Accurate and Objective Preoperative Diagnosis of Thyroid Papillary

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[CANCER RESEARCH 58. 491.1-4917, November I. 1998] Accurate and Objective Preoperative Diagnosis of Thyroid Papillary Carcinomas by Reverse Transcription-PCR Detection of Oncofetal Fibronectin Messenger RNA in Fine-Needle Aspiration Biopsies1

Toru Takano,2 Akira Miyauchi, Tamotsu Yokozawa, Fumio Matsuzuka, Gang Liu, Takuya Higashiyama, Shinji Morita, Kanji Kuma, and Nobuyuki Amino Departments of Laboratory Medicine Â¡T.T., G. L, N. A.] and Surgical Oncology IT. HJ, Osaka University Medical School. Osaka 565-0871. and Kuma Hospital Kobe. Hyngn 650-0011. Â¡A.M.. T. Y., F.M.. S. M.. K. KJ. Japan

ABSTRACT cannot be used for this purpose because substantial contamination of blood cells often occurs in FNAB. Recently, the restricted expression of oncofetal fibronectin mRNA was In our previous study (13), we used a PCR-based method. Sequence reported in thyroid papillary and anaplastic carcinomas. In this study, by Specific Differential Display, to screen for specific mRNAs in thyroid extracting RNA from the leftover cells inside the needles used for fine- carcinomas, and succeeded in finding several mRNAs the expression needle aspiration biopsy, we establish a new method for gene diagnosis of of which is restricted in cancer tissues. One such gene was oncofetal these carcinomas without further invasiveness to the patient (aspiration fibronectin. Fibronectins are high-molecular-mass adhesive glycopro- biopsy-reverse transcription-PCR, ABRP). RNA was extracted from 177 fine-needle aspiration biopsies of thyroid nodules that were suspicious for teins present in the extracellular matrix and in body fluids (14). malignancy, and then the gene diagnoses made by reverse transciption- Oncofetal fibronectin is characterized by the presence of the oncofetal PCR detection of oncofetal fibronectin mRNA were compared with cyto- domain (IIICS domain), which is absent in normal fibronectin (15). logical diagnoses. Thirty-five (94.6%) of 37 samples that were diagnosed Many researchers have used this antibody in Â¡mmunohistochemical as papillary or anaplastic carcinomas by cytological examination showed analysis to indicate the existence of oncofetal fibronectin in such a positive result by gene diagnosis, whereas only 4 (3.7%) of 109 samples malignant tissues as oral, breast, colon, and gastric cancers (16-19); that were cytologically diagnosed negative for both carcinomas showed a in addition, a recent study (20) showed the expression of intracellular positive result. Among all of the cases, 50 patients underwent surgery, and fibronectin in thyroid carcinomas. In our previous study of tissues a histolÃ³gica! diagnosis was consequently made. The sensitivity and spec from 98 thyroid tissue samples (13), we found that papillary and ificity of this method were 96.9 and 100%, respectively. A combined anaplastic carcinomas could be distinguished from normal thyroid examination using both genetic and cytological approaches may contrib tissues, follicular adenomas, and follicular carcinomas by RT-PCR ute to a more precise preoperative diagnosis of papillary and anaplastic amplifying oncofetal fibronectin cDNA. Subsequently, the restricted carcinomas. expression of oncofetal fibronectin mRNA in papillary and anaplastic carcinomas was further confirmed by in situ hybridization (21 ). Thus, INTRODUCTION oncofetal fibronectin is considered to be an ideal target for gene diagnosis and therapy of these two carcinomas, which together com Thyroid tumors are often diagnosed by FNAB3 as well as by prise about 90% of all malignant thyroid tumors in iodide-sufficient ultrasonography. Cytological examination of FNAB by a skillful counties (22). Accurate preoperative diagnosis of papillary carcino pathologist who is an expert in thyroid tumors provides the most mas is clinically important because they usually indicate the need for reliable information for the diagnosis of thyroid neoplasms (1-3). In surgical dissection. some clinical situations, however, slide samples are not adequate for In a previous study (23), we introduced a new method of preoper cytological examination because of poor fixation, and a well-trained ative gene diagnosis of thyroid carcinomas. This technique, ABRP. expert pathologist is not always available for diagnosis. In such cases, allows us to perform cytological and gene diagnoses simultaneously. a more objective method is required for exact diagnosis. Gene diag After preparing a slide glass for cytological examination, we extract nosis by analyzing nucleic acids from aspirates may be used for this RNA from leftover cells within the needle used for FNAB for RT- purpose. Most previous trials, however, have failed to provide much PCR analysis. ABRP thus provides both RNA information and a information on the clinical features of thyroid tumors (4, 5). Thyroid cytological diagnosis without further invasiveness to the patient. The cancers are known to overexpress a number of genes, such as EOF RNA extracted from a single FNAB provides us with sufficient cDNA receptor, c-erbB, IGF-I, p53, dipeptidyl aminopeptidase IV, c-Met, for as many as 20 PCR examinations. Furthermore, the results of and CD44E (6-12). However, mRNAs of these genes are not ade cytological and gene diagnoses using the same FNAB can be com quate for gene diagnosis by RT-PCR because their expression is not pared. Most importantly, only a few minutes are required to produce restricted in cancer cells and because not all of the cancer tissues both a slide glass and a RNA sample. Thus, it is possible to collect overexpress their mRNAs. As for genes expressed in blood cells, they samples for gene diagnosis in the midst of clinical work. In this study, we first performed ABRP using the 72 surgically dissected benign and malignant thyroid tissues to confirm the useful Received 4/2/98; accepted 8/31/98. The costs of publication of this article were defrayed in part by the payment of page ness of this method. Next, to establish the method of preoperative charges. This article must therefore be hereby marked advertisement in accordance with gene diagnosis of thyroid papillary and anaplastic carcinomas, ABRP 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported hy a Grant-in-Aid 10771346 for Encouragement of Young was performed in 155 patients, and the results of the cytological and Scientists (to T. T.) from the Ministry of Education, Science. Sports and Culture of Japan gene diagnoses were compared. Finally, the sensitivity and specificity and Granls-in-Aid from Osaka Cancer Research Foundation and Clinical Pathology of ABRP were determined postoperatively. Research Foundation of Japan. 2 To whom requests for reprints should be addressed. Department of Laboratory Medicine. Osaka University Medical School. 2-2 Yamadaoka. Suita. Osaka 565-0871. MATERIALS AND METHODS Japan. 3 The abbreviations used are: FNAB. fine-needle aspiration biopsy; ABRP. aspiration biopsy-reverse transcription-polymerase chain reaction; dNTP. deoxynucleotide triphos- ABRP Using Surgical Tissues. Seventy-two thyroid tissues (23 normal phate: RT-PCR. reverse transcription-PCR; UG-FNAB, ultrasound-guided FNAB. thyroid tissues from the opposite lobe of the thyroid carcinomas. 14 adeno- 4913

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1998 American Association for Cancer Research. DIAGNOSIS OF THYROID CARCINOMAS BY RT-PCR matous goiters, 13 tollicular adenomas, 3 follicular carcinomas, 18 papillary carcinomas, and 1 anaplastic carcinoma) were surgically dissected and used immediately. ABRP was performed as previously described Ret. 23; Fig. 1). A syringe with a 22-gauge needle was used for FNAB from each tissue sample. onfFN Samples were prepared on a slide glass for cytological examinations, and then leftover cells inside the needle were lysed rapidly and flushed into a 1.5-ml tube using a denaturing solution containing 4 M guanidine thiocyanate. 25 mM sodium citrate (pH 7.0), 0.5% sarcosyl. and O.I M2-mercaptoethanol. The tube was stored at 4Â°C.Total cellular RNA was extracted by the method of TG Chomc/ynski and Sacchi (24). After Papanicolaou staining, slide glasses were used to make sure the tumor cells were aspirated from the tissues (25). Reverse transcription was performed using the whole RNA extracted in a reverse transcription mixture containing 50 mM Tris-HCl (pH 8.3), 75 mM KC1, 10 mM M 1 2 3 456 7 8 91011 DTT. 3 mM MgCl2, 0.5 mM dNTPs, 200 units M-MLV reverse transcriptase N AG FA (Life Technologies. Gaithersburg. MD), 2 units//Â¿lRNase inhibitor (Takara. Shiga, Japan), and 2.5 /J.Moligo dT (Life Technologies) in a total volume of 20 Ã•Â¿1at37Â°Cfor 60 min. One ^1 of first strand cDNA was used as a template for the PCR reaction with specific primers for either oncofetal fibronectin (26) or onfFN thyroglobulin (27). For the amplification of oncofetal fibronectin cDNA, a polyl A (-anchor primer (13) was used instead of a 3' specific primerio prevent false positive results. The oligonucleotides used as primers were: (

The sensitivity of cytology was calculated as:

B) C) Number of samples of positive and suspicious cytology from papillary and anaplastic carcinomas Number of papillary and anaplastic carcinomas

and the specificity of cytology was calculated as:

Number of samples of negative cytology from other tumors than papillary and anaplaslic carcinomas Number of other tumors than papillary and anaplastic carcinomas

Fig. 1. Procedures of ABRP. Tumor cells were aspirated by UG-FNAB using a syringe with a 22-gauge needle (A}. After preparing a sample on a slide glass for cytological RESULTS examinations (lÃ¬),theneedle was dipped into a 1.5-ml tube containing denaturing solution (O. The denaturing solution was aspirated in and out of the tube three times to lyse the To confirm the reliability of ABRP, we aspirated 72 thyroid tissues cells inside the needle, and then the tube was stored at 4Â°C. and examined the expression of oncofetal fibronectin and thyroglob- 4914

materials were 28 (15.8%) for the cytological diagnosis and 9 (5.1%) for the gene diagnosis. Among the 155 patients, 50 underwent sur gery, and, thus, histolÃ³gica! diagnoses were made in these cases onfFN (Table 3). Both the gene and cytological diagnoses agreed with the histological diagnosis in 47 of 48 cases. The sensitivity and specificity were 96.9 and 100% for the gene diagnosis, and 100 and 93.8% for the cytological diagnosis, respectively (Table 4). TG DISCUSSION

M ' 234567 Nodular thyroid disease is a frequently observed condition, espe cially in women. The prevalence of palpable thyroid nodules is reported to be 4 to 7% in adults (30). FNAB has been used as a common method of preoperative diagnosis of thyroid nodules, and its safety and reliability have been established by many studies. For a successful diagnosis, FNAB requires the following three conditions: onfFN (a) accurate aspiration by a well-trained thyroidologist: (b) quick and accurate preparation of slide samples; and (c) intensive cytological examination by a skilled pathologist. Accurate aspiration may be performed by the use of UG-FNAB (2). The training for the prepa ration of slide samples is relatively easy. However, the final step, that of the cytological examination, is often problematic. Diagnosis by a TG pathologist specializing in thyroid tumors may not always be possible. Thus, in many cases, screeners who are not familiar with thyroid diseases are engaged in the diagnosis. In fact, the requirement for an M12131415161718 19 2021 22 expert cytopathologist has discouraged the wide use of FNAB in a number of countries, and the wide variance in the previously reported sensitivity and specificity of FNAB may be the result, at least in part, Fig. 3. Representative gel image of ABRP gene diagnosis. After FNAB. total RNA was extracted from leftover cells inside the needle. Then RT-PCR analysis was performed to of varying skills among the cytopathologists (31). Objective diagnosis detect either oncofetal ftbronectin (onfFW)or thyroglobulin (TG) mRNA as described in by FNAB may be established by genetic analysis. However, many "Materials and Methods." The PCR products were run on l% agarose gel. The gel was previous trials failed to yield a sensitivity and specificity comparable then stained with ethidium bromide. Arrows, the expected positions of each PCR product. M. PHY DNA size markers (Takara);(-), negative cytology: (Â±).suspicious cytology: to that of cytological examinations because no target genes capable of and (+ ). positive cytology. clearly distinguishing malignant and benign thyroid tumors had yet been found. Our recent finding of the restricted expression of oncofe tal fibronectin mRNA in thyroid papillary and anaplastic carcinomas ulin mRNA by RT-PCR. A representative gel image is shown in Fig. enabled us to establish an efficient method of gene diagnosis by 2. Thyroglobulin mRNA was detected in all of the samples, including ABRP. one anaplastic carcinoma sample. Oncofetal fibronectin mRNA was In the present study, the sensitivity and specificity of ABRP in the detected in all of the papillary and anaplastic carcinomas but not in diagnosis of papillary and anaplastic carcinomas were 96.9 and 100%, normal thyroid tissues, follicular adenomas, adenomatous goiters, or respectively. These results are almost the same as those derived from follicular carcinomas (Table 1). Next, 177 aspirates from 155 patients cytological examination by a skillful pathologist. Previous studies were examined. A representative gel image is shown in Fig. 3. reported the sensitivity and the specificity of FNAB for thyroid Oncofetal fibronectin mRNA was detected in 94.6, 66.7, and 3.7% of malignancy as 65-98% and 72-100%, respectively (31). However, it the samples that were diagnosed as positive, suspicious, and negative is difficult to directly compare these previous results and our present cytology, respectively (Table 2). The total numbers of the inadequate results for two reasons: (a) in most of the reports, 10-30% of the samples are cytologically Table 1 ABRPfrom H.v.vi/c1sample* determined as "intermediate" or "suspicious" (32). Therefore, the

TissueNormal number2314133181onfFN(+)"0(10(I181onfFN(-)231413300 results differ considerably depending on whether these samples were seen as negative or positive (for example, including intermediate thyroidAdenomatous goiterFollicular samples in the positive group increases the sensitivity but decreases adenomaFollicular the specificity); and carcinomaPapillary (b) especially in the reports from iodide-deficient countries, a carcinomaAnaplastic carcinomaTotal considerable number of cases of follicular carcinomas, which show a 1onfFN. oncofetal fibronectin; -K detected: -. not detected. negative result by ABRP, are included in the category of "malignant"

Table 2 Comparison between the cytology tinti ABRP Tg<+ )" Tg(-)

Cytology (n =177)Positive of cases(%)37 +)35 +)0 (20.9) (94.6%) (2.7%) (2.7%) Suspicious 3(1.7) 2 (66.7%) 1(33.3%) 0 0 Negative 109(61.6) 4 (3.7%) 101(92.7%) 00onfFN(-)1 4 (3.7%) Inadequateno. 28(15.8)onfFN( 4(14.3%)onlFN(-)1 20(71.4%)onfFN( 4(14.3%) 'Tg. thyroglobulin: onfFN. oncofetal; +. delected; -. undetected. 4915

Table 3 Comparison between histology and ABRP Tg( + )" Tg(-)

Histology n = 50 (no. of cases] Cytology (no. of cases) onfFN( + ) onfFN(-) onfFN( + ) onfFN(-) Papillary(33)Anaplaslic carcinoma (29)Suspicious (3.0%)0001 (3.0%)01 (2)Inadequate (2)Positive (3.0%)1 (3.0%)000000 ]Follicular carcinoma [1 (1)Positive (100%)000001 [4|Kollicularcarcinoma (1)Negative (25.0%)3 (3)Negative (75.0%)6(100%)5(100%)1 [6]Adcnomatousadenoma (6)Negative [5]Graves' goiter (5)Negative disease [ 1]Positive ( 1)27(81.8%)2(6.1%)1 (100%)0000000(101 'Tg. thyroglobulin: onfFN. oncofetal fibronectin; + , detected; -, undetected. tumors without classification by the histolÃ³gica! typing of papillary, samples (5.1%) have been determined to be inadequate compared with tbllicular. and anaplastic carcinomas. cytological examination (15.8%). This result indicates that gene di Differentiation of tbllicular adenomas and carcinomas by ABRP is agnosis requires a smaller number of cells than does cytological not possible at present. However, we do not consider this to be a examination. The method of ultra fine-needle biopsy using 24- to serious limitation of this method, because the preoperative diagnosis 26-gauge needles ought to be established because it reduces invasion of tbllicular carcinomas is also quite difficult by cytological exami into the patients. Furthermore, up to six multiple aspirations from a nation (33). This method may be more useful in iodide-sufficient single nodule to prevent false negative results, as recommended by countries like Japan because the prevalence of follicular carcinomas is Hamburger et al. (36), may not be necessary when a combined very low. diagnosis using genetic and cytological examinations is made possi In the present study, 1 of the 50 cases operated on was misdiag- ble. nosed by ABRP. We have not been able to determine the reason for ABRP requires only a few minutes to produce both a slide glass and the differences between the cytology and the gene diagnosis. Possibly, RNA samples, far less than the time required in previous studies in the majority of cancer cells aspirated by FNAB were washed out with which aspirated materials were divided into aliquots (35). The total the blood onto the slide glass, as sometimes occurred when we cost of RT-PCR analysis of both thyroglobulin and oncofetal fi aspirated a large volume of blood or cystic fluid by FNAB. This is one bronectin mRNAs is only $4.00 (500 yen), much cheaper than that of of the major causes of the unsuccessful detection of thyroglobulin most other tests of thyroid carcinomas. Therefore, RT-PCR detection mRNA in the ABRP samples and renders these samples inadequate of oncofetal fibronectin mRNA may be used for the first screening of for gene diagnosis. One case could not be diagnosed by cytological papillary and anaplastic carcinomas. examination because of poor fixation but was correctly diagnosed by ABRP. We, therefore, consider ABRP to be as useful and accurate as cytological examination. ACKNOWLEDGMENTS Four of 109 samples that diagnosed as negative cytology showed a We thank Mayumi Hisago for technical assistance. positive result in the gene diagnosis. We could not clarify the reason for this discrepancy because these patients had not undergone an operation. Repeated FNAB may be needed in these cases for a more REFERENCES accurate diagnosis to decide whether an operation is warranted. The 1. Hamburger. J. 1. Diagnosis of thyroid nodules by fine needle biopsy: use and abuse. carcinoma cells may have remained inside the needle, failing to J. Clin. Endocrinol. Metab., 79: 335-339, 1994. transfer onto the slide glass, which sometimes happens when only a 2. Yokozawa. T., Miyauchi, A., Kuma. K., and Sugawara, M. 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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1998 American Association for Cancer Research. Accurate and Objective Preoperative Diagnosis of Thyroid Papillary Carcinomas by Reverse Transcription-PCR Detection of Oncofetal Fibronectin Messenger RNA in Fine-Needle Aspiration Biopsies

Toru Takano, Akira Miyauchi, Tamotsu Yokozawa, et al.

Cancer Res 1998;58:4913-4917.

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