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The Journal of Clinical Endocrinology & Metabolism 87(1):292–296 Copyright © 2002 by The Endocrine Society
RT-PCR-Based Detection of Circulating CalcitoninProducing Cells in Patients with Advanced Medullary Thyroid Cancer B. SALLER, G. FELDMANN, K. HAUPT, M. BROECKER, O. E. JANSSEN, M. ROGGENDORF, K. MANN, AND M. LU Division of Endocrinology, Department of Internal Medicine, Department of Clinical Chemistry (K.H., M.B.), and Institute of Virology (M.R., M.L.), University of Essen, 45122 Essen, Germany 70 – 46,787 pg/ml; median, 932 pg/ml (n ⴝ 13); P ⴝ 0.006]. CT mRNA was detectable in 5 of 8 patients with distant metastases, in 1 of 6 patients with local/regional lymph node metastases, but in none of the patients with newly diagnosed, organ-confined MTC (n ⴝ 2) or surgically treated MTC without tumor manifestation by various imaging studies (n ⴝ 3). In peripheral blood from 10 healthy volunteers and 21 patients with benign thyroid nodules, no CT RNA could be detected. In conclusion, an RT-PCR-based procedure was established to detect circulating CT-producing cells in the peripheral blood of patients with MTC. RT-PCR results seem to reflect tumor spread and aggressiveness and thus may help with early identification of patients with disseminated and rapidly progressive disease. (J Clin Endocrinol Metab 87: 292–296, 2002)
In patients with medullary thyroid carcinoma (MTC) the clinical course of disease ranges from rapid tumor progression to long-lasting stable disease. The purpose of the present study was to investigate whether circulating tumor cells can be detected in the peripheral blood of patients with MTC by RTPCR targeted to calcitonin (CT) mRNA and whether the results of this method are correlated with disease manifestation and metastatic potential. Blood samples from 19 consecutive patients with MTC and elevated CT levels were analyzed. Four had newly diagnosed MTC, and 15 patients had undergone total thyroidectomy. Six of 19 patients had detectable CT mRNA by RT-PCR. CT levels in the CT mRNA-positive patients were significantly higher than those in CT mRNA-negative patients [2,239 –265,313 pg/ml; median 80,921 pg/ml (n ⴝ 6) vs.
M
EDULLARY THYROID carcinoma (MTC) is a malignant tumor that arises from thyroid parafollicular C cells secreting calcitonin (CT). MTC is responsible for 5–10% of all thyroid carcinomas. It may occur sporadically, as a familial form without associated endocrinopathies, or together with other endocrinopathies in MEN2 (1, 2). The prognosis for MTC is intermediary between differentiated and anaplastic thyroid carcinoma (3– 8). Age, tumor size, stage of disease, histological features, and tumor form have been established as significant prognostic factors. In addition, postsurgical normalization of basal and stimulated serum CT levels is a very powerful predictor of long-term survival (4, 5). The majority of patients with sporadic MTC have nonorgan-confined disease and involvement of at least one lymph node compartment at first presentation. Consequently, approximately 60% of patients have elevated postoperative CT levels, indicating residual disease (9, 10). The clinical course of disease for these patients is currently difficult to predict. Some survive untreated for many years with localized disease, but, in contrast, in other patients the tumor metastasizes rapidly, causing death of the patient within a few years. New criteria to define the aggressiveness and metastatic potential of a tumor are therefore needed to separate patients who have localized disease and may benefit from reoperation and extensive lymph node dissection from those with generalized and rapid progressive disease. The formation of metastases involves multisequential
events, including tumor cell detachment from the primary lesion into the peripheral blood circulation. During the last years, many studies based on RT-PCR could sensitively identify hematogeneous spreading of tumor cells in patients with various malignancies, including prostate cancer (11, 12), breast cancer (13), malignant melanoma (14), germ cell tumors (15), and nonmedullary thyroid cancer (16). For some tumors, first evidence has been provided for an association between the detection of circulating tumor cells and a high risk of disease progression (17–20). The primary objective of the present study was to investigate whether circulating tumor cells can be detected in patients with newly diagnosed or residual MTC by RT-PCR targeted to CT mRNA and to obtain first data on the correlation of this method with tumor spread and metastatic potential. Subjects and Methods Subjects Nineteen consecutive patients with MTC and elevated basal CT levels who had been admitted at our institution between June 1999 and May 2000 were examined (12 women and 7 men; age, 49 ⫾ 17 yr). We included only patients with elevated CT, because tumor progression and development of metastases can almost be completely excluded in patients with normal CT levels. Four patients had newly diagnosed MTC and were included before they had primary surgery. Two of these patients had organ-confined disease, and 2 had distant metastases at the time of diagnosis. Fifteen patients had undergone total thyroidectomy between 1–22 yr (median, 5 yr) before inclusion in the study. Tumors were staged by ultrasonography of the neck; high resolution computerized axial tomography scanning of the neck, thorax, and abdomen; and [99mTc]diphosphonate bone scintigraphy. In addition, 8 patients underwent a
Abbreviations: CT, Calcitonin; MTC, medullary thyroid carcinoma.
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whole body positron emission tomography study with [18F]fluorodeoxy glucose (21). CT was measured in all subjects by commercial two-site immunoradiometric assay (Scantibodies Laboratory, Santee, CA) with a lower limit of detection of 0.7 pg/ml. Normal ranges with this assay had been established in 100 healthy controls and are 5 pg/ml or less for females and 10 pg/ml or less for males. The patients’ characteristics are summarized in Table 1. Five patients had received chemotherapy for rapidly progressive disease, but had been off cytostatic drugs for at least 3 wk before sample collection. Thirty-one controls [21 with benign thyroid nodules (18 women and 3 men; age, 48 ⫾ 17 yr) and 10 healthy individuals (5 women and 5 men; age, 35 ⫾ 15 yr] were also tested. CT levels were within the normal ranges in all control subjects. In patients with benign thyroid nodules, thyroid malignancy was ruled out by fine needle aspiration biopsy.
Cell cultures The TT cell line (American Type Culture Collection, Manassas, VA; CRL 1803), derived from a human MTC and known to express high levels of CT, was used to obtain positive control mRNA and to set up an in vitro sensitivity test of circulating tumor cell detection.
Sample collection and RNA extraction Blood samples (6 ml) were immediately mixed with EDTA and were stored at 4 C for no more than 3 h. Storage experiments were performed in two patients and could document that RT-PCR results are not effected by a storage of blood samples at 4 C up to 5 h (patient 1) and 9 h (patient 2), respectively. For selective erythrocyte lysis by osmotic shock, the standard protocol was scaled up as recommended by the manufacturer (QIAGEN, Hilden, Germany). The remaining cells, mainly containing leukocytes and the potential tumor cells, were pelleted by centrifugation at 350 ⫻ g for 10 min, then lysed using 2.4 ml lysis buffer and homogenized using the Qiashredder spin columns, both supplied by QIAGEN. Total RNA was extracted from 300 l of the lysate using an RNA extraction kit (QIAGEN). The resulting 3.6 – 6.0 g RNA were dissolved in 50 l ribonuclease-free water and stored at ⫺80 C until used. Concentration was determined by analysis of 28S and 18S rRNA on ethidium bromide-stained agarose gel.
RT Nine microliters of the obtained solution corresponding to approximately 0.6 –1.0 g total RNA were reverse transcribed into cDNA at 42
C for 60 min using 0.15 g oligo-(deoxythymidine)(12–18) primers (purchased from Life Technologies, Inc., Eggenstein, Germany) and Expand Reverse Transcriptase (Roche, Mannheim, Germany) in a final volume of 20 l [1 mm dNTP, 10 mm dithiothreitol, 50 mm Tris-HCl, 40 mm KCl, 5 mm MgCl2, 0.5% Tween 20 (vol/vol), 20 U ribonuclease inhibitor (Life Technologies, Inc.), and 50 U Expand reverse transcriptase].
PCR protocol Ten microliters of the cDNA were used as PCR template, which was carried out in a final volume of 20 l. We chose sequence specific primers corresponding to nucleotide sequences in exons 3 and 4 of the human CT ␣-gene, which are separated by an intron containing about 950 bp, to be able to discriminate between mRNA amplification leading to a 180-bp cDNA fragment and potential amplification of contaminating genomic DNA or unspliced mRNA resulting in a fragment of about 1.1 kb (22). The nucleotide sequences of the primers used are shown in Table 2. The 20-l reaction mix contained 10 pmol of both primers, 0.9 mm of each dNTP, 4 mm MgCl2, and 1.75 U Expand High Fidelity DNA polymerase mix (Roche). PCR was carried out over 40 cycles with a first denaturation step at 94 C for 2 min, then the denaturation step was at 94 C for 15 sec, followed by annealing at 58 C for 30 sec and extension at 72 C for 45 sec. During cycles 11– 40 the extension time was prolonged by 5 sec/cycle as recommended by Roche to get higher yields of DNA. A final extension of 7 min was performed at 72 C. Five microliters of the PCR product were visualized by ethidium bromide-stained agarose gel electrophoresis. All PCRs were carried out together with a positive control RNA sample and water as a negative control using a Mastercycler Gradient-Thermocycler (Eppendorf, Hamburg, Germany) and thin-walled autoclaved 500-l reaction tubes. RTPCR was repeated at least once for each patient sample. To confirm positive PCR results, DNA sequencing was carried out for three of the PCR-positive samples. Amplification of -actin mRNA was carried out from the same RT reaction to ensure the presence of intact mRNA and adequate cDNA synthesis in the RT procedure as well as in CT mRNA-negative samples.
Statistics Differences were analyzed using the unpaired t test, performed by Prism (version 3.00 for Windows, GraphPad Software, Inc., San Diego, CA). All data are expressed as the mean ⫾ sd or the range and median. The level of significance was set at P ⬍ 0.05.
TABLE 1. Patients’ characteristics Patient no.
Gender
Age (yr)
Sporadic/familial MTC
1
M
28
Sporadic
108
2 3
W W
29 73
MEN 2A Sporadic
265313 932
4 5 6 7 8 9 10 11 12
M W W W W M M W W
60 59 54 70 57 57 33 17 62
Sporadic Sporadic Sporadic Sporadic Sporadic Sporadic Sporadic MEN 2B Sporadic
27656 95224 70 176 180 3304 46787 3241 66618
13
M
24
Sporadic
41249
14 15 16 17 18 19
W M W M W W
62 51 60 30 51 54
Sporadic Sporadic Sporadic MEN 2B MEN 2A Sporadic
193 104249 110 8038 2239 2152
The patients’ numbers refer to the results given in Fig. 2.
Ct (pg/ml)
Tumor manifestation
MTC before primary surgery, no extrathyroid manifestation Distant metastases MTC before primary surgery, no extrathyroid manifestation Distant metastases Distant metastases Negative imaging studies Negative imaging studies Distant metastases Local/regional lymph nodes Distant metastases Local/regional lymph nodes MTC before primary surgery, distant metastases MTC before primary surgery, distant metastases Negative imaging studies Distant metastases Local/regional lymph nodes Local/regional lymph nodes Local/regional lymph nodes Local/regional lymph nodes
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Saller et al. • RT-PCR-Based Detection of Circulating MTC Cells
TABLE 2. Sequences and locations of the primers used (22) Primer
Sequence
Location
Calcitonin, sense Calcitonin, antisense -actin, sense -actin, antisense
5⬘-CACGCTCAGTGAGGACGAAG-3⬘ 5⬘-AAGTCCTGCGTGTATGTGCC-3⬘ 5⬘-TGGAGTCCTGTGGCATCCACGAAA-3⬘ 5⬘-TGTAACGCAACTAAGTCATAGTCCG-3⬘
2308–2327 3426–3407 2566–2589 3026–3002
Results CT RT-PCR assay sensitivity
We analyzed in vitro assay sensitivity by mixing cultured TT cells with 6 ml whole blood from a healthy person in different MTC cell/lymphocyte ratios. RNA was extracted from these mixtures. A positive result was obtained from one single TT cell in 6 ml whole blood, which corresponds to a theoretical sensitivity of one tumor cell of 4 ⫻ 107 peripheral blood lymphocytes (Fig. 1). Clinical evaluation of RT-PCR assay for CT mRNA
Six of 19 patients with MTC and elevated basal serum CT levels had detectable CT mRNA (Fig. 2). In these patients, basal CT levels were significantly higher than in those with undetectable CT mRNA (n ⫽ 13) (mRNA negative, 70 – 46,787 pg/ml; median, 932 pg/ml; mRNA positive, 2,239 –265,313 pg/ml; median, 80,921 pg/ml; P ⫽ 0.006; Fig. 3). The association between tumor manifestation and RT-PCR results is given in Table 3. Circulating CT mRNA was undetectable in patients with organ-confined newly diagnosed MTC (n ⫽ 2) and in those patients with negative imaging studies in the presence of elevated serum CT levels (n ⫽ 3). In contrast, 5 of 8 patients with distant metastases had positive RT-PCR results. RT-PCR positivity in patients with metastatic disease was related to the presence of bone metastases. Skeletal tumor manifestations were seen in 4 of 6 RT-PCR positive patients, but in only 1 of 13 RT-PCR negative patients (P ⫽ 0.017, by Fisher’s exact test). Interestingly, the RT-PCR-negative patient with bone metastases had been treated by polychemotherapy and had a significant tumor regression before study inclusion. In vivo specificity of the RT-PCR assay has been evaluated by blood samples from 10 healthy volunteers and 21 patients with benign thyroid nodules. In none of these samples could CT RNA be detected. Discussion
In our study we report on a RT-PCR-based procedure to detect circulating CT-producing cells in the peripheral blood of patients with MTC and provide the first evidence that this test can give valuable clinical information on tumor spread and metastatic potential. In a first step we have optimized all technical steps of the RT-PCR protocol, such as mRNA isolation from peripheral blood, choice of primers to ensure cDNA-specific amplification, and PCR conditions to exclude PCR products from illegitimate transcription and to reproducibly obtain a threshold in vitro sensitivity of one TT cell in 6 ml peripheral blood. Due to the high expression of CT mRNA in TT cells, this high sensitivity does not necessarily reflect in vivo assay sensitivity in patients with MTC.
FIG. 1. Sensitivity of CT (Ct) mRNA analysis. Cultured TT cells were mixed with 6 ml whole blood from a healthy subject. Lane M, Size marker; lanes 2– 4, RT-PCR results with 100, 10, and 1 TT cells/6 ml whole blood.
As RNA is very unstable in the extracellular environment, and only the cellular compartment has been used for RT-PCR analysis, there is good evidence that the detection of CT mRNA in our assay really indicates the presence of circulating tumor cells. Nevertheless, the amplification of naked CT mRNA cannot be completely ruled out. Using the optimized RT-PCR procedure, we have tested 19 patients with MTC. As normal serum CT concentrations are almost always associated with complete tumor removal and long-term disease-free survival (6), only patients with elevated basal CT concentrations either before primary surgery or during follow-up were included in the study. Circulating CT-producing cells were detected in 6 of 19 CT-positive MTC patients and in none of 31 control subjects, including 21 patients with benign thyroid nodules. Despite the relatively small number of patients with MTC, it can be concluded from these results that our RT-PCR test seems to be specific for the detection of circulating CT-producing cells and that positive RT-PCR results are associated with MTC. Moreover, the association between RT-PCR results and serum CT levels as well as the association between RT-PCR results and the extent of tumor manifestation indicate that the detection of CT mRNA by RT-PCR may be considered a marker of metastatic disease. The highest association was found between positive RT-PCR results and the presence of bone metastases. Nevertheless, there were three patients with negative RTPCR results in the presence of metastatic disease and highly elevated serum CT levels. There were no clinical differences between RT-PCR-positive and RT-PCR-negative patients with metastatic disease with the exception that bone metastases were found in four of five RT-PCR positive patients, but in only one of three RT-PCR-negative patients. The RT-PCR assay, however, will only become a useful tool in the follow-up of patients with MTC if it can provide information on the biological behavior and metastatic potential of the tumor additional to the measurement of serum CT. Future prospective studies have to clarify whether negative RT-PCR results in patients with elevated serum CT levels are asso-
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FIG. 2. Analysis of CT (Ct) expression in peripheral blood samples from 19 patients with MTC and elevated basal CT levels (lanes 1–19). Patients’ characteristics are given in Table 1, and the number of patients in Table 1 refers to the numbers given in the figure. Lane M, Size marker; lane W, distilled water. Expression of -actin has been evaluated in all patient samples to ensure the presence of intact mRNA.
FIG. 3. Basal serum CT (Ct) levels in patients with negative and positive CT mRNA in peripheral blood. Statistical analysis was performed by Mann-Whitney test. TABLE 3. Relationship between extent of tumor manifestation and peripheral blood RT-PCR results Tumor manifestation
MTC before primary surgery No extrathyroid manifestation Distant metastases MTC after primary surgery Elevated Ct, negative imaging studies Local/regional lymph node metastases Distant metastases
RT-PCR positive (%)
0/2 (0%) 2/2 (100%) 0/3 (0%) 1/6 (17%) 3/6 (50%)
ciated with a more favorable course of disease and whether a positive RT-PCR result in a patient with clinically localized disease can be regarded as an early indicator of metastatic tumor spread. There is now good evidence that the information given by RT-PCR is not generally improved by presentation of quantitative, rather than qualitative, analysis of PCR data as in the present study. This is primarily due to the fact that the amount of target mRNA considerably varies between individual tumor cells, i.e. the results of RT-PCR are more closely related to the transcription rate in the individual tumor cells than to the amount of tumor cells itself (23). In addition, only a few milliliters of peripheral blood are analyzed at a certain time, and therefore, sampling problems due to intermittent
shedding of tumor cells into the circulation cannot be excluded (15). Detection of CT expression as a tool to detect circulating MTC cells was used, because CT is produced by almost all MTC cells and is generally expressed at a high level (24). There is a recent report on the detection of circulating thyroid carcinoma cells in peripheral blood by RT-PCR of cytokeratin 20 expression. The study included eight patients with MTC, and three of them turned out to be RT-PCR positive (25). Although these results are similar to those from our study, future studies have to clarify whether the expression of CT mRNA or the expression of cytokeratin 20 mRNA offers the more sensitive approach to detect patients with the most aggressive disease. A point that has to be taken into account is that five of our patients had been treated by chemotherapy before study inclusion. All of them had disseminated and rapidly progressive disease, and four had positive RT-PCR results. Sabbatini et al. (17) recently reported that chemotherapy may increase the number of RT-PCR-positive results 4 and 8 d after chemotherapy in patients with breast cancer. In our study cytostatic drugs had been discontinued for at least 3 wk at the time of sampling, and therefore, a significant impact of chemotherapy on RT-PCR results seems unlikely. In conclusion, an RT-PCR-based procedure was established to detect circulating CT-producing cells in the peripheral blood of patients with MTC. Based on our preliminary results, RT-PCR seems to reflect tumor spread and aggressiveness and thus may help in the early identification of patients with disseminated and rapidly progressive disease. The prognostic impact of CT RT-PCR-based tumor cell detection must be further evaluated in prospective studies. Acknowledgments Received April 16, 2001. Accepted October 19, 2001. Address all correspondence and requests for reprints to: B. Saller, M.D., Division of Endocrinology, Department of Medicine, University of Essen, Hufelandstrasse 55, 45122 Essen, Germany. E-mail: bernhard.
[email protected]. This work was supported by a grant from Forum Schilddru¨ se e.V. (to B.S.).
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