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The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 3 951-955
Copyright © 1999 by The Endocrine Society

Original Studies

Preoperative Diagnosis of Medullary Thyroid Carcinoma by RT-PCR Using RNA Extracted from Leftover Cells within a Needle Used for Fine Needle Aspiration Biopsy1

Toru Takano, Akira Miyauchi, Fumio Matsuzuka, Gang Liu, Takuya Higashiyama, Tamotsu Yokozawa, Kanji Kuma and Nobuyuki Amino

Department of Laboratory Medicine (T.T., G.L., N.A.) and Surgical Oncology (T.H.), Osaka University Medical School, Osaka 565-0871, and Kuma Hospital (A.M., F.M., T.Y., K.K.), Simoyamate-Dori, Kobe, Hyogo 650-0011, Japan

Address correspondence and requests for reprints to: Toru Takano, Department of Laboratory Medicine, Osaka University Medical School, D2, 2-2 Yamadaoka, Suita, Osaka, Japan 565-0871.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Fine needle aspiration Biopsy (FNAB) is commonly used to diagnose thyroid tumors. In some clinical situations, however, accurate diagnosis requires a more objective method than cytological examination alone. Medullary thyroid carcinomas (MTC) derive from C cells in the thyroid and express some specific messenger RNAs (mRNA), such as those transcribed from the RET proto-oncogene, the calcitonin gene, and the gene for carcinoembryonic antigen (CEA), which usually do not exist in normal thyroid follicular cells or thyroid tumors of follicular epithelial descent. Recently, we established a new method for the molecular diagnosis of thyroid tumors without additional invasion to the patient by extracting RNA for RT-PCR from the leftover cells inside the needles used for fine needle aspiration biopsy (Aspiration Biopsy-Reverse Transcription-Polymerase Chain Reaction, ABRP). By applying the ABRP method to the detection of RET, calcitonin, and CEA mRNAs, an accurate molecular-based diagnosis for MTC may be established as an adjunct to cytological diagnosis. In this study, 35 aspirates were obtained at the time of surgery from thyroid tumors, including 11 MTCs. The expression of these mRNAs in the leftover cells inside the needles used for the aspiration was then examined. Transcripts from all three genes were detected in the samples from all 11 MTCs, but none of these mRNAs were detected in the other tumors or normal thyroid tissues. Furthermore, MTC was preoperatively diagnosed in three patients by ABRP detection of these mRNAs, and these diagnoses were confirmed by subsequent cytological and histopathological analyses. Thus RT-PCR detection of RET, calcitonin, and CEA mRNAs in FNABs may be an efficient molecular adjunct for diagnosing MTC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Medullary thyroid carcinoma (MTC) is a rare tumor derived from the parafollicular C cells of the thyroid (1, 2, 3). Although this carcinoma accounts for less than 5% of all malignant thyroid tumors, it is of special interest for the following reasons. First, calcitonin and carcinoembryonic antigen (CEA) secreted by carcinoma cells can be used as effective markers for diagnosis. Second, in 20–25% of all cases, the disease is inherited, either alone as familial MTC or as a part of multiple endocrine neoplasia syndrome type 2A or 2B (MEN 2A, 2B). Third, germ-line mutations of the RET proto-oncogene have been identified as the cause of this carcinoma in familial cases; thus, DNA analysis to detect this mutation is increasingly used to screen high risk individuals (4, 5, 6, 7, 8, 9, 10, 11, 12, 13).

Thyroid tumors are often diagnosed by fine needle aspiration biopsy (FNAB) as well as by ultrasonography (14, 15, 16), and a cytological examination of FNAB by a skillful pathologist who is an expert in thyroid tumors provides the most reliable means of diagnosing thyroid neoplasms. In some clinical situations, however, accurate cytological examination is impossible because of the inadequacy of the samples. This inadequacy can be due to a lack of epithelial cells or poor fixation, and diagnosis by an expert pathologist is not always available. In such situations, a more objective method is needed to assure reliable diagnosis. A molecular-based diagnosis using RNA extracted from aspirates and RT-PCR may be used for this purpose, provided there exist messenger RNAs (mRNA) that are expressed only in cancer tissues (17, 18). To establish a method of preoperative molecular-based diagnosis of thyroid carcinomas, we have introduced a new technique, aspiration biopsy-RT-PCR (ABRP). In this technique, leftover cells within the needle used for FNAB are used for RT-PCR (19). In this way, ABRP provides additional RNA analysis data to augment the results of cytological diagnosis without further invasion to the patient. The RNA extracted from an FNAB provides sufficient cDNA for as many as 20 PCR examinations. Further, the results of cytological and genetic diagnoses using cells from the same FNAB can be compared.

We have recently reported the restricted expression of oncofetal fibronectin mRNA in thyroid papillary and anaplastic carcinomas (20, 21), which make up 90% of thyroid malignancies in iodide-sufficient countries. We have also demonstrated the clinical usefulness of preoperative genetic diagnosis of these carcinomas by the ABRP detection of oncofetal fibronectin mRNA (22, 23).

Previous reports have shown the existence of some mRNAs, such as those from the RET proto-oncogene, calcitonin, and carcinoembryonic antigen (CEA), whose expression is restricted to MTCs (24, 25, 26). Like papillary and anaplastic carcinomas, MTCs may be diagnosed by RT-PCR analysis using FNABs when these mRNAs are expressed specifically enough to distinguish MTCs from other thyroid tumors and normal thyroid tissues.

In this study, in order to examine the possibility of establishing a method of preoperative diagnosis of MTC by ABRP, we obtained aspirates from 35 surgically dissected thyroid tissues, including 11 MTCs, and performed RT-PCR to amplify RET, calcitonin, and CEA cDNAs. Further, in three patients, ABRP was utilized for the preoperative diagnosis of MTC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Thirty-five thyroid tissue samples (6 normal thyroid tissue samples from the opposite lobe of thyroid carcinomas, 3 adenomatous goiters, 6 follicular adenomas, 7 papillary carcinomas, 2 follicular carcinomas, and 11 MTCs) were obtained immediately after their surgical dissection (TableGo I) for use in this study. ABRP was performed as previously described (19) (Fig. 1Go). In brief, a syringe with a 22-gauge needle was used to obtain an FNAB from the tissue sample. A sample of the FNAB was prepared on a slide glass for cytological examination, and leftover cells inside the needle were then lysed with denaturing solution containing 4 M guanidine thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol into a 1.5-ml tube. The tubes were then stored at 4 C. Total cellular RNA was extracted according to the method of Chomczynski and Sacchi (27). Papanicolaou staining was then applied to the samples on slide glasses to certify that the tumor cells were aspirated from the tissues. A portion of the tissue samples were dissected simultaneously, then immediately frozen in liquid nitrogen. Total RNA was extracted as previously described for use in the following study. Similarly, RNA samples were obtained preoperatively by ABRP from three patients suspected of MTC.


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  		Table 1. Tissues of MTC used in this study

 


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  		Figure 1. Procedures of ABRP. Tumor cells were aspirated by FNAB using a syringe with a 22-gauge needle (A). After preparing a sample on a slide glass for cytological examinations (B), the needle was dipped into the denaturing solution in a 1.5-ml tube (C). The denaturing solution was aspirated and pushed back into the tube three times to lyse the cell inside the needle. The tube was then stored at 4 C.

 
Reverse transcription was performed using either the whole RNA extracted by ABRP or 1 µg of total RNA from tissue samples in an RT mixture containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 10 mM dithiothreitol, 3 mM MgCl2, 0.5 mM deoxynucleotide triphosphates (dNTPs), 200 U M-MLV reverse transcriptase (Gibco, Gaithersburg, MD), 2 U/µl RNase inhibitor (Takara, Shiga, Japan), and 2.5 µM oligo dT (Gibco) in a total volume of 20 µl at 37 C for 60 min. One microliter of first-strand cDNA was used as a template for the PCR reaction with specific primers for either RET (28), calcitonin (29), CEA (30), thyroglobulin (31), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA (32). The sequences of the primers were as follows: Go


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  		Table 2.

 
All primers were purchased from Gibco. Each reaction mixture consisted of 1 µl of cDNA, 0.5 µM of each primer, 2 µl of 10 x Ex Taq Buffer, 1.6 µl of dNTP mix, 0.5 U of Ex Taq polymerase, and nuclease-free water to a final volume of 20 µl. 10 x Ex Taq Buffer, dNTP mix, and Ex Taq polymerase were obtained from Perkin-Elmer Cetus (Emeryville, CA). The reaction mixture was subjected to the PCR reaction. The conditions were as follows: for RET, calcitonin, and CEA, 30 cycles of denaturation (94 C, 30 sec), annealing (60 C, 30 sec), and extension (72 C, 30 sec); for thyroglobulin and GAPDH, 35 cycles of denaturation (94 C, 1 min), annealing (55 C, 1 min), and extension (72 C, 1 min). After PCR amplification, 5 µl of the reaction mixture was run on a 1.5% SeaKem GTG agarose gel (Takara) in Tris-HCl/acetic acid/EDTA (TAE) buffer. The gel was stained with Sybr Green I (Takara), and the fluorescence image was analyzed with a Fluor Imager (Molecular Dynamics, Sunnyvale, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
After aspirates were obtained from the 35 surgically dissected thyroid tissues, RNA extraction was performed followed by RT-PCR to amplify RET, calcitonin, CEA, thyroglobulin, and GAPDH cDNAs (Fig. 2Go). GAPDH mRNA was detected in all samples. Thyroglobulin mRNA was detected in all normal thyroids, adenomatous goiters, follicular adenomas, papillary carcinomas, and follicular carcinomas. It was also weakly detected in four of 11 MTCs and was hardly detectable in the rest of the MTC samples. The expression of RET, calcitonin, and CEA mRNAs was detected in the aspirates from all MTCs but not in those from non-MTC tumors. Furthermore, to clarify the difference between the results using the tissues and the ABRP samples, RT-PCR analysis using RNAs from the tumor tissues was also performed. The results showed no difference (Fig. 3Go).



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  		Figure 2. ABRP analysis of the expression of RET, calcitonin, and CEA mRNAs. Samples of six normal thyroid tissues (N) from the opposite lobe of thyroid carcinomas, three adenomatous goiters (A), six follicular adenomas (FA), seven papillary carcinomas (P), two follicular carcinomas (FC), and 11 MTCs (M) were surgically dissected, followed by immediate performance of ABRP as described in Materials and Methods. Arrows indicate the expected positions of the PCR products.

 


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  		Figure 3. Expression of RET, calcitonin, and CEA mRNAs in thyroid tumors. A portion of the tissue samples used in Fig. 2Go were dissected, and then RT-PCR analyses of RET, calcitonin, and CEA mRNAs were performed. Arrows indicate the expected positions of the PCR products.

 
Aspirates were obtained from a thyroid nodule (case 1: a 72-yr-old woman) that was suspected of being an MTC based on ultrasonography findings, and from two metastatic lymph nodes of MTC (case 2: a 17-yr-old man, MEN 2B; and case 3: a 45-yr-old woman). RNA samples that were screened by ABRP detected RET, calcitonin, and CEA mRNAs in all three patients’ aspirates, leading to a preoperative diagnosis of MTC. These diagnoses agreed with the subsequent cytological and histological findings (Fig. 4Go).



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  		Figure 4. Expression of RET, calcitonin, CEA, and thyroglobulin mRNAs in preoperative aspirates from MTCs. Arrows indicate the expected positions of the PCR products. P, an aspirate from a papillary carcinoma as a negative control; M1, an aspirate from an MTC; M2 and M3, aspirates from recurrent lymph nodes of MTCs.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Like most other tumors occurring in the thyroid, MTC are usually diagnosed by palpation, ultrasonography, and FNAB followed by cytological examination. Also, calcitonin and CEA in patient sera are useful tumor markers, both pre- and postoperatively. Further, sequence analysis to detect germ-line mutations in the RET proto-oncogene is a powerful tool for screening the members of high risk families to determine who will develop MEN 2.

In addition to the above examination, immunohistochemistry to stain calcitonin using FNAB-derived samples is sometimes useful to confirm a diagnosis of MTC (33, 34). In the present study, RET, calcitonin, and CEA mRNAs were clearly detected in the aspirates from the tissues of all 11 MTCs, but not in other tumors or normal thyroid tissues. They were also detected in three RNA samples from MTCs preoperatively obtained by ABRP. ABRP provides both a slide sample for cytological examination and RNA for molecular diagnosis, and the whole procedure for the preparation of both samples takes only a few minutes. ABRP provides cDNA for as many as 20 RT-PCR analyses, with a single RT-PCR analysis taking only 6 h. Thus, ABRP analysis for the detection of either RET, calcitonin, or CEA mRNA, can be considered more sensitive and perhaps more clinically convenient than immunohistochemistry alone.

While calcitonin and CEA mRNAs were clearly detected in all MTCs, RET mRNA was only weakly detected in two of them, probably due to the lower expression of RET, mRNA than of calcitonin or CEA. Thus calcitonin and CEA mRNAs may be more suitable targets than RET mRNA for use in ABRP diagnosis of MTC. However, we must qualify this conclusion by emphasizing that the sensitivity of the PCR analysis depends on the primer design, the reaction conditions, and the area from which the needle is sampling.

Thyroglobulin mRNA was weakly detected in aspirates from 4 of 11 tissues of MTC and 1 of 3 preoperatively obtained aspirates. These results are consistent with previous immunohistochemical and Northern blot studies that have demonstrated thyroglobulin mRNA expression in only a few MTCs (35, 36). By analyzing the relative expression levels of RET, calcitonin, or CEA mRNA by competitive RT-PCR analysis with thyroglobulin mRNA, we may be able to establish an accurate molecular-based method for the diagnosis of MTC (37).

Using ABRP, multiple genetic analyses by RT-PCR are possible. The combination of cytological examination and genetic analyses, the latter detecting the expression of RET, calcitonin, or CEA mRNA in those samples in which thyroglobulin mRNA is absent or only weakly detected, might be an efficient preoperative method to screen for MTC.


   Footnotes
 
1 This work was supported by a Grant-in-Aid for Encouragement of Young Scientists (to T.T.; No. 10771346) from the Ministry of Education, Science, Sports and Culture of Japan, and a Grant-in-Aid from Clinical Pathology Research Foundation of Japan. Back

Received December 2, 1997.

Revised September 16, 1998.

Revised December 15, 1998.

Accepted December 17, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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