagnostica, Dietzenbach, Germany). All patients had values of both antibodies no greater than 35 U/liter (normal value, <100 in both assays). Tg levels were measured by means of two different immunoradiometric assays with a sensitivity of 0.9 (Immulite 2000; Medical System) and 0.1 (Delfia hTg; Wallac, Inc., Turku, Finland) µg/liter, respectively. The first assay system was defined as nonhighly sensitive (nhs) and the second as hs. Ten replicates of an internal control Tg sample showed a mean concentration (±SD) of 2.1 ± 0.45 µg/liter (intra-assay coefficient of variation, 21.4%) in nhs-Tg assay and of 1.54 ± 0.08 µg/liter (coefficient of variation, 5.2%) in hs-Tg assay. As far as hs-Tg assay is concerned, we determined the lower limit of detection in our laboratory as the Tg value corresponding to the mean + 2.5 SD of 10 standard 0 replicates. This limit ranged from 0.14–0.17 µg/liter in five different assays, and we set the cut-off value for detectable Tg at 0.18 µg/liter in the hs assay. In Delfia system, a recovery test of Tg immunoreactivity was performed by adding the Tg assay standard 1000 µg/liter at the dilution 1:11 to each serum sample, to further exclude the presence of endogenous anti-Tg antibodies. The recovery test ranged from 87–112% of added Tg, therefore giving indirect confirmation of the absence in all serum samples of possible interference factors in Tg immunoassay.
Each patient underwent the following study protocol: d 1, basal determination of fT3, fT4, TSH, Tg, Ab-Tg, Ab-TPO; Tg mRNA sampling (see below); and first rhTSH injection; d 2, TSH, Tg, Ab-Tg, Ab-TPO; Tg mRNA sampling; and second rhTSH injection; d 3, TSH, Tg, Ab-Tg, Ab-TPO; Tg mRNA sampling; and tracing 131I dose; d 4, TSH, Tg, Ab-Tg, Ab-TPO; and Tg mRNA sampling; d 5, TSH, Tg, Ab-Tg, Ab-TPO; Tg mRNA sampling; TBS; and ultrasound; and d 6, TSH, Tg, Ab-Tg, Ab-TPO, and Tg mRNA sampling.
Detection of Tg mRNA from blood samples
RNA extraction. Blood samples were obtained for each patient before the first rhTSH administration and on d 2–6. The method for sample collection was chosen to match the needs of routine sampling. To avoid RNA degradation, 1 ml of blood was immediately placed in sterile tubes containing 9 ml of RNAzol B (Tel-Test, Inc., Friendswood, TX) and stored at -80 C. Total RNAs were then prepared from whole blood according to the manufacturer’s recommendations. The same technique was used to prepare total RNA from cells derived from different human cell lines (K562-chronic myelogenous leukemia cells, HEK293-embryonic kidney cells, SK-N-SH-neuroblastoma cells) and from liver and pituitary gland obtained at autopsy. These samples were selected as those having a high probability to lack Tg mRNA expression and were used to optimize the RT-PCR conditions for amplification of the Tg mRNA from peripheral blood avoiding the detection of illegitimate transcription.
Seven normal subjects, three patients with Graves’ disease, and one patient with thyroid agenesia and undetectable serum Tg levels were also evaluated. Peripheral-blood mononuclear cells (PBMCs) were separated from granulocytes and erythrocytes using Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) starting from 10 ml of fresh blood; thereafter, total RNA was extracted from both the PBMC phase and the granulocyte/erythrocyte phase, as described above.
RT-PCR. The RT-PCR conditions for amplification of the Tg mRNA from peripheral blood were optimized to avoid detection of illegitimate transcription. We established the amount of cDNA of K562 cells that, in our conditions, does not give rise to Tg amplification products. Thereafter, using densitometer analysis of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) amplification products, all cDNA obtained from different patients, cell lines, or tissues was normalized to this amount of K562 cDNA.
In particular, 4 µg of total RNA was heated for 10 min at 70 C in the presence of 1 µg of random primers in a total volume of 50 µl. Subsequently, the RNA and the random primers mixture was incubated for 60 min at 37 C in the presence of reverse transcription buffer, 200 U Moloney murine leukemia virus reverse transcriptase, 0.125 µM of each deoxynucleotide triphosphate, and 25 U rRNasin ribonuclease inhibitor (Promega Corp., Madison, WI). PCR for HGPRT was performed using 2.5 µl first strand cDNA in a 50-µl volume containing Taq polymerase buffer (1.5 mM magnesium chloride), 0.2 µM of each deoxynucleotide triphosphate, 0.5 µM of each HGPRT primer, and 2.5 U Taq polymerase (Promega Corp.). The HGPRT PCR conditions were: initial denaturation at 94 C for 5 min, followed by 28 cycles consisting of denaturation for 30 sec at 94 C, annealing for 30 sec at 52 C, and extension for 30 sec at 72 C. Final extension lasted 5 min at 72 C. HGPRT-specific primers were designed to amplify a 97-bp product from cDNA and a 267-bp product from genomic DNA to visualize the eventual genomic contamination. HGPRT primers were: forward 5'-GCTTGCTGGTGAAAAGGACC-3'; reverse 5'-GTCAAGGGCATATCCTACAAC-3'. PCR products were electrophoresed on a 4% agarose gel and visualized with ethidium bromide. Fluorescence of HGPRT amplification products was analyzed by a densitometer to normalize cDNA levels. After this test, normalized quantities of cDNA were used, and a PCR was repeated to verify that the amount of the PCR products was equal in all samples.
Tg PCR was performed using normalized cDNA levels in a 50-µl volume containing Taq polymerase buffer (1.5 mM magnesium chloride), 0.2 µM of each deoxynucleotide triphosphate, 0.25 µM of each Tg primer, 10% dimethylsulfoxide, and 2.5 U Taq polymerase (Promega Corp.). The amplification was performed using specific primers previously described (9). PCR conditions were: initial denaturation at 94 C for 5 min followed by 39 cycles consisting of denaturation for 45 sec at 94 C, annealing for 45 sec at 63 C, and extension for 45 sec at 72 C. Final extension was for 5 min at 72 C. RT-PCR products were electrophoresed on a 3% agarose gel and visualized with ethidium bromide. The identity of the 348-bp RT-PCR was further confirmed by automatic sequencing using the ABI Prism 310 (PE Applied Biosystems, Foster City, CA) and the DyeDeoxy Terminator Cycle Sequencing Kit (Perkin-Elmer Corp., Wellesley, MA).
hs-Basal Tg testing and clinical evaluation. All patients have been classified on the basis of a hs-basal Tg testing by taking into account the results of their baseline samples in hs-immunoassay and RT-PCR method; hs-basal Tg testing was considered positive when the marker was detectable in at least one of the two tests. Results of hs-basal Tg testing have then been compared with those of a global clinical evaluation, including serum Tg in hs assay after stimulation with rhTSH, and reports of contemporary 131I-TBS and neck ultrasound. The clinical evaluation was positive and indicative of recurrent/metastatic disease or residual tissue when at least one of these three parameters yielded positive results. As far as neck ultrasound is concerned, no clinical value was assigned to SLN showing no 131I uptake at TBS (as in case 35), because biopsy was not performed due to the small volume of the lesion.
Results
Clinical and biochemical evaluation
The 131I TBS showed thyroid residues in 6 of 36 cases (Table 2). The residual thyroid tissue was also visualized at ultrasonography in three patients (12, 32, and 36). In four patients (14, 15, 24, 35), ultrasonography identified SLNs (Table 2).
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Basal nhs-Tg levels below the limit of detectability (<1 µg/liter) were found in 34 cases (32 patients), whereas values ranged from 2.0–3.0 µg/liter in the remaining 4 patients. An increase in nhs serum Tg concentrations after rhTSH was found in 13 patients with levels ranging between 2.0 and 25.0 µg/liter (Table 2
). Basal hs-Tg levels below 0.18 µg/liter were recorded in 27 cases, whereas in the remaining 11 determinations, hs-Tg values ranged between 0.20 and 1.13 µg/liter. An increase in hs serum Tg levels after rhTSH was detected in 20 cases, with values of 0.21–14.5 µg/liter (Table 2). Serum Tg peak was obtained at d 4 and 5 in the majority of cases (61.5% nhs and 72.7% hs; data not shown).
Serum basal nhs and hs-Tg values were compared with the clinical evaluation (obtained by the evaluation of the results given by hs-Tg levels after stimulus, TBS, and ultrasonography; see Patients and Methods). Results were considered to be concordant when undetectable levels of serum Tg were found in a patient with a negative clinical evaluation or when detectable levels of Tg were associated to a positive clinical evaluation. Basal Tg in nhs measurement was poorly concordant (53%; 20 of 38 determinations), whereas basal hs-Tg evaluation was concordant with the clinical evaluation in 71% of cases (27 of 38 determinations) (Table 3).
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Tg mRNA amplification was performed using cDNA levels that were normalized as described in Patients and Methods. Under these conditions, no amplification product was obtained from liver, pituitary gland, or different human cell lines (K562, SK-N-SH, HEK293) (Fig. 1, A and B). Moreover, we did not obtain any amplification product in the material derived from the blood of a patient with congenital hypothyroidism due to thyroid agenesia (Fig. 1C) and from PBMCs obtained from controls (Fig. 1A). In contrast, we amplified Tg mRNA from whole blood and from the granulocyte/erythrocyte phase of controls (Fig. 1A) and of the patient with Graves’ disease (Fig. 1C).
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). Both basal and rhTSH-stimulated Tg mRNA detection were concordant with the clinical evaluation in 66% of cases (25 of 38 determinations) (Table 3). We thus consider, in the evaluation of the hs-basal Tg testing, only the basal Tg mRNA determination. Clinical evaluation and hs-basal Tg testing
The individual results of hs-basal Tg testing (i.e. basal hs-Tg plus basal Tg mRNA) and clinical evaluation (i.e. peak hs-Tg plus TBS plus ultrasound) are reported in Table 4. These results and their concordance are summarized in Fig. 2. When compared with the clinical evaluation, Tg mRNA detection alone showed 10 apparently false-positive results, whereas serum basal hs-Tg gave false-negative results in 11 additional patients. Nevertheless, an improved predictability was found by combining these two parameters in the hs-basal Tg testing that was concordant with a negative clinical evaluation in 6 of 6 patients and with a positive clinical evaluation in 22 of 32 patients. When both basal hs serum Tg and Tg mRNA were positive, the hs-basal Tg testing was always associated with a positive clinical evaluation (patients 5, 6, 14, 21, 24, 28b, 30, and 34).
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The present study deals with the tentative determination of a basal parameter able to assess the disease status in DTC patients apparently disease free on the basis of previous clinical history. Although DTC patients in clinical and biochemical remission after appropriate treatment represent the large majority, there is still controversy about the more adequate management of these cases, mainly due to the fact that available parameters do not frequently allow the definite distinction between low risk of recurrence and complete remission.
Our experience confirms that rhTSH is a valuable alternative to hypothyroidism after L-T4 withdrawal in the preparation for the TBS and stimulated Tg evaluation. This method has been very well accepted by all patients, and no major adverse effects or technical difficulties were recorded. As already reported in the literature (18), TSH and Tg peak values were obtained in the majority of cases at 24 h and at 48–72 h since the time of the second rhTSH injection, respectively.
In previous studies, both Tg mRNA and serum Tg determinations have been obtained during L-T4 suppression in the majority of the cases, and the results compared with previous TBS performed several months before (7, 8, 9, 10). In contrast, we compare the predictive value of the hs-basal Tg testing, including serum hs-Tg and Tg mRNA determinations at d 1 of rhTSH test, with the clinical evaluation obtained at d 5 on the basis of the negative/positive results after stimulation with rhTSH.
Circulating Tg mRNA was detected by means of a RT-PCR protocol that prevents the possible interference by illegitimate transcription and includes the normalization of cDNA input; we have standardized our protocol to avoid amplification product from human cells of nonthyroidal origin, yet achieving a good sensitivity. Furthermore, particular care was paid to the collection of blood samples and to RNA extraction. Several findings here reported support the view that Tg mRNA detected in blood arises from circulating thyroid cells rather than from lymphocytes (9). Under the present experimental conditions, we did not detect circulating Tg transcripts in a patient with thyroid agenesia and in control samples, such as liver, pituitary gland, or different human cell lines (K562, SK-N-SH, HEK293). Furthermore, Tg mRNA has been detected after just one round of amplification, whereas identification of illegitimate transcription typically requires two rounds of amplification (19). Finally, Tg mRNA has been detected only in the granulocyte/erytrocyte phase, where the thyroid cells are predicted to localize (9). In previous studies (9, 10), Tg mRNA detection has been found to reflect more accurately than serum Tg the presence of thyroid residue or recurrence. However, circulating Tg mRNA detection was compared with serum Tg immunoassays with the limit of sensitivity set at 0.9 µg/liter. The comparison with conventional serum Tg measurement gave the same result in our series, whereas hs-Tg assay has been proven to possess a similar or even higher predictive value with respect to qualitative Tg mRNA evaluation in patients without anti-Tg autoantibodies (Table 3). The superior performance of hs-Tg measurement when compared with the conventional Tg assays is highlighted by the ability to detect basal Tg secretion or minor serum Tg increases after rhTSH in patients with small residues, such as cases 6, 12, and 36 (Table 4), in whom conventional Tg assay was negative. Indeed, when changing to more sensitive assays, one should revise previous cut-off values because serum Tg levels below 0.9 µg/liter or minor increments after rhTSH acquire some significance and may indicate minimal residual disease. Therefore, only the results obtained with the hs method have been considered to assess the hs-basal Tg testing.
Despite the relative concordance with the clinical evaluation (66 and 71%; Table 3), circulating Tg mRNA detection showed false-positive results (n = 10), and serum hs-Tg measurement at baseline showed a comparable number (n = 11) of false-negative results. These two Tg parameters could be considered complementary to each other and their combination in the hs-basal Tg testing was then compared with the clinical evaluation. Indeed, hs-basal Tg testing and clinical evaluation were concordantly negative in 6 of 6 cases and concordantly positive in 22 of 32 cases. The discrepancy was exclusively due to the apparently false-positive results derived from Tg mRNA detection (Fig. 2). However, it is worth noting that combined positive results of Tg mRNA detection and serum hs-Tg determination were always associated with a positive clinical evaluation (eight cases; Fig. 2, surrounded with dotted line). Such results indicate the potential role of hs-basal Tg testing in the management of patients with DTC. In particular, a negative hs-basal Tg testing would indicate that these patients may be considered disease free, thus allowing to lengthen the intervals between the rhTSH testing and TBS, with a consequent saving of time and money for both patient and institution and a reduced exposure of the patient and the environment to the radioactive isotope. Furthermore, the negative hs-basal Tg testing would indicate the possibility to maintain these patients on a less profound TSH suppression by reducing L-T4 daily doses or even switching them to L-T4 replacement treatment. This can result in a reduced number of possible untoward effects, including those on cardiac function (20). On the other hand, the positive hs-basal Tg testing, particularly when both determinants are positive, was correlated with the persistence of thyroid tissue remnants and with the risk of residual or metastatic disease, thus indicating the need to perform a full clinical evaluation. In these cases the direct administration of ablative 131I doses, instead of the tracing ones, may be envisaged.
In 10 of 24 cases, the positive hs-basal Tg testing resulted from detectable circulating Tg mRNA in the absence of any other evidence of thyroid residue. This discrepancy, already reported in the literature with both the qualitative (7, 10) and quantitative methods (11), is likely due to a false-positive result of Tg mRNA and suggests that caution should be taken in the routine use of these evaluations in the clinical practice. However, because the interference by ectopic or illegitimate transcription was avoided by our methodological protocol, the possibility that a positive Tg mRNA could reflect the presence of micrometastases that will become apparent after many years, should be considered. Indeed, among all the recurrences, over 50% appear in the first 5 yr. In a minority of patients, metastases may become evident years, and sometimes decades, after initial therapy (21, 22). Accordingly, Biscolla et al. (10) found recurrence of the tumor in three patients that were negative for serum Tg and TBS and positive for Tg mRNA. In the present series, similar results were found in patients 15 and 28. Patient 15a had, at the first evaluation, a negative clinical evaluation with negative Tg mRNA, whereas after 1 yr basal Tg mRNA became positive and hs-Tg response to stimulus was documented. A similar behavior was found in patient 28, who had a positive Tg mRNA and negative hs-Tg with a positive clinical evaluation (hs-Tg peak, 0.47 µg/liter with negative TBS) at the first control. One year later, the positive Tg mRNA result was confirmed, as was the elevation of hs-Tg (baseline, 0.4 µg/liter; after rhTSH stimulus, 1.9 µg/liter). To date, no clinical recurrence has been identified with imaging techniques in the above patients. Nevertheless, because definite evidence of complete remission is lacking, the clinical management of these patients would remain unchanged.
In conclusion, the hs-basal Tg testing, which implies the simultaneous blood recollection for hs-Tg measurement and for qualitative Tg mRNA detection, has been proven to be simple and reproducible if routinely performed. Thus, it may provide a new strategy for monitoring patients with DTC, possibly leading to an improved clinical management and to economic advantages. However, long-term follow-up is necessary to establish the clinical relevance of a positive hs-basal Tg testing in patients with no evidence of residual disease.
Footnotes
This work was partially supported by Funds of Ricerca Corrente (Project 05C010) of IRCCS Istituto Auxologico Italiano (to L.P.), Milan; and by Funds of Ricerca Finalizzata of the Italian Ministry of Health (Project TUMOTI), Rome, Italy.
Abbreviations: DTC, Differentiated thyroid cancer; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; hs, highly sensitive; nhs, nonhighly sensitive; PBMC, peripheral-blood mononuclear cell; rhTSH, recombinant human TSH; SLN, suspicious lymph node; TBS, total body scan; Tg, thyroglobulin; TNM, tumor node metastasis; TPO, thyroperoxidase.
Received November 19, 2001.
Accepted March 25, 2002.
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); no increase of hs-Tg levels after rhTSH with positive TBS and/or US (
). Patients with negative clinical evaluation are indicated with a white square (
). The hs-basal Tg testing was defined on the basis of baseline evaluations by hs-immunoassay and circulating Tg mRNA detection. It was considered positive when the marker was detectable with one or both of these methods. The hs-basal Tg testing correctly identified 6 of 6 patients with a negative clinical evaluation and 22 of 32 patients with a positive clinical evaluation. The 10 apparently false-positive results of hs-basal Tg testing were all due to detectable Tg transcripts. When both serum Tg levels in hs immunoassay and circulating Tg mRNA were positive, as in the eight patients surrounded with a dotted line, the hs-basal Tg testing was always associated to a positive clinical evaluation.