Expression of Oncofetal Fibronectin Messenger Ribonucleic Acid in

0021-972X/00/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 2000 by The Endocrine Society

Vol. 85, No. 2 Printed in U.S.A.

Expression of Oncofetal Fibronectin Messenger Ribonucleic Acid in Fibroblasts in the Thyroid: A Possible Cause of False Positive Results in MolecularBased Diagnosis of Thyroid Carcinomas* TORU TAKANO, AKIRA MIYAUCHI, FUMIO MATSUZUKA, KANJI KUMA, NOBUYUKI AMINO

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Department of Laboratory Medicine, Osaka University Medical School (T.T., N.A.), D2, 2–2 Yamadaoka, Suita, Osaka 565-0871; and Kuma Hospital (A.M., F.M., K.K.), 8 –2-35, SimoyamateDori, Chuo-Ku, Kobe, Hyogo 650-0011, Japan ABSTRACT Oncofetal fibronectin (onfFN) messenger ribonucleic acid (mRNA) is abundantly expressed in thyroid papillary and anaplastic carcinomas. These carcinomas can be preoperatively diagnosed by the detection of onfFN mRNA in fine needle aspiration biopsies (FNABs). However, previous reports have noted that the expression of onfFN mRNA was observed in 3.7% of the FNABs that were diagnosed as negative cytology. To clarify this discrepancy, we examined the ex-

pression of onfFN mRNA in fibroblasts in the thyroid. By RT-PCR and real-time quantitative RT-PCR analyses, we detected a high copy number of onfFN mRNA in cultured fibroblasts obtained from the normal thyroid tissues dissected surgically. Thus, we conclude that the contaminated fibroblasts in FNABs due to tumor necrosis or acute or chronic inflammation may be a cause of false positive results in molecular-based diagnosis of thyroid carcinomas. (J Clin Endocrinol Metab 85: 765–768, 2000)

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NCOFETAL fibronectin (onfFN) is characterized by the presence of the oncofetal domain (IIICS domain), and the existence of onfFN is reported in various kinds of malignant tissue (1–3). We have reported the restricted expression of onfFN messenger ribonucleic acid (mRNA) in thyroid papillary and anaplastic carcinomas and confirmed this by Northern blot analysis, RT-PCR, and in situ hybridization (4, 5). In addition, Ryu et al. reported the expression of intracellular fibronectin in thyroid carcinomas detected immunohistochemically (6, 7). Thus, onfFN mRNA as well as human telomerase reverse transcriptase and galectin-3 (8, 9) are considered to be ideal markers for molecular-based diagnosis of thyroid papillary and anaplastic carcinomas. Our recent study demonstrated the clinical usefulness of preoperative diagnosis of these carcinomas by the detection of onfFN mRNA in leftover cells inside the needles used for fine needle aspiration biopsies (FNABs) (10 –12). This method, aspiration biopsy-RT-PCR (ABRP), showed a diagnostic accuracy as high as that of an expert cytopathologist. In the previous study, however, the expression of onfFN mRNA was observed in 3.7% of the FNABs that were diagnosed as negative for papillary or anaplastic carcinoma even though no other cells besides papillary and anaplastic carcinoma cells express onfFN mRNA in in situ hybridization

studies (5). onfFN is sometimes detected by RT-PCR in FNABs from diffuse goiters with acute or chronic inflammation or cystic regions in the thyroid (data not shown), and a previous study reported the expression of fibronectin mRNA with the IIICS sequence in human fetal lung fibroblasts (13). Considering these facts, it is likely that proliferating fibroblasts in connective tissue in the thyroid express fibronectin mRNA with the IIICS sequence, and aspiration of a considerable number of fibroblasts by FNAB results in the false positive results in ABRP analysis. In light of this information, we examined the expression of onfFN mRNA in the fibroblast cultures from normal thyroid tissues by RT-PCR and quantified its copy number by realtime quantitative RT-PCR (14, 15) to estimate the effect of onfFN mRNA detection on the molecular-based diagnosis of thyroid carcinomas. Materials and Methods RNA extraction and cell culture Tissue samples from 10 follicular adenomas, 10 papillary carcinomas, and 7 thyroid tissues (3 from patients with Graves’ disease, 2 from the adjacent tissues of follicular adenomas, and 2 from the opposite lobes of medullary carcinomas) were obtained by surgery after informed consent was obtained. All tissues were frozen in liquid nitrogen immediately after resection. Total RNA was extracted according to the method of Chomczynski and Sacchi (16). Before freezing, part of the 7 thyroid tissues (⬃500 mg each) was cut into small fragments, and the fragments were digested at 37 C in Ham’s F-12 medium containing 10% FCS (Life Technologies, Inc., Gaithersburg, MD) and 2 mg/mL collagenase (CLS2, Funakoshi, Tokyo, Japan) for 3 h. Cells were collected by centrifugation at 500 ⫻ g for 10 min and were sparsely seeded in three 10-cm culture dishes. Cells were cultured at 37 C in Ham’s F-12 medium containing 10% FCS, and the medium was replaced every 3 days. After 10 days, when most of the surface of each dish was covered by proliferating

Received June 10, 1999. Revision received October 6, 1999. Accepted October 22, 1999. Address all correspondence and requests for reprints to: Dr. Toru Takano, Department of Laboratory Medicine, D2, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka 565-0871, Japan. * This work was supported by 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 Kurozumi Medical Foundation.

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fibroblasts, cells were scraped and collected by centrifugation at 500 ⫻ g for 10 min. Total RNA was extracted as described above. Total RNA was also extracted from human peripheral blood by Isogen-LS (Wako, Osaka, Japan) according to the manufacturer’s protocol.

RT RT was performed using 1 ␮g total RNA in a RT mixture containing 50 mmol/L Tris-HCl (pH 8.3), 75 mmol/L KCl, 10 mmol/L dithiothreitol, 3 mmol/L MgCl2, 0.5 mmol/L deoxy-NTPs (dNTPs), 200 U Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.), 2 U/␮L ribonuclease inhibitor (Takara, Shiga, Japan), and 2.5 ␮mol/L oligo(deoxythymidine) (Life Technologies, Inc.) in a total volume of 20 ␮L at 37 C for 60 min.

RT-PCR analysis The PCR reaction to amplify onfFN complementary DNA (cDNA) with the IIICS sequence was performed as previously described (11). cDNAs from FNABs from a follicular adenoma and a papillary carcinoma were used for a negative and positive control, respectively. The oligonucleotides used as intron-spanning primers were 5⬘-AAGGCATAGGCCAAGACCATAC-3⬘ (bases 6127– 6148) and 5⬘-ATGCGAATTCGTTTTTTTTTTTTTTTTTTT-3⬘ (poly A-anchor primer). All primers were purchased from Life Technologies, Inc.. Each reaction mixture consisted of 1 ␮L cDNA, 0.5 ␮mol/L of each primer, 1 ␮L of 10 ⫻ Ex Taq buffer, 0.8 ␮L dNTP mix, 0.5 U Ex Taq polymerase, and nuclease-free water to a final volume of 10 ␮L. Ten ⫻ Ex Taq buffer, dNTP mix, and Ex Taq polymerase were obtained from Takara. The reaction mixture was subjected to 35 cycles of denaturation (94 C, 1 min), annealing (55 C, 1 min), and extension (72 C, 1 min). After PCR amplification, the reaction mixture was run on 1% SeaKem GTG agarose gel (Takara). The gel was then stained with ethidium bromide. Direct sequencing of the positive bands was performed using a Suprec-01 (Takara) and a Dye Terminator Cycle Sequencing FS Ready Reaction Kit (Perkin-Elmer Corp., Foster City, CA) according to the manufacturer’s protocol.

Real-time quantitative PCR Real-time quantitative PCR (TaqMan PCR) using an ABI PRISM 7700 Sequence Detection System and a TaqMan PCR Core Reagent Kit (Perkin-Elmer Corp.) was performed according to the manufacturer’s protocol. One microliter of the first strand cDNA was used in the following assay. The copy number of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as an internal control. The two primers and one TaqMan probe used for the quantification of onfFN (IIICS sequence), thyroglobulin, and GAPDH mRNA were (17–19): onfFN5 (0.5 ␮mol/L), 5⬘-TCTTCATGGACCAGAGATCT-3⬘ (bases 5932–5951); onfFN3 (0.5 ␮mol/L), 5⬘-TATGGTCTTGGCCTATGCCT-3⬘ (bases 6128 – 6147); onfFN-TM (10 pmol), 5⬘-FAM-AGCAACCCAGTGTTGGGCAACATAMRA-3⬘ (bases 6045– 6066); Tg5 (0.5 ␮mol/L), 5⬘- GAGAAGAGCCTGTCGCTGAA-3⬘ (bases 7980 –7999); Tg3 (0.5 ␮mol/L), 5⬘-CAGCTCACTGAACTCCTTGT-3⬘ (bases 8128 – 8147); and Tg-TM (10 pmol), 5⬘-FAM-TGAGTTCTCACGGAAAGTACCCA-TAMRA-3⬘ (bases 8054 – 8076); and GAPDH5 (0.5 ␮mol/L), 5⬘-TCCATGACAACTTTGGTATC-3⬘ (bases 551–570); GAPDH3 (0.5 ␮mol/L), 5⬘-AAGGTCATCCCTGAGCTAGA-3⬘ (bases 715–734); and GAPDH-TM (10 pmol), 5⬘-FAMAGAACATCATCCCTGCCTCTACT-TAMRA-3⬘ (bases 671– 693), respectively. The copy number of each cDNA was measured in the separate plate. The conditions for the TaqMan PCR were as follows: 95 C for 10 min, followed by 40 cycles of 95 C for 15 s and 60 C for 1 min. A recombinant pGEM T-vector (Promega Corp., Tokyo, Japan) containing onfFN, thyroglobulin, or GAPDH cDNA was constructed by PCR cloning with the same set of primers used in the TaqMan PCR and was used as the standard sample. The representative amplification plots of onfFN cDNA are shown in Fig. 1A. These plots were used to determine the threshold cycle (CT). The CT value represented the PCR cycle at which an increase in reporter fluorescence (⌬Rn) above the line of the optimal value (optimal ⌬Rn) was first detected. A plot of the CT against the input target quantity (common log scale) is shown in Fig. 1B. The initial copy number of the target mRNA was calculated by this plot. No

FIG. 1. Measurement of the copy number of onfFN mRNA by realtime quantitative PCR. A, Representative amplification plots of onfFN cDNA. Five different dilutions, including 41 pg (f), 5.12 pg (䡺), 640 fg (Œ), 80 fg (‚), and 10 fg (), of the plasmid DNA containing the sequence of onfFN cDNA and cDNAs from cultured fibroblasts (F) and from a papillary carcinoma (ƒ) were tested. The horizontal line shows the optimal ⌬Rn. B, A plot of the threshold cycle (CT) against the input target quantity (common log scale). F, Fibroblasts; ƒ, papillary carcinoma.

increase in ⌬Rn was observed in the samples without RT or with no template cDNA.

Results

Expression of onfFN mRNA was detected in all seven cultures of fibroblasts from the thyroid tissues by RT-PCR, whereas onfFN mRNA was not detected in the peripheral blood (Fig. 2). Some positive bands were confirmed to be derived from the fibronectin cDNA by direct sequencing (data not shown). The positive bands were not detected in the samples without RT (data not shown). The expression levels of thyroglobulin and onfFN mRNAs in thyroid tissues and cultured fibroblasts were measured by real-time quantitative RT-PCR. The copy number of GAPDH cDNA was about 106 (data not shown). The expression levels of thyroglobulin mRNA in the tissues samples were about 102–104 times more than those in the cultured fibroblasts, showing there was only a small remnant of the thyroid fol-

FIBRONECTIN IN FIBROBLASTS IN THE THYROID

licular cells in the cultures. On the other hand, onfFN mRNA greatly increased in the fibroblast cultures (Fig. 3). The onfFN/GAPDH mRNA ratio in fibroblasts was compared with that in follicular adenomas and papillary carci-

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nomas (Fig. 4). Fibroblasts expressed a much higher copy number of onfFN mRNA than all follicular adenomas did. They also expressed onfFN mRNA at a higher level than 6 of 10 papillary carcinomas did. Discussion

FIG. 2. Expression of onfFN mRNA in cultured fibroblasts. cDNAs of cultured fibroblasts from the tissues from patients with Graves’ disease (lanes 4 – 6), the adjacent tissues of follicular adenomas (lanes 7 and 8), and the opposite lobes of medullary carcinomas (lanes 9 and 10) were amplified by PCR with specific primers for the onfFN gene. The PCR products were run on 1% agarose gel. The gel was then stained with ethidium bromide. The arrow indicates the expected position of the PCR product. M, PHY DNA size markers (Takara); lanes 1–3, PCR products using cDNAs of the peripheral blood (lane 1) and aspirates from a follicular adenoma (lane 2) and a papillary carcinoma (lane 3).

FIG. 3. The thyroglobulin/GAPDH mRNA ratio (A) and onfFN/ GAPDH mRNA ratio (B) measured by real-time quantitative RT-PCR in thyroid tissues and cultured fibroblasts. Each circle and square shows the mean of a duplicate assay. F, Tissues from patients with Graves’ disease and their cultures; E, adjacent tissues of follicular adenomas and their cultures; f, tissues and cultures form the opposite thyroid lobe of medullary carcinomas.

Thyroid carcinomas are usually diagnosed by palpation, ultrasonography, and FNAB followed by cytological examination. In addition to these diagnostic methods, our recent study examined the clinical usefulness of the preoperative diagnosis of papillary and anaplastic carcinomas, which make up 90% of thyroid malignancies in iodide-sufficient countries, by ABRP detection of oncofetal fibronectin mRNA, which has previously been thought to be expressed only in these two carcinomas (11, 20). In the present study the cultured fibroblasts derived from normal thyroid tissues were shown to express a high copy number of onfFN mRNA, although the possibility remains that the expression of onfFN mRNA was enhanced by the culture conditions, such as the high concentration of FBS. Further, a previous study reported the lack of expression of the IIICS domain in lymphocytes that frequently contaminate FNA samples (21). Thus, the contamination of fibroblasts in FNABs can be a cause of false positive results in ABRP analysis, because a FNAB sample in which onfFN mRNA is detected by RT-PCR might be diagnosed as papillary or anaplastic carcinoma. In FNABs of nodular goiters, however, the false positive rate of ABRP is limited to less than 3.7%, which indicates that there is generally a low level of fibroblast contamination in FNABs. This is because connective tissues, including fibro-

FIG. 4. The onfFN/GAPDH mRNA ratio measured by real-time quantitative RT-PCR in thyroid tumors and cultured fibroblasts. Each closed circle shows the mean of a duplicate assay. FB, Fibroblasts; FA, follicular adenomas; PC, papillary carcinomas.

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blasts, are usually hard to aspirate by FNAB compared with thyroid tumor cells or blood cells. However, when focal acute or chronic inflammation or tumor necrosis occurs in the thyroid, especially when the patient suffers from chronic thyroiditis, subacute thyroiditis, or the degradation caused by an adenomatous goiter, connective tissues containing fibroblasts may be easily aspirated by FNAB. Thus, in these cases, detection of the specifically expressed genes, such as collagen genes in FNABs, may help to prevent a false positive result, and by quantifying the expression levels of these fibroblast-specific genes together with onfFN and thyroglobulin genes in FNABs by real-time quantitative RT-PCR, we may be able to establish a more accurate system of molecularbased diagnosis of thyroid carcinomas (22). References 1. Matsuura H, Hakomori S. 1985 The oncofetal domain of fibronectin defined by monoclonal antibody FDC-6: its presence in fibronectins from fetal and tumor tissues and its absence in those from normal adult tissues and plasma. Proc Natl Acad Sci USA. 82:6517– 6521. 2. Loridon-Rosa B, Vielh P, Matsuura H, Clausen H, Cuadrado C, Burtin P. 1990 Distribution of oncofetal fibronectin in human mammary tumors: immunofluorescence study on histological sections. Cancer Res. 50:1608 –1612. 3. Inufusa H, Nakamura M, Adachi T, et al. 1995 Localization of oncofetal and normal fibronectin in colorectal cancer. Correlation with histologic grade, liver metastasis, and prognosis. Cancer. 75:2802–2808. 4. Takano T, Matsuzuka F, Sumizaki H, Kuma K, Amino N. 1997 Rapid detection of specific messenger RNAs in thyroid carcinomas by reverse transcription-PCR with degenerate primers: specific expression of oncofetal fibronectin messenger RNA in papillary carcinoma. Cancer Res. 57:3792–3797. 5. Takano T, Matsuzuka F, Miyauchi A, et al. 1998 Restricted expression of oncofetal fibronectin mRNA in thyroid papillary and anaplastic carcinoma: an in situ hybridization study. Br J Cancer. 78:221–224. 6. Ryu S, Jimi S, Takebayashi S. 1997 Thyroid carcinoma distinctively expresses intracellular fibronectin in vivo. Cancer Lett. 121:189 –193. 7. Ryu S, Jimi S, Eura Y, Kato T, Takebayashi S. 1998 Retention of intracellular fibronectin expression in primary and metastatic thyroid carcinoma: an immunohistochemical study. Cancer Lett. 133:215–222.

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